**Part 1**

## **Novel Approaches to Cancer Treatment**

**1** 

*USA* 

**Discovery and Optimization of** 

**Novel Drugs for Cancer Therapy** 

Jakyung Yoo and José L. Medina-Franco *Torrey Pines Institute for Molecular Studies* 

**Inhibitors of DNA Methyltransferase as** 

The genome contains genetic and epigenetic information. While the genome provides the blueprint for the manufacture of all the proteins required to create a living thing, the epigenetic information provides instruction on how, where, and when the genetic information should be used (Robertson, 2001). The major form of epigenetic information in mammalian cells is DNA methylation that is the covalent addition of a methyl group to the 5-position of cytosine, mostly within the CpG dinucleotide (Robertson, 2001). DNA methylation is involved in the control of gene expression, regulation of parental imprinting and stabilization of X chromosome inactivation as well as maintenance of the genome integrity. It is also implicated in the development of the immune system, cellular reprogramming and brain function and behaviour (Jurkowska et al., 2011). DNA methylation is mediated by a family of DNA methyltransferase enzymes (DNMTs). In mammals, three DNMTs have been identified so far in the human genome, including the two *de novo* methyltransferases (DNMT3A and DNMT3B) and the maintenance methyltransferase (DNMT1), which is generally the most abundant and active of the three (Goll and Bestor, 2005; Robertson, 2001; Yokochi and Robertson, 2002). DNMT3L is a related protein that has high sequence similarity with DNMT3A, but it lacks any catalytic activity owing to the absence of conserved catalytic residues. However, DNMT3L is required for the catalytic activity of DNMT3A and 3B (Cheng and Blumenthal, 2008). The protein DNMT2 can also be found in mammalian cells. Despite the fact that the structure of DNMT2 is very similar to other DNMTs, its role is comparably less understood (Schaefer and Lyko, 2010). It has been reported that DNMT2 does not methylate DNA but instead methylates aspartic acid transfer RNA (tRNAAsp) (Goll et al., 2006). Recent evidence suggests that DNMT2 activity is not limited to tRNAAsp and that DNMT2 represents a noncanonical enzyme of the

DNMT1 is responsible for duplicating patterns of DNA methylation during replication and is essential for mammalian development and cancer cell growth (Chen et al., 2007). These enzymes are key regulators of gene transcription, and their roles in carcinogenesis have been the subject of considerable interest over the last decade (Jones and Baylin, 2007; Robertson, 2001). Therefore, specific inhibition of DNA methylation is an attractive and novel approach for cancer therapy (Kelly et al., 2010; Lyko and Brown, 2005; Portela and

**1. Introduction** 

DNMT family (Schaefer and Lyko, 2010).

## **Discovery and Optimization of Inhibitors of DNA Methyltransferase as Novel Drugs for Cancer Therapy**

Jakyung Yoo and José L. Medina-Franco *Torrey Pines Institute for Molecular Studies USA* 

## **1. Introduction**

The genome contains genetic and epigenetic information. While the genome provides the blueprint for the manufacture of all the proteins required to create a living thing, the epigenetic information provides instruction on how, where, and when the genetic information should be used (Robertson, 2001). The major form of epigenetic information in mammalian cells is DNA methylation that is the covalent addition of a methyl group to the 5-position of cytosine, mostly within the CpG dinucleotide (Robertson, 2001). DNA methylation is involved in the control of gene expression, regulation of parental imprinting and stabilization of X chromosome inactivation as well as maintenance of the genome integrity. It is also implicated in the development of the immune system, cellular reprogramming and brain function and behaviour (Jurkowska et al., 2011). DNA methylation is mediated by a family of DNA methyltransferase enzymes (DNMTs). In mammals, three DNMTs have been identified so far in the human genome, including the two *de novo* methyltransferases (DNMT3A and DNMT3B) and the maintenance methyltransferase (DNMT1), which is generally the most abundant and active of the three (Goll and Bestor, 2005; Robertson, 2001; Yokochi and Robertson, 2002). DNMT3L is a related protein that has high sequence similarity with DNMT3A, but it lacks any catalytic activity owing to the absence of conserved catalytic residues. However, DNMT3L is required for the catalytic activity of DNMT3A and 3B (Cheng and Blumenthal, 2008). The protein DNMT2 can also be found in mammalian cells. Despite the fact that the structure of DNMT2 is very similar to other DNMTs, its role is comparably less understood (Schaefer and Lyko, 2010). It has been reported that DNMT2 does not methylate DNA but instead methylates aspartic acid transfer RNA (tRNAAsp) (Goll et al., 2006). Recent evidence suggests that DNMT2 activity is not limited to tRNAAsp and that DNMT2 represents a noncanonical enzyme of the DNMT family (Schaefer and Lyko, 2010).

DNMT1 is responsible for duplicating patterns of DNA methylation during replication and is essential for mammalian development and cancer cell growth (Chen et al., 2007). These enzymes are key regulators of gene transcription, and their roles in carcinogenesis have been the subject of considerable interest over the last decade (Jones and Baylin, 2007; Robertson, 2001). Therefore, specific inhibition of DNA methylation is an attractive and novel approach for cancer therapy (Kelly et al., 2010; Lyko and Brown, 2005; Portela and

Discovery and Optimization of Inhibitors

crystal structure.

do not function via incorporation into DNA.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 5

Consequently, there is clear need to identify novel and more specific DNMT inhibitors that

Fig. 1. Mechanism of DNA cytosine-C5 methylation. Amino acid residue numbers are based on the homology model. Equivalent residue numbers in parentheses correspond to the

There is now an increasing number of substances that are reported to inhibit DNMTs (Lyko and Brown, 2005). Selected DNMT inhibitors and other candidate demethylating agents are depicted in Fig. 2. Some of these compounds are approved drugs for other indications; i.e., the antihypertensive drug hydralazine (Segura-Pacheco et al., 2003), the local anaesthetic procaine (Villar-Garea et al., 2003), and the antiarrhythmic drug procainamide (Lee et al., 2005a). Others like the L-tryptophan derivative RG108, NSC 14778 (Fig. 2) have been identified by docking-based virtual screening (Kuck et al., 2010a; Siedlecki et al., 2006). Several natural products have been implicated in DNA methylation inhibition. Selected examples are the main polyphenol compound from green tea, (-)-epigallocathechin-3-gallate (EGCG) (Fang et al., 2003; Lee et al., 2005b), other tea polyphenols such as catechin and epicatechin, and the bioflavonoids quercetin, fisetin, and myricetin. Curcumin, the major component of the Indian curry spice turmeric, has been reported to inhibit the DNA cytosine C5 methyltransferase M.SssI, an analogue of DNMT1 (Liu et al., 2009). However, more recent studies showed that curcumin did not cause DNA demethylation in three arbitrarily chosen human cancer cell lines (Medina-Franco et al., 2011). Mahanine, a plantderived carbazole alkaloid, and a fluorescent carbazole analogue, has been reported to induce the Ras-association domain family 1 (RASSF1) gene in human prostate cancer cells, presumably by inhibiting DNMT activity (Jagadeesh et al., 2007; Sheikh et al., 2010). Nanaomycin A, a quinone antibiotic isolated from a culture of *Streptomyces*, has been described as the first non *S*-adenosyl-L-homocysteine (AdoHcy/SAH) analogue acting as a DNMT3B-selective inhibitor that induces genomic demethylation. Nanaomycin A treatment reduced the global methylation levels in three cell lines and reactivated transcription of the RASSF1A tumor suppressor gene (Kuck et al., 2010b). These and several other natural products as putative demethylating agents are extensively reviewed elsewhere (Gilbert and Liu, 2010; Hauser and Jung, 2008; Li and Tollefsbol, 2010; Medina-Franco and Caulfield, 2011). While the substantial number of recent reports may suggest that many natural products inhibit DNA methylation, it should be noted that only a few reports provide compelling evidence for DNMT inhibition in biochemical and in cellular assays. As such, it

Esteller, 2010; Robertson, 2001). It is worth noting that DNA methylation inhibitors have also emerged as a promising strategy for the treatment of immunodeficiency and brain disorders (Miller et al., 2010; Zawia et al., 2009).

The structure of mammalian DNMTs can be divided into two major parts, a large Nterminal regulatory domain of variable size, which has regulatory functions, and a Cterminal catalytic domain which is conserved in eukaryotic and prokaryotic carbon-5 DNMTs. The N-terminal domain guides the nuclear localization of the enzymes and mediates their interactions with other proteins, DNA, and chromatine. The smaller Cterminal domain harbors the active center of the enzyme and contains ten amino acids motifs diagnostic for all carbon-5 DNMTs (Jurkowska et al., 2011). Motifs I-III form the cofactor binding pocket, motif IV has the catalytic cysteine, motifs VI, VIII, and X compose the substrate binding site, and motifs V and VII form the target recognition domain (Sippl and Jung, 2009). Human DNMT1 has 1616 amino acids for which limited three-dimensional structural information is available. For example, just recently a crystal structure of human DNMT1 bound to duplex DNA containing unmethylated cytosine-guanine (CG) sites was published (Song et al., 2011). Further details of the structure of DNMTs and other available crystal structures of DNMTs are extensively reviewed elsewhere (Cheng and Blumenthal, 2008; Lan et al., 2010; Sippl and Jung, 2009).

The proposed mechanism of DNA cytosine-C5 methylation is summarized in Fig. 1 (Schermelleh et al., 2005; Sippl and Jung, 2009; Vilkaitis et al., 2001). DNMT forms a complex with DNA, and the cytosine which will be methylated flips out from the DNA. The thiol of the catalytic cysteine from motif IV acts as a nucleophile that attacks the 6-position of the target cytosine to generate a covalent intermediate. The 5-position of the cytosine is activated and conducts a nucleophilic attack on the cofactor S-adenosyl-L-methionine (AdoMet) to form the 5-methyl covalent adduct and *S*-adenosyl-L-homocysteine (AdoHcy). The attack on the 6-position is assisted by a transient protonation of the cytosine ring at the endocyclic nitrogen atom N3, which is stabilized by a glutamate residue from motif VI. The same residue also contacts the exocyclic N4 amino group and stabilizes the flipped base. The carbanion may also be stabilized by resonance, where an arginine residue from motif VIII may participate in the stabilization of the cytosine base. The covalent complex between the methylated base and the DNA is resolved by deprotonation at the 5-position to generate the methylated cytosine and the free enzyme.

DNA methylation inhibitors have been well characterized and tested in clinical trials (Issa and Kantarjian, 2009). To date, only 5-azacytidine and 5-aza-2'-deoxycytidine (Fig. 2) have been developed clinically. These two drugs are nucleoside analogues, which, after incorporation into DNA, cause covalent trapping and subsequent depletion of DNA methyltransferases (Schermelleh et al., 2005; Stresemann and Lyko, 2008). Aza nucleosides are approved by the Food and Drug Administration of the United States for the treatment of myelodysplastic syndrome, where they demonstrate significant, although usually transient improvement in patient survival and are currently being tested in many solid cancers (Issa et al., 2005; Schrump et al., 2006). Despite the clinical successes achieved with DNA methylation inhibitors, there is still need for improvement since aza nucleosides have relatively low specificity and are characterized by substantial cellular and clinical toxicity (Stresemann and Lyko, 2008). Their exact mechanism of antitumor action – demethylation of aberrantly silenced growth regulatory genes, induction of DNA damage, or other mechanism also remains unclear (Fandy et al., 2009; Issa, 2005; Palii et al., 2008).

Esteller, 2010; Robertson, 2001). It is worth noting that DNA methylation inhibitors have also emerged as a promising strategy for the treatment of immunodeficiency and brain

The structure of mammalian DNMTs can be divided into two major parts, a large Nterminal regulatory domain of variable size, which has regulatory functions, and a Cterminal catalytic domain which is conserved in eukaryotic and prokaryotic carbon-5 DNMTs. The N-terminal domain guides the nuclear localization of the enzymes and mediates their interactions with other proteins, DNA, and chromatine. The smaller Cterminal domain harbors the active center of the enzyme and contains ten amino acids motifs diagnostic for all carbon-5 DNMTs (Jurkowska et al., 2011). Motifs I-III form the cofactor binding pocket, motif IV has the catalytic cysteine, motifs VI, VIII, and X compose the substrate binding site, and motifs V and VII form the target recognition domain (Sippl and Jung, 2009). Human DNMT1 has 1616 amino acids for which limited three-dimensional structural information is available. For example, just recently a crystal structure of human DNMT1 bound to duplex DNA containing unmethylated cytosine-guanine (CG) sites was published (Song et al., 2011). Further details of the structure of DNMTs and other available crystal structures of DNMTs are extensively reviewed elsewhere (Cheng and Blumenthal,

The proposed mechanism of DNA cytosine-C5 methylation is summarized in Fig. 1 (Schermelleh et al., 2005; Sippl and Jung, 2009; Vilkaitis et al., 2001). DNMT forms a complex with DNA, and the cytosine which will be methylated flips out from the DNA. The thiol of the catalytic cysteine from motif IV acts as a nucleophile that attacks the 6-position of the target cytosine to generate a covalent intermediate. The 5-position of the cytosine is activated and conducts a nucleophilic attack on the cofactor S-adenosyl-L-methionine (AdoMet) to form the 5-methyl covalent adduct and *S*-adenosyl-L-homocysteine (AdoHcy). The attack on the 6-position is assisted by a transient protonation of the cytosine ring at the endocyclic nitrogen atom N3, which is stabilized by a glutamate residue from motif VI. The same residue also contacts the exocyclic N4 amino group and stabilizes the flipped base. The carbanion may also be stabilized by resonance, where an arginine residue from motif VIII may participate in the stabilization of the cytosine base. The covalent complex between the methylated base and the DNA is resolved by deprotonation at the 5-position to generate the

DNA methylation inhibitors have been well characterized and tested in clinical trials (Issa and Kantarjian, 2009). To date, only 5-azacytidine and 5-aza-2'-deoxycytidine (Fig. 2) have been developed clinically. These two drugs are nucleoside analogues, which, after incorporation into DNA, cause covalent trapping and subsequent depletion of DNA methyltransferases (Schermelleh et al., 2005; Stresemann and Lyko, 2008). Aza nucleosides are approved by the Food and Drug Administration of the United States for the treatment of myelodysplastic syndrome, where they demonstrate significant, although usually transient improvement in patient survival and are currently being tested in many solid cancers (Issa et al., 2005; Schrump et al., 2006). Despite the clinical successes achieved with DNA methylation inhibitors, there is still need for improvement since aza nucleosides have relatively low specificity and are characterized by substantial cellular and clinical toxicity (Stresemann and Lyko, 2008). Their exact mechanism of antitumor action – demethylation of aberrantly silenced growth regulatory genes, induction of DNA damage, or other mechanism also remains unclear (Fandy et al., 2009; Issa, 2005; Palii et al., 2008).

disorders (Miller et al., 2010; Zawia et al., 2009).

2008; Lan et al., 2010; Sippl and Jung, 2009).

methylated cytosine and the free enzyme.

Consequently, there is clear need to identify novel and more specific DNMT inhibitors that do not function via incorporation into DNA.

Fig. 1. Mechanism of DNA cytosine-C5 methylation. Amino acid residue numbers are based on the homology model. Equivalent residue numbers in parentheses correspond to the crystal structure.

There is now an increasing number of substances that are reported to inhibit DNMTs (Lyko and Brown, 2005). Selected DNMT inhibitors and other candidate demethylating agents are depicted in Fig. 2. Some of these compounds are approved drugs for other indications; i.e., the antihypertensive drug hydralazine (Segura-Pacheco et al., 2003), the local anaesthetic procaine (Villar-Garea et al., 2003), and the antiarrhythmic drug procainamide (Lee et al., 2005a). Others like the L-tryptophan derivative RG108, NSC 14778 (Fig. 2) have been identified by docking-based virtual screening (Kuck et al., 2010a; Siedlecki et al., 2006). Several natural products have been implicated in DNA methylation inhibition. Selected examples are the main polyphenol compound from green tea, (-)-epigallocathechin-3-gallate (EGCG) (Fang et al., 2003; Lee et al., 2005b), other tea polyphenols such as catechin and epicatechin, and the bioflavonoids quercetin, fisetin, and myricetin. Curcumin, the major component of the Indian curry spice turmeric, has been reported to inhibit the DNA cytosine C5 methyltransferase M.SssI, an analogue of DNMT1 (Liu et al., 2009). However, more recent studies showed that curcumin did not cause DNA demethylation in three arbitrarily chosen human cancer cell lines (Medina-Franco et al., 2011). Mahanine, a plantderived carbazole alkaloid, and a fluorescent carbazole analogue, has been reported to induce the Ras-association domain family 1 (RASSF1) gene in human prostate cancer cells, presumably by inhibiting DNMT activity (Jagadeesh et al., 2007; Sheikh et al., 2010). Nanaomycin A, a quinone antibiotic isolated from a culture of *Streptomyces*, has been described as the first non *S*-adenosyl-L-homocysteine (AdoHcy/SAH) analogue acting as a DNMT3B-selective inhibitor that induces genomic demethylation. Nanaomycin A treatment reduced the global methylation levels in three cell lines and reactivated transcription of the RASSF1A tumor suppressor gene (Kuck et al., 2010b). These and several other natural products as putative demethylating agents are extensively reviewed elsewhere (Gilbert and Liu, 2010; Hauser and Jung, 2008; Li and Tollefsbol, 2010; Medina-Franco and Caulfield, 2011). While the substantial number of recent reports may suggest that many natural products inhibit DNA methylation, it should be noted that only a few reports provide compelling evidence for DNMT inhibition in biochemical and in cellular assays. As such, it

Discovery and Optimization of Inhibitors

**2.1.1 Natural product databases** 

and Walters, 2002).

**2.1.2 Combinatorial libraries** 

**2.1 Screening databases** 

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 7

A number of compound databases from different sources can be used in *in silico* screening. These libraries may contain existing or hypothetical; i.e., virtual compounds. Libraries of existing compounds may be proprietary; e.g., in-house libraries, commercial, or public. The sources of screening libraries, with emphasis on libraries in the public domain, have been reviewed (Bender, 2010; Scior et al., 2007). Currently, the ZINC database is one of the most used libraries (Irwin and Shoichet, 2005). The type of screening library utilized should be closely associated with the objective of the particular screening campaign (Shelat and Guy, 2007). Chemically diverse libraries are particular attractive for identifying novel scaffolds for new or relatively unexplored targets such as DNMTs. If the goal is lead optimization, e.g., optimize the activity of known DNMT inhibitors (Fig. 2), focused libraries or collections

with high inter-molecular similarity (highly dense libraries) are an attractive source.

The presence of DNMT inhibitors in dietary products and commonly used herbal remedies (Gilbert and Liu, 2010; Hauser and Jung, 2008; Li and Tollefsbol, 2010) demonstrates the feasibility of identifying additional inhibitors of natural origin. Natural products have unique characteristics attractive for drug discovery. For example, the chemical structures of natural products are, in general, different from the chemical structures of synthetic compounds occupying different areas of chemical space (Ganesan, 2008; Medina-Franco et al., 2008; Singh et al., 2009b). In addition, natural products may be drug candidates themselves or may be the starting point for an optimization program (Ganesan, 2008; Hauser and Jung, 2008). Indeed several natural products are bioavailable, and the rationale of these observations has been recently provided (Ganesan, 2008). Fig. 3 shows a visual representation of the chemical space of natural products, drugs, and DNMT inhibitors. To compare the chemical space, a subset of 1,000 compounds was randomly selected from each database. The visual representation was obtained with principal component analysis (PCA) of the similarity matrix of the databases computed using Molecular ACCess System (MACCS) keys (166 bits) and the Tanimoto coefficient (Maggiora and Shanmugasundaram, 2011). The first three principal components are displayed in Fig. 3 and account for 79% of the variance. This figure clearly shows that most of the DNMT inhibitors, e.g., nucleoside analogues, RG108, RG108-1, procaine, procainamide, SG1027, and hydralazine, share the same chemical space of drugs. This observation is expected from inhibitors such as procaine, procainamide, and hydralazine. In contrast, NSC14778 and DNMT inhibitors from natural origin, EGCG and curcumin, are in a less-dense populated area of the chemical space of drugs. These compounds are characterized by containing one or more hydroxyl groups. Fig. 3b shows that most of the compounds in the natural product database also occupy this second region. Before conducting virtual and experimental screening, it is feasible to filter out natural products with potential toxicity issues using drug- or lead-like filters (Charifson

Combinatorial libraries, either existing or virtual, are important sources of compound collections that can be used for *in silico* screening (López-Vallejo et al., 2011). Advances in synthetic approaches can generate *libraries from libraries*, *target-oriented* libraries, and *diversity-oriented* libraries which explore the chemical space in different ways (López-Vallejo

remains possible that many of these compounds have an indirect and fortuitous effect on DNA methylation, but do not show a pharmacologically relevant activity that can be developed further for therapeutic purposes (Medina-Franco et al., 2011).

Fig. 2. Chemical structures of selected DNA methyltransferase inhibitors and other compounds with putative demethylating activity.

Until now most compounds associated with DNA methylation inhibition have been identified fortuitously. Remarkable exceptions are RG108 and 5,5'-methylenedisalicylic acid (NSC 14778) that were identified by computational screening followed by experimental evaluation (Kuck et al., 2010a; Siedlecki et al., 2006). In order to accelerate the discovery and optimization of new DNMT inhibitors, rational approaches are increasingly being used. To this end, *in silico* studies have significantly helped to understand the structure and function of DNMTs and the mechanism of DNMT inhibition (Medina-Franco and Caulfield, 2011). This chapter focuses on the different strategies ongoing in our and other research groups for the discovery and optimization of inhibitors of DNMTs with particular emphasis on *in silico* screening (section two) and *in silico* design (section three).

#### **2.** *In silico* **screening of compound collections to identify novel inhibitors**

*In silico* screening, also called in the literature, computational or virtual screening, consists of the computational evaluation of databases aiming to select a small number of reliable and experimentally testable candidate compounds that have a high probability of being active (Muegge, 2008; Shoichet, 2004). *In silico* screening is one of the most common rational approaches to guide the identification of new hits from large compound libraries. Hit identification using this approach requires several interactive steps that include (1) the compound collection, (2) the computational methods used for screening, and (3) the analysis of the output (López-Vallejo et al., 2011).

## **2.1 Screening databases**

6 Drug Development – A Case Study Based Insight into Modern Strategies

remains possible that many of these compounds have an indirect and fortuitous effect on DNA methylation, but do not show a pharmacologically relevant activity that can be

developed further for therapeutic purposes (Medina-Franco et al., 2011).

Fig. 2. Chemical structures of selected DNA methyltransferase inhibitors and other

Until now most compounds associated with DNA methylation inhibition have been identified fortuitously. Remarkable exceptions are RG108 and 5,5'-methylenedisalicylic acid (NSC 14778) that were identified by computational screening followed by experimental evaluation (Kuck et al., 2010a; Siedlecki et al., 2006). In order to accelerate the discovery and optimization of new DNMT inhibitors, rational approaches are increasingly being used. To this end, *in silico* studies have significantly helped to understand the structure and function of DNMTs and the mechanism of DNMT inhibition (Medina-Franco and Caulfield, 2011). This chapter focuses on the different strategies ongoing in our and other research groups for the discovery and optimization of inhibitors of DNMTs with particular emphasis on *in silico*

**2.** *In silico* **screening of compound collections to identify novel inhibitors** 

*In silico* screening, also called in the literature, computational or virtual screening, consists of the computational evaluation of databases aiming to select a small number of reliable and experimentally testable candidate compounds that have a high probability of being active (Muegge, 2008; Shoichet, 2004). *In silico* screening is one of the most common rational approaches to guide the identification of new hits from large compound libraries. Hit identification using this approach requires several interactive steps that include (1) the compound collection, (2) the computational methods used for screening, and (3) the analysis

compounds with putative demethylating activity.

screening (section two) and *in silico* design (section three).

of the output (López-Vallejo et al., 2011).

A number of compound databases from different sources can be used in *in silico* screening. These libraries may contain existing or hypothetical; i.e., virtual compounds. Libraries of existing compounds may be proprietary; e.g., in-house libraries, commercial, or public. The sources of screening libraries, with emphasis on libraries in the public domain, have been reviewed (Bender, 2010; Scior et al., 2007). Currently, the ZINC database is one of the most used libraries (Irwin and Shoichet, 2005). The type of screening library utilized should be closely associated with the objective of the particular screening campaign (Shelat and Guy, 2007). Chemically diverse libraries are particular attractive for identifying novel scaffolds for new or relatively unexplored targets such as DNMTs. If the goal is lead optimization, e.g., optimize the activity of known DNMT inhibitors (Fig. 2), focused libraries or collections with high inter-molecular similarity (highly dense libraries) are an attractive source.

## **2.1.1 Natural product databases**

The presence of DNMT inhibitors in dietary products and commonly used herbal remedies (Gilbert and Liu, 2010; Hauser and Jung, 2008; Li and Tollefsbol, 2010) demonstrates the feasibility of identifying additional inhibitors of natural origin. Natural products have unique characteristics attractive for drug discovery. For example, the chemical structures of natural products are, in general, different from the chemical structures of synthetic compounds occupying different areas of chemical space (Ganesan, 2008; Medina-Franco et al., 2008; Singh et al., 2009b). In addition, natural products may be drug candidates themselves or may be the starting point for an optimization program (Ganesan, 2008; Hauser and Jung, 2008). Indeed several natural products are bioavailable, and the rationale of these observations has been recently provided (Ganesan, 2008). Fig. 3 shows a visual representation of the chemical space of natural products, drugs, and DNMT inhibitors. To compare the chemical space, a subset of 1,000 compounds was randomly selected from each database. The visual representation was obtained with principal component analysis (PCA) of the similarity matrix of the databases computed using Molecular ACCess System (MACCS) keys (166 bits) and the Tanimoto coefficient (Maggiora and Shanmugasundaram, 2011). The first three principal components are displayed in Fig. 3 and account for 79% of the variance. This figure clearly shows that most of the DNMT inhibitors, e.g., nucleoside analogues, RG108, RG108-1, procaine, procainamide, SG1027, and hydralazine, share the same chemical space of drugs. This observation is expected from inhibitors such as procaine, procainamide, and hydralazine. In contrast, NSC14778 and DNMT inhibitors from natural origin, EGCG and curcumin, are in a less-dense populated area of the chemical space of drugs. These compounds are characterized by containing one or more hydroxyl groups. Fig. 3b shows that most of the compounds in the natural product database also occupy this second region. Before conducting virtual and experimental screening, it is feasible to filter out natural products with potential toxicity issues using drug- or lead-like filters (Charifson and Walters, 2002).

### **2.1.2 Combinatorial libraries**

Combinatorial libraries, either existing or virtual, are important sources of compound collections that can be used for *in silico* screening (López-Vallejo et al., 2011). Advances in synthetic approaches can generate *libraries from libraries*, *target-oriented* libraries, and *diversity-oriented* libraries which explore the chemical space in different ways (López-Vallejo

Discovery and Optimization of Inhibitors

(Medina-Franco and Caulfield, 2011).

residues: Gln1226, Ser1229, Gly1230, Arg1268, and Arg1310.

agreement with the proposed catalytic mechanism of DNA methylation.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 9

construction of useful homology models has been facilitated by the extensive conservation of the catalytic domain of DNMTs (Kumar et al., 1994). Crystal structures of other methyltransferases such as bacterial DNA cytosine C5 methyltransferase from *Haemophilus hemolyticus* (M.HhaI), bacterial cytosine C5 methyltransferase M.HaeIII, and the human DNMT2 (Siedlecki et al., 2003; Yoo and Medina-Franco, 2011) have been used as templates

We have recently developed two homology models of the catalytic domain of DNMT1. In one model (Yoo and Medina-Franco, 2011), the crystal structures of the DNMTs M.HhaI, M.HaeIII, and DNMT2 were used as templates. The first structure is a ternary complex of M.HhaI, *S*-adenosyl methionine (AdoMet), and DNA containing flipped 4'-thio-2' deoxcytidine with partial methylation at C5. The crystal structure of M.HaeIII is bound covalently to DNA. In this complex, the substrate cytosine is extruded from the DNA and it is inserted into the active site. The structure of human DNMT2 complexed with *S*-adenosyl-L-homocysteine (AdoHcy/SAH) has high similarities to methyltransferases of both prokaryotes and eukaryotes. A second homology model was developed using only the structure of M.HhaI as template. Both models contain DNA and the conserved residues which are involved in the catalytic mechanism. The target cytosine which is flipped out of the embedded DNA is inserted into the active site. The catalytic loop containing the catalytic cysteine is located above the cytosine as an active site "lid". The target cytosine lies between the nucleophile cysteine residue (Cys1225) and the sulfur atom of AdoHcy. The distance of cytosine C6 to the sulfur atom of Cys1225 is 3.3 Å. The cytosine C5 atom is 3.0 Å away from the sulfur atom of AdoHcy. In the reactive state of Cys1225, the distance between Oε1 of Glu1265 and N3 of cytidine is 2.8 Å, where the N3 atom is proposed to be protonated making a hydrogen bond with the acidic side chain of Glu1265. In addition, the N3 protonated form of cytosine can make hydrogen bonds with Arg1311 and Pro1223. These key interactions in the catalytic site are commonly observed in both homology models. More specifically, in the first homology model, the α-phosphate backbone and 3'-OH of the sugar moiety of deoxycytidine make a hydrogen bond network with Arg1311, Arg1461, Ser1229, Gly1230, and Gln1396; in the second model, the interactions are observed with the following

Fig. 4 shows a superimposition of the first homology model of the catalytic site of hDNMT1 (Yoo and Medina-Franco, 2011) with the recently published crystal structure of unmethylated human DNMT1 (Song et al., 2011). The catalytic cores of their methyltransferase domains have similar features, but unmethylated DNA in the crystallographic structure is positioned further away from the active site. In the crystal structure of the human DNMT complex, the key amino acid residues Glu1265 and Arg1311 are positioned in very similar place. In contrast, the catalytic loop adopts a different conformation with respect to the homology model. The catalytic loop has an open conformation, and the catalytic cysteine is far from the binding site, e.g., the distance of superimposed cytosine C6 to the sulfur atom of Cys1225 is 9.5 Å. Taken together, the structural characterization of the catalytic site supports that our homology model is in full

*In silico* screening has been successfully used to identify novel small molecule inhibitors of DNMT1. In one study, 1990 compounds in the Diversity Set available from the National Cancer Institute were the starting point of a screening using docking with a validated homology model of human DNMT1. Compounds with undesirable size, hydrophobicity, and uncommon atom types were filtered out. Two of the top scoring compounds were

et al., 2011) and can be used in lead optimization or hit-identification, depending on the goals of the screening campaign.

Fig. 3. Comparison of 486 natural products (black triangles), 1,000 drugs (red triangles), and 14 DNMT1 inhibitors (blue spheres). Depiction of a visual representation of the chemical space obtained by PCA of the similarity matrix computed using MACCS keys and Tanimoto similarity. The first three PCs account for 79% of the variance. (a) Comparison of drugs and DNMT1 inhibitors. (b) Comparison of drugs, natural products, and DNMT inhibitors.

#### **2.2 Development and validation of computational approaches**

*In silico* screening can be divided into two general strategies: ligand-based and structurebased (Medina-Franco et al., 2006; Ooms, 2000). Ligand-based approaches use the structural information and biological activity data from a set of known active compounds to select promising candidates for experimental screening. When the three-dimensional structure of the target is known, structure-based methods can be used. Three-dimensional structure information of the target is usually obtained from X-ray crystallography or nuclear magnetic resonance. In the absence of three-dimensional structural information of the receptor, homology models have been successfully used (Grant, 2009; Villoutreix et al., 2009). Perhaps the most common structure-based approach is molecular docking. Docking aims to find the best position and orientation of a molecule within a binding site and gives a score for each docked pose (Hernández-Campos et al., 2010; Kitchen et al., 2004; Villoutreix et al., 2009). Ligand- and structure-based methods can be combined if information for both the experimentally active compounds and the three-dimensional structure of the target are available (Sperandio et al., 2008). The selection of a particular method is generally based on the goal of the project, the information available for the system, and the computational resources available. For structure-based and ligand-based methods, it is highly advisable to validate the virtual screening protocol prior to the selection of compounds for experimental testing. However, the experimental results of the tested candidates will provide full validation of the virtual screening approach.

### **2.2.1 Structure-based screening**

Structure-based screening for novel DNMT inhibitors performed so far has been conducted with homology models of the catalytic domain (Kuck et al., 2010a; Siedlecki et al., 2006). The

et al., 2011) and can be used in lead optimization or hit-identification, depending on the

Fig. 3. Comparison of 486 natural products (black triangles), 1,000 drugs (red triangles), and 14 DNMT1 inhibitors (blue spheres). Depiction of a visual representation of the chemical space obtained by PCA of the similarity matrix computed using MACCS keys and Tanimoto similarity. The first three PCs account for 79% of the variance. (a) Comparison of drugs and DNMT1 inhibitors. (b) Comparison of drugs, natural products, and DNMT inhibitors.

*In silico* screening can be divided into two general strategies: ligand-based and structurebased (Medina-Franco et al., 2006; Ooms, 2000). Ligand-based approaches use the structural information and biological activity data from a set of known active compounds to select promising candidates for experimental screening. When the three-dimensional structure of the target is known, structure-based methods can be used. Three-dimensional structure information of the target is usually obtained from X-ray crystallography or nuclear magnetic resonance. In the absence of three-dimensional structural information of the receptor, homology models have been successfully used (Grant, 2009; Villoutreix et al., 2009). Perhaps the most common structure-based approach is molecular docking. Docking aims to find the best position and orientation of a molecule within a binding site and gives a score for each docked pose (Hernández-Campos et al., 2010; Kitchen et al., 2004; Villoutreix et al., 2009). Ligand- and structure-based methods can be combined if information for both the experimentally active compounds and the three-dimensional structure of the target are available (Sperandio et al., 2008). The selection of a particular method is generally based on the goal of the project, the information available for the system, and the computational resources available. For structure-based and ligand-based methods, it is highly advisable to validate the virtual screening protocol prior to the selection of compounds for experimental testing. However, the experimental results of the tested candidates will provide full

Structure-based screening for novel DNMT inhibitors performed so far has been conducted with homology models of the catalytic domain (Kuck et al., 2010a; Siedlecki et al., 2006). The

**2.2 Development and validation of computational approaches** 

validation of the virtual screening approach.

**2.2.1 Structure-based screening** 

goals of the screening campaign.

construction of useful homology models has been facilitated by the extensive conservation of the catalytic domain of DNMTs (Kumar et al., 1994). Crystal structures of other methyltransferases such as bacterial DNA cytosine C5 methyltransferase from *Haemophilus hemolyticus* (M.HhaI), bacterial cytosine C5 methyltransferase M.HaeIII, and the human DNMT2 (Siedlecki et al., 2003; Yoo and Medina-Franco, 2011) have been used as templates (Medina-Franco and Caulfield, 2011).

We have recently developed two homology models of the catalytic domain of DNMT1. In one model (Yoo and Medina-Franco, 2011), the crystal structures of the DNMTs M.HhaI, M.HaeIII, and DNMT2 were used as templates. The first structure is a ternary complex of M.HhaI, *S*-adenosyl methionine (AdoMet), and DNA containing flipped 4'-thio-2' deoxcytidine with partial methylation at C5. The crystal structure of M.HaeIII is bound covalently to DNA. In this complex, the substrate cytosine is extruded from the DNA and it is inserted into the active site. The structure of human DNMT2 complexed with *S*-adenosyl-L-homocysteine (AdoHcy/SAH) has high similarities to methyltransferases of both prokaryotes and eukaryotes. A second homology model was developed using only the structure of M.HhaI as template. Both models contain DNA and the conserved residues which are involved in the catalytic mechanism. The target cytosine which is flipped out of the embedded DNA is inserted into the active site. The catalytic loop containing the catalytic cysteine is located above the cytosine as an active site "lid". The target cytosine lies between the nucleophile cysteine residue (Cys1225) and the sulfur atom of AdoHcy. The distance of cytosine C6 to the sulfur atom of Cys1225 is 3.3 Å. The cytosine C5 atom is 3.0 Å away from the sulfur atom of AdoHcy. In the reactive state of Cys1225, the distance between Oε1 of Glu1265 and N3 of cytidine is 2.8 Å, where the N3 atom is proposed to be protonated making a hydrogen bond with the acidic side chain of Glu1265. In addition, the N3 protonated form of cytosine can make hydrogen bonds with Arg1311 and Pro1223. These key interactions in the catalytic site are commonly observed in both homology models. More specifically, in the first homology model, the α-phosphate backbone and 3'-OH of the sugar moiety of deoxycytidine make a hydrogen bond network with Arg1311, Arg1461, Ser1229, Gly1230, and Gln1396; in the second model, the interactions are observed with the following residues: Gln1226, Ser1229, Gly1230, Arg1268, and Arg1310.

Fig. 4 shows a superimposition of the first homology model of the catalytic site of hDNMT1 (Yoo and Medina-Franco, 2011) with the recently published crystal structure of unmethylated human DNMT1 (Song et al., 2011). The catalytic cores of their methyltransferase domains have similar features, but unmethylated DNA in the crystallographic structure is positioned further away from the active site. In the crystal structure of the human DNMT complex, the key amino acid residues Glu1265 and Arg1311 are positioned in very similar place. In contrast, the catalytic loop adopts a different conformation with respect to the homology model. The catalytic loop has an open conformation, and the catalytic cysteine is far from the binding site, e.g., the distance of superimposed cytosine C6 to the sulfur atom of Cys1225 is 9.5 Å. Taken together, the structural characterization of the catalytic site supports that our homology model is in full agreement with the proposed catalytic mechanism of DNA methylation.

*In silico* screening has been successfully used to identify novel small molecule inhibitors of DNMT1. In one study, 1990 compounds in the Diversity Set available from the National Cancer Institute were the starting point of a screening using docking with a validated homology model of human DNMT1. Compounds with undesirable size, hydrophobicity, and uncommon atom types were filtered out. Two of the top scoring compounds were

Discovery and Optimization of Inhibitors

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 11

Fig. 5. (a) Structure-based pharmacophore model proposed for human DNMT1 inhibitors. *Red sphere*: negative ionizable, *pink sphere*: hydrogen bond acceptor, *blue sphere*: hydrogen bond donors, and *orange ring*: aromatic ring. Selected amino acid residues in the catalytic site of homology model are schematically depicted for reference. Comparison between the binding mode and pharmacophore hypothesis for representative DNMT inhibitors, (b) 5-

Recently, our group developed a structure-based pharmacophore hypothesis for inhibitors of DNMT1 (Yoo and Medina-Franco, 2011). Using the energy optimized hypothesis, 'epharmacophore' method (Salam et al., 2009) the pharmacophore model was developed based on the scores and predicted binding modes of 14 known DNMT1 inhibitors docked with a homology model of DNMT1. Fig. 5a shows the pharmacophore model for the 14 DNMT1 inhibitors. The model contains five features which represent the most important interactions of the inhibitors with the catalytic domain. The energetic value assigned to each pharmacophoric feature is displayed in the figure. Nearby amino acids are schematically depicted for reference. The best-scoring feature is a negative charge which is close to the side chains of Ser1229, Gly1230, and Arg1311. The second most favorable feature is an acceptor site that is in close proximity with the side chains of Arg1311 and Arg1461. The third ranked features are an aromatic ring that stabilizes the binding conformation of ligands between AdoHcy and Cys1225, and a donor site that is close to the side chain of Gln1396. The fifth-ranked feature is a donor site that is nearby the side chain of Glu1265 which is a residue implicated in the methylation mechanism. Fig. 5b shows the alignment of representative DNMT inhibitors to the pharmacophore hypothesis. It remains to evaluate

the performance of the pharmacophore model in prospective *in silico* screening.

Ligand-based screening can be performed as an alternative approach when the relevant crystal structures are not available on the molecular target. Ligand-based approaches

azacytidine, (c) NSC14778, (d) hydralazine, and (e) RG108.

**2.2.2 Ligand-based screening** 

tested experimentally showing activity both *in vitro* and *in vivo*, probably by binding into the DNMT1 catalytic pocket (Siedlecki et al., 2006). In that work, RG108 (Fig. 2) showed an IC50 = 0.60 M with M.SssI (Siedlecki et al., 2006). Additional characterization showed that this L-tryptophan derivative did not cause covalent enzyme trapping and that the carboxylate group plays an essential role in the binding with the enzyme since the analogue without this moiety is inactive (Brueckner et al., 2005).

Fig. 4. (a) Superposition of the homology model (green) of the catalytic domain of human DNMT1 with the crystallographic structure (pink) of the unmethylated human DNMT1. The catalytic loops are marked with arrows. AdoHcy and the flipped cytosine in the homology model are shown in space-filling view. (b) Binding model of deoxycytidine (black) with key amino acid residues of homology model (carbon atoms in green) and crystal structure (carbon atoms in pink). Hydrogen bonding interactions are represented by dotted lines.

In a follow-up study, our group screened a larger set of the National Cancer Institute database containing more than 260,000 compounds (Kuck et al., 2010a). In order to focus the screening on compounds that could be promising for further development, we selected a subset of approximately 65,000 lead-like molecules (Charifson and Walters, 2002). The leadlike set was further filtered using a high-throughput *in silico* screening. As part of the screening, three docking programs were used. Favorable docking scores from all three docking approaches were combined to create a total of 24 consensus compounds. Of the 24 molecules that were identified, thirteen were obtained for experimental testing. Seven out of the thirteen consensus hits had detectable DNMT1 inhibitory activity in biochemical assays. Further experimental characterization of active compounds showed that six out of the seven inhibitors appeared selective for DNMT1. The methylenedisalicylic acid derivative, NSC 14778 (Fig. 2), showed an IC50 = 92 M with DNMT1 and an IC50 = 17 M with DNMT3B. The observed potency was comparably low for most test compounds, which was partially attributed to the high amount of protein used in the biochemical assay. In fact, it is wellknown that DNMTs are weak catalysts and are difficult to assay (Hemeon et al., (2011 - ASAP)). Despite the low potency, the *in silico* screening was successful in that it identified diverse scaffolds that were not previously reported for DNMT inhibitors. The new scaffolds represent excellent candidates for optimizing their inhibitory activity and selectivity.

tested experimentally showing activity both *in vitro* and *in vivo*, probably by binding into the DNMT1 catalytic pocket (Siedlecki et al., 2006). In that work, RG108 (Fig. 2) showed an IC50 = 0.60 M with M.SssI (Siedlecki et al., 2006). Additional characterization showed that this L-tryptophan derivative did not cause covalent enzyme trapping and that the carboxylate group plays an essential role in the binding with the enzyme since the analogue without this

Fig. 4. (a) Superposition of the homology model (green) of the catalytic domain of human DNMT1 with the crystallographic structure (pink) of the unmethylated human DNMT1. The catalytic loops are marked with arrows. AdoHcy and the flipped cytosine in the homology model are shown in space-filling view. (b) Binding model of deoxycytidine (black) with key amino acid residues of homology model (carbon atoms in green) and crystal structure (carbon atoms in pink). Hydrogen bonding interactions are represented by dotted lines.

In a follow-up study, our group screened a larger set of the National Cancer Institute database containing more than 260,000 compounds (Kuck et al., 2010a). In order to focus the screening on compounds that could be promising for further development, we selected a subset of approximately 65,000 lead-like molecules (Charifson and Walters, 2002). The leadlike set was further filtered using a high-throughput *in silico* screening. As part of the screening, three docking programs were used. Favorable docking scores from all three docking approaches were combined to create a total of 24 consensus compounds. Of the 24 molecules that were identified, thirteen were obtained for experimental testing. Seven out of the thirteen consensus hits had detectable DNMT1 inhibitory activity in biochemical assays. Further experimental characterization of active compounds showed that six out of the seven inhibitors appeared selective for DNMT1. The methylenedisalicylic acid derivative, NSC 14778 (Fig. 2), showed an IC50 = 92 M with DNMT1 and an IC50 = 17 M with DNMT3B. The observed potency was comparably low for most test compounds, which was partially attributed to the high amount of protein used in the biochemical assay. In fact, it is wellknown that DNMTs are weak catalysts and are difficult to assay (Hemeon et al., (2011 - ASAP)). Despite the low potency, the *in silico* screening was successful in that it identified diverse scaffolds that were not previously reported for DNMT inhibitors. The new scaffolds

represent excellent candidates for optimizing their inhibitory activity and selectivity.

moiety is inactive (Brueckner et al., 2005).

Fig. 5. (a) Structure-based pharmacophore model proposed for human DNMT1 inhibitors. *Red sphere*: negative ionizable, *pink sphere*: hydrogen bond acceptor, *blue sphere*: hydrogen bond donors, and *orange ring*: aromatic ring. Selected amino acid residues in the catalytic site of homology model are schematically depicted for reference. Comparison between the binding mode and pharmacophore hypothesis for representative DNMT inhibitors, (b) 5 azacytidine, (c) NSC14778, (d) hydralazine, and (e) RG108.

Recently, our group developed a structure-based pharmacophore hypothesis for inhibitors of DNMT1 (Yoo and Medina-Franco, 2011). Using the energy optimized hypothesis, 'epharmacophore' method (Salam et al., 2009) the pharmacophore model was developed based on the scores and predicted binding modes of 14 known DNMT1 inhibitors docked with a homology model of DNMT1. Fig. 5a shows the pharmacophore model for the 14 DNMT1 inhibitors. The model contains five features which represent the most important interactions of the inhibitors with the catalytic domain. The energetic value assigned to each pharmacophoric feature is displayed in the figure. Nearby amino acids are schematically depicted for reference. The best-scoring feature is a negative charge which is close to the side chains of Ser1229, Gly1230, and Arg1311. The second most favorable feature is an acceptor site that is in close proximity with the side chains of Arg1311 and Arg1461. The third ranked features are an aromatic ring that stabilizes the binding conformation of ligands between AdoHcy and Cys1225, and a donor site that is close to the side chain of Gln1396. The fifth-ranked feature is a donor site that is nearby the side chain of Glu1265 which is a residue implicated in the methylation mechanism. Fig. 5b shows the alignment of representative DNMT inhibitors to the pharmacophore hypothesis. It remains to evaluate the performance of the pharmacophore model in prospective *in silico* screening.

## **2.2.2 Ligand-based screening**

Ligand-based screening can be performed as an alternative approach when the relevant crystal structures are not available on the molecular target. Ligand-based approaches

Discovery and Optimization of Inhibitors

restoration of RASSF1A expression (Sheikh et al., 2010).

**3.2 Structure-based optimization of hydralazine** 

(Singh et al., 2009a; Yoo and Medina-Franco, 2011).

inhibition assays.

2011).

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 13

has the same interaction with Arg1311, Ser1229, and Gly1230 (Yoo and Medina-Franco,

The natural product mahanine (Fig. 2) has the ability to restore RASSF1A expression, and it is a potent inhibitor of androgen dependent (LNCaP) and androgen independent (PC-3) human prostate cancer cell proliferation (Jagadeesh et al., 2007). The antiproliferative activity of mahanine is associated with inhibition of the DNMT activity. Recently, fluorescent carbazole analogues of mahanine were designed and synthesized to find a novel and more potent small molecule with a mechanistic profile similar to that of the parent compound. Compound '9' in Fig. 2 inhibited human prostate cancer cell proliferation at 1.5 µM and also showed DNMT inhibition activity without the cytotoxic effects seen with mahanine treatment. Inhibition of DNMT was proposed as the event leading to the

Hydralazine, a potent arterial vasodilator, has been used for the management of hypertensive disorders and heart failure. Using a drug repurposing strategy (Duenas-Gonzalez et al., 2008), clinical trials have demonstrated the antitumor effect of the combination of hydralazine with valporic acid (a histone deacetylase inhibitor). Hydralazine and procainamide were first reported to have DNA methylation inhibition effect in 1988. Despite the fact that numerous studies were conducted with hydralazine, its molecular mechanism has remained unknown. In order to help understand the activity of hydralazine at the molecular level, we developed a binding mode of this compound with a validated homology model of the catalytic domain of DNMT1 using docking and molecular dynamics

In molecular modeling studies, hydralazine showed similar interactions within the binding pocket as nucleoside analogues including a complex network of hydrogen bonds with arginine and glutamic acid residues that play a major role in the mechanism of DNA methylation (Yoo and Medina-Franco, 2011). Fig. 6 shows the comparison of the binding modes of hydralazine with 5-azacytidine. The amino group of hydralazine matched well with the amino group of 5-azacytidine, and it is capable of making hydrogen bonds with Glu1265 and Pro1223. The nitrogen of the phthalazine ring overlapped with the carbonyl oxygen of 5-azacytidine and formed hydrogen bonds with Arg1311 and Arg1461. We also identified that the small structure of hydralazine could not occupy the site of the sugar ring and phosphate backbone of nucleoside analogues. This result also suggests that hydralazine can be substituted at the C4 position to yield analogues with enhanced affinity with the enzyme. In contrast, there is a small empty pocket nearby the carbocyclic aromatic ring of hydralazine (C5-C8) that can be occupied by a substituent (Yoo and Medina-Franco, 2011). The molecule shown in Fig. 6 was designed based on the structure and binding mode of hydralazine. Molecular modeling indicates that the addition of a phenyl group in the C4 position of hydralazine improves the calculated binding affinity with DNMT1. Moreover, adding polar substituents at various positions of the phenyl group can provide additional favorable interactions with the catalytic site. Also based on our molecular modeling analysis, the binding affinity is expected to increase by the addition of polar groups to the carbocyclic aromatic ring of phthalazine. It is *expected* that new compounds with increased calculated affinity with the enzyme will show increased potency in the DNMT1 enzyme

include similarity searching, substructure, clustering, quantitative structure-activity relationships (QSAR), pharmacophore-, or three-dimensional shape matching techniques (Villoutreix et al., 2007). Several ligand-based methods, including similarity searching and QSAR, can roughly be divided into two- or three-dimensional approaches. Ligand-based virtual screening may be applied even if a single known-ligand has been identified through similarity-based screening. Interestingly, although many more successful structure-based than ligand-based virtual screening applications are reported to date, recent reviews indicate that the potency of hits identified by ligand-based approaches is on average considerably higher than for structure-based methods (Ripphausen et al., 2011; Ripphausen et al., 2010).

If multiple active compounds are known, it is possible to apply QSAR using two- or threedimensional information of the ligands. One of the main goals of QSAR is to derive statistical models that can be used to predict the activity of molecules not previously tested in the biological assay. Despite the fact that QSAR is a valuable tool, there are potential pitfalls to develop predictive QSAR models (Scior et al., 2009). A major pitfall can occur when the compounds were assayed using different experimental conditions. Other major pitfall is due to the presence of "activity cliffs," i.e., compounds with very high structural similarity but very different biological activity (Maggiora, 2006). Activity cliffs give rise to QSAR with poor predictive ability (Guha and Van Drie, 2008).

## **3.** *In silico* **design and optimization of established inhibitors**

Concerns about severe toxicity of nucleoside analogues have strongly encouraged not only identifying novel DNMT inhibitors but also developing further established non-nucleoside inhibitors. To this end, medicinal chemistry approaches, either alone or in combination with *in silico* strategies, are being pursued.

#### **3.1 Optimization of RG108, procaine, and mahanine**

As mentioned above, procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation (Fig. 2). In a recent report, constrained analogues of procaine were synthesized and tested for their inhibition against DNMT1 (Castellano et al., 2008). Procaine as a lead structure was modified to partially reduce the high flexibility which can have a detrimental effect for drug-likeness. The most potent inhibitor in an *in vitro* methylation assay also showed demethylation activity in HL60 human myeloid leukemia cells, and it was suggested as a lead compound for further studies (Castellano et al., 2008).

In a separate work, a series of maleimide derivatives of RG108 were reported (Suzuki et al., 2010). In that work, design, chemical synthesis, inhibitory activity assays, and automated docking methods were used. The most active compound of the series was RG108-1 (Fig. 2). A binding model of RG108-1 with the crystal structure of bacterial M.HhaI suggested that this compound could be a covalent blocker of the catalytic cysteine. A more recent molecular modelling study using a model of human DNMT1 (Yoo and Medina-Franco, 2011) supported this hypothesis. Interestingly, in the model obtained with human DNMT, the maleimide moiety of RG108-1 interacts with Arg1311, Arg1461, and lies next to Cys1225, where the conjugate addition of the thiol group of the catalytic cysteine to the maleimide can occur. In addition, the carboxylate anion of RG108-1 overlaps with that of RG108 and

include similarity searching, substructure, clustering, quantitative structure-activity relationships (QSAR), pharmacophore-, or three-dimensional shape matching techniques (Villoutreix et al., 2007). Several ligand-based methods, including similarity searching and QSAR, can roughly be divided into two- or three-dimensional approaches. Ligand-based virtual screening may be applied even if a single known-ligand has been identified through similarity-based screening. Interestingly, although many more successful structure-based than ligand-based virtual screening applications are reported to date, recent reviews indicate that the potency of hits identified by ligand-based approaches is on average considerably higher than for structure-based methods (Ripphausen et al., 2011; Ripphausen

If multiple active compounds are known, it is possible to apply QSAR using two- or threedimensional information of the ligands. One of the main goals of QSAR is to derive statistical models that can be used to predict the activity of molecules not previously tested in the biological assay. Despite the fact that QSAR is a valuable tool, there are potential pitfalls to develop predictive QSAR models (Scior et al., 2009). A major pitfall can occur when the compounds were assayed using different experimental conditions. Other major pitfall is due to the presence of "activity cliffs," i.e., compounds with very high structural similarity but very different biological activity (Maggiora, 2006). Activity cliffs give rise to

Concerns about severe toxicity of nucleoside analogues have strongly encouraged not only identifying novel DNMT inhibitors but also developing further established non-nucleoside inhibitors. To this end, medicinal chemistry approaches, either alone or in combination with

As mentioned above, procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation (Fig. 2). In a recent report, constrained analogues of procaine were synthesized and tested for their inhibition against DNMT1 (Castellano et al., 2008). Procaine as a lead structure was modified to partially reduce the high flexibility which can have a detrimental effect for drug-likeness. The most potent inhibitor in an *in vitro* methylation assay also showed demethylation activity in HL60 human myeloid leukemia cells, and it was suggested as a

In a separate work, a series of maleimide derivatives of RG108 were reported (Suzuki et al., 2010). In that work, design, chemical synthesis, inhibitory activity assays, and automated docking methods were used. The most active compound of the series was RG108-1 (Fig. 2). A binding model of RG108-1 with the crystal structure of bacterial M.HhaI suggested that this compound could be a covalent blocker of the catalytic cysteine. A more recent molecular modelling study using a model of human DNMT1 (Yoo and Medina-Franco, 2011) supported this hypothesis. Interestingly, in the model obtained with human DNMT, the maleimide moiety of RG108-1 interacts with Arg1311, Arg1461, and lies next to Cys1225, where the conjugate addition of the thiol group of the catalytic cysteine to the maleimide can occur. In addition, the carboxylate anion of RG108-1 overlaps with that of RG108 and

QSAR with poor predictive ability (Guha and Van Drie, 2008).

**3.1 Optimization of RG108, procaine, and mahanine** 

lead compound for further studies (Castellano et al., 2008).

*in silico* strategies, are being pursued.

**3.** *In silico* **design and optimization of established inhibitors** 

et al., 2010).

has the same interaction with Arg1311, Ser1229, and Gly1230 (Yoo and Medina-Franco, 2011).

The natural product mahanine (Fig. 2) has the ability to restore RASSF1A expression, and it is a potent inhibitor of androgen dependent (LNCaP) and androgen independent (PC-3) human prostate cancer cell proliferation (Jagadeesh et al., 2007). The antiproliferative activity of mahanine is associated with inhibition of the DNMT activity. Recently, fluorescent carbazole analogues of mahanine were designed and synthesized to find a novel and more potent small molecule with a mechanistic profile similar to that of the parent compound. Compound '9' in Fig. 2 inhibited human prostate cancer cell proliferation at 1.5 µM and also showed DNMT inhibition activity without the cytotoxic effects seen with mahanine treatment. Inhibition of DNMT was proposed as the event leading to the restoration of RASSF1A expression (Sheikh et al., 2010).

## **3.2 Structure-based optimization of hydralazine**

Hydralazine, a potent arterial vasodilator, has been used for the management of hypertensive disorders and heart failure. Using a drug repurposing strategy (Duenas-Gonzalez et al., 2008), clinical trials have demonstrated the antitumor effect of the combination of hydralazine with valporic acid (a histone deacetylase inhibitor). Hydralazine and procainamide were first reported to have DNA methylation inhibition effect in 1988. Despite the fact that numerous studies were conducted with hydralazine, its molecular mechanism has remained unknown. In order to help understand the activity of hydralazine at the molecular level, we developed a binding mode of this compound with a validated homology model of the catalytic domain of DNMT1 using docking and molecular dynamics (Singh et al., 2009a; Yoo and Medina-Franco, 2011).

In molecular modeling studies, hydralazine showed similar interactions within the binding pocket as nucleoside analogues including a complex network of hydrogen bonds with arginine and glutamic acid residues that play a major role in the mechanism of DNA methylation (Yoo and Medina-Franco, 2011). Fig. 6 shows the comparison of the binding modes of hydralazine with 5-azacytidine. The amino group of hydralazine matched well with the amino group of 5-azacytidine, and it is capable of making hydrogen bonds with Glu1265 and Pro1223. The nitrogen of the phthalazine ring overlapped with the carbonyl oxygen of 5-azacytidine and formed hydrogen bonds with Arg1311 and Arg1461. We also identified that the small structure of hydralazine could not occupy the site of the sugar ring and phosphate backbone of nucleoside analogues. This result also suggests that hydralazine can be substituted at the C4 position to yield analogues with enhanced affinity with the enzyme. In contrast, there is a small empty pocket nearby the carbocyclic aromatic ring of hydralazine (C5-C8) that can be occupied by a substituent (Yoo and Medina-Franco, 2011).

The molecule shown in Fig. 6 was designed based on the structure and binding mode of hydralazine. Molecular modeling indicates that the addition of a phenyl group in the C4 position of hydralazine improves the calculated binding affinity with DNMT1. Moreover, adding polar substituents at various positions of the phenyl group can provide additional favorable interactions with the catalytic site. Also based on our molecular modeling analysis, the binding affinity is expected to increase by the addition of polar groups to the carbocyclic aromatic ring of phthalazine. It is *expected* that new compounds with increased calculated affinity with the enzyme will show increased potency in the DNMT1 enzyme inhibition assays.

Discovery and Optimization of Inhibitors

demethylating activity.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 15

binding affinity with the enzyme. Further chemical modifications to the structures of the lead DNMT inhibitors will be suggested toward the improvement of the *in vitro* and *in vivo*

Fig. 7. Binding mode of hydralazine analogues (carbon atoms in pink) designed by scaffold hopping. The carbon atoms of new core scaffold are in green. Analogues with (a) ortho-

Currently, DNMT inhibitors have been screened in different assays using different conditions, and QSAR studies may not be reliable. However, once quality activity data has been gathered for several compounds assayed under comparable experimental conditions, it is feasible to conduct structure-activity relationships (SAR) of the compounds tested. When there is a significant amount of data, for example, activity data for more than 100 or 200 compounds, systematic analysis of the SAR can be performed via chemoinformatic approaches using the concept of "activity landscape modelling" (Wawer et al., 2010). The goal of activity landscape modeling of molecular data sets is to help rationalize the underlying SAR identifying key compounds and structural features for further exploration. The concept of activity landscape is strongly associated with the basic relationships between molecular structure and biological activity. While predictive SAR methods, such as pharmacophore modelling and traditional QSAR, focus on specific molecular descriptors or arrangements of substructures or functional groups associated with activity, descriptive activity landscape models rely on the "similarity property principle," i.e., similar structures should have similar biological properties (Bender and Glen, 2004; Maggiora and Shanmugasundaram, 2011) and employ whole-molecular similarity measures (Wawer et al., 2010). Systematic approaches to model activity landscapes and to detect "activity cliffs" using multiple representations are published elsewhere (Medina-Franco et al., 2009; Pérez-

DNA methyltransferases are promising epigenetic targets for the treatment of cancer and other diseases. Clinical data demonstrates the potential of DNMT inhibitors for the therapeutic treatment of cancer. This is evidenced by the two DNMT inhibitors approved by the Food and Drug Administration of the United States for the treatment of patients with high-risk myelodysplastic syndrome. However, current approved drugs are nucleoside analogues that are not specific and still present issues such as cellular and clinical toxicity. A

carboxylate substitution and (b) meta-acetyl substitution on the phenyl moiety.

**3.4 Characterization of structure-activity relationships** 

Villanueva et al., 2010; Pérez-Villanueva et al., 2011).

**4. Conclusion** 

Fig. 6. Design of analogues of hydralazine. (a) comparison of the binding modes of hydralazine (carbon atoms in pink) with 5-azacytidine (carbon atoms in green) (b) structure-guided design of a representative hydralazine analogue (carbon atoms in green).

### **3.3 Design of focused combinatorial libraries**

Computer-assisted combinatorial library design is a powerful tool frequently used in the discovery and optimization of new lead compounds. Molecular diversity has played a critical role in designing combinatorial libraries for screening (Tommasi and Cornella, 2006; Zheng and Johnson, 2008). However, the core chemical scaffolds of some currently used diverse libraries might be inadequate to provide drug-like compounds for new targets. Library design based on bioisosteric replacement or scaffold hopping methods can be used as an alternative to diversity oriented synthesis. Bioisostere searching involves swapping functional groups of a molecule with other functional groups that have similar biological properties. Scaffold hopping is an approach to discover structurally novel compounds starting from known active compounds by modifying the central core structure of the molecule (Brown and Jacoby, 2006). Scaffold hopping is an important drug design strategy to develop novel molecules with potent activity, altered physicochemical attributes, and Absorption, Distribution, Metabolism, Excretion and Toxicity –ADMET- properties. An example of application is phosphodiesterase 5 inhibitors for the treatment of erectile dysfunction. Sildenafil and vardenafil represent a case of heteroaromatic core scaffolds hopping with a small change in the scaffold (Jordan and Roughley, 2009). In contrast, tadalafil has a very different core scaffold, but it has the same biological activity. Computational design of focused libraries or compounds designed using any other strategy has to be in agreement with the experimental synthetic feasibility of the compounds proposed. Ideally, synthetic routes should follow short and easy steps.

Fig. 7 shows additional hydralazine analogues that have been proposed based on the knowledge gained in our previous molecular modeling studies of DNMT inhibitors. Starting from the 1-hydrazinyl-4-phenylphthalazine, polar groups are introduced into the carbocyclic aromatic ring of phthalazine. Based on molecular modeling analysis, substitution at ortho-, meta-, and para- position of the phenyl group with e.g., carboxyl, cyanide, and acetyl, showed a significant improvement in the calculated binding of the new compounds with DNMT1. A carboxyl group introduced into the ortho position plays a key role to make hydrogen bonds with Ser1229 or Gly1230. In contrast, substitution at C8 position of the phthalazine did not fit well into the catalytic site because of the narrow pocket size. Addition of polar groups to other positions slightly increases the predicted

Fig. 6. Design of analogues of hydralazine. (a) comparison of the binding modes of hydralazine (carbon atoms in pink) with 5-azacytidine (carbon atoms in green)

proposed. Ideally, synthetic routes should follow short and easy steps.

Fig. 7 shows additional hydralazine analogues that have been proposed based on the knowledge gained in our previous molecular modeling studies of DNMT inhibitors. Starting from the 1-hydrazinyl-4-phenylphthalazine, polar groups are introduced into the carbocyclic aromatic ring of phthalazine. Based on molecular modeling analysis, substitution at ortho-, meta-, and para- position of the phenyl group with e.g., carboxyl, cyanide, and acetyl, showed a significant improvement in the calculated binding of the new compounds with DNMT1. A carboxyl group introduced into the ortho position plays a key role to make hydrogen bonds with Ser1229 or Gly1230. In contrast, substitution at C8 position of the phthalazine did not fit well into the catalytic site because of the narrow pocket size. Addition of polar groups to other positions slightly increases the predicted

**3.3 Design of focused combinatorial libraries** 

green).

(b) structure-guided design of a representative hydralazine analogue (carbon atoms in

Computer-assisted combinatorial library design is a powerful tool frequently used in the discovery and optimization of new lead compounds. Molecular diversity has played a critical role in designing combinatorial libraries for screening (Tommasi and Cornella, 2006; Zheng and Johnson, 2008). However, the core chemical scaffolds of some currently used diverse libraries might be inadequate to provide drug-like compounds for new targets. Library design based on bioisosteric replacement or scaffold hopping methods can be used as an alternative to diversity oriented synthesis. Bioisostere searching involves swapping functional groups of a molecule with other functional groups that have similar biological properties. Scaffold hopping is an approach to discover structurally novel compounds starting from known active compounds by modifying the central core structure of the molecule (Brown and Jacoby, 2006). Scaffold hopping is an important drug design strategy to develop novel molecules with potent activity, altered physicochemical attributes, and Absorption, Distribution, Metabolism, Excretion and Toxicity –ADMET- properties. An example of application is phosphodiesterase 5 inhibitors for the treatment of erectile dysfunction. Sildenafil and vardenafil represent a case of heteroaromatic core scaffolds hopping with a small change in the scaffold (Jordan and Roughley, 2009). In contrast, tadalafil has a very different core scaffold, but it has the same biological activity. Computational design of focused libraries or compounds designed using any other strategy has to be in agreement with the experimental synthetic feasibility of the compounds binding affinity with the enzyme. Further chemical modifications to the structures of the lead DNMT inhibitors will be suggested toward the improvement of the *in vitro* and *in vivo* demethylating activity.

Fig. 7. Binding mode of hydralazine analogues (carbon atoms in pink) designed by scaffold hopping. The carbon atoms of new core scaffold are in green. Analogues with (a) orthocarboxylate substitution and (b) meta-acetyl substitution on the phenyl moiety.

## **3.4 Characterization of structure-activity relationships**

Currently, DNMT inhibitors have been screened in different assays using different conditions, and QSAR studies may not be reliable. However, once quality activity data has been gathered for several compounds assayed under comparable experimental conditions, it is feasible to conduct structure-activity relationships (SAR) of the compounds tested. When there is a significant amount of data, for example, activity data for more than 100 or 200 compounds, systematic analysis of the SAR can be performed via chemoinformatic approaches using the concept of "activity landscape modelling" (Wawer et al., 2010). The goal of activity landscape modeling of molecular data sets is to help rationalize the underlying SAR identifying key compounds and structural features for further exploration. The concept of activity landscape is strongly associated with the basic relationships between molecular structure and biological activity. While predictive SAR methods, such as pharmacophore modelling and traditional QSAR, focus on specific molecular descriptors or arrangements of substructures or functional groups associated with activity, descriptive activity landscape models rely on the "similarity property principle," i.e., similar structures should have similar biological properties (Bender and Glen, 2004; Maggiora and Shanmugasundaram, 2011) and employ whole-molecular similarity measures (Wawer et al., 2010). Systematic approaches to model activity landscapes and to detect "activity cliffs" using multiple representations are published elsewhere (Medina-Franco et al., 2009; Pérez-Villanueva et al., 2010; Pérez-Villanueva et al., 2011).

## **4. Conclusion**

DNA methyltransferases are promising epigenetic targets for the treatment of cancer and other diseases. Clinical data demonstrates the potential of DNMT inhibitors for the therapeutic treatment of cancer. This is evidenced by the two DNMT inhibitors approved by the Food and Drug Administration of the United States for the treatment of patients with high-risk myelodysplastic syndrome. However, current approved drugs are nucleoside analogues that are not specific and still present issues such as cellular and clinical toxicity. A

Discovery and Optimization of Inhibitors

perspective. *Structure* 16, 341-350.

agents. *Mol. Cancer* 7, 33.

*Blood* 114, 2764-2773.

*Chem. Biol.* 12, 306-317.

*Chem.* 17, 1756-1768.

*Chem.*, 83, 4996-5004.

*Drug Des.* 76, 269-276.

S24-9.

74, 481-514.

7570.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 17

Cheng, X.D., Blumenthal, R.M., 2008. Mammalian DNA methyltransferases: A structural

Dueñas-González, A., García-López, P., Herrera, L.A., Medina-Franco, J.L., González-Fierro,

Fandy, T.E., Herman, J.G., Kerns, P., Jiemjit, A., Sugar, E.A., Choi, S.H., Yang, A.S., Aucott,

Fang, M.Z., Wang, Y.M., Ai, N., Hou, Z., Sun, Y., Lu, H., Welsh, W., Yang, C.S., 2003. Tea

Ganesan, A., 2008. The impact of natural products upon modern drug discovery. *Curr. Opin.* 

Gilbert, E.R., Liu, D., 2010. Flavonoids influence epigenetic-modifying enzyme activity:

Goll, M.G., Bestor, T.H., 2005. Eukaryotic cytosine methyltransferases. *Annu. Rev. Biochem.*

Goll, M.G., Kirpekar, F., Maggert, K.A., Yoder, J.A., Hsieh, C.-L., Zhang, X., Golic, K.G.,

Grant, M.A., 2009. Protein structure prediction in structure-based ligand design and virtual

Guha, R., Van Drie, J.H., 2008. Assessing how well a modeling protocol captures a structure-

Hauser, A.T., Jung, M., 2008. Targeting epigenetic mechanisms: Potential of natural products

Hemeon, I., Gutierrez, J.A., Ho, M.-C., Schramm, V.L., (2011). Characterizing DNA

Hernández-Campos, A., Velázquez-Martínez, I., Castillo, R., López-Vallejo, F., Jia, P., Yu, Y.,

Irwin, J.J., Shoichet, B.K., 2005. ZINC - A free database of commercially available

Issa, J.-P., 2005. Optimizing therapy with methylation inhibitors in myelodysplastic

compounds for virtual screening. *J. Chem. Inf. Model.* 45, 177-182.

methyltransferases with an ultrasensitive luciferase-linked continuous assay. *Anal.* 

Giulianotti, M.A., Medina-Franco, J.L., 2010. Docking of protein kinase B inhibitors: Implications in the structure-based optimization of a novel scaffold. *Chem. Biol.* 

syndromes: Dose, duration, and patient selection. *Nat. Clin. Pract. Oncol.* 2 Suppl 1,

methyltransferase homolog Dnmt2. *Science* 311, 395-398.

activity landscape. *J. Chem. Inf. Model.* 48, 1716-1728.

in cancer chemoprevention. *Planta Med.* 74, 1593-1601.

screening. *Comb. Chem. High Throughput Screening* 12, 940-960.

A., Candelaria, M., 2008. The prince and the pauper. A tale of anticancer targeted

T., Dauses, T., Odchimar-Reissig, R., Licht, J., Mcconnell, M.J., Nasrallah, C., Kim, M.K.H., Zhang, W.J., Sun, Y.Z., Murgo, A., Espinoza-Delgado, I., Oteiza, K., Owoeye, I., Silverman, L.R., Gore, S.D., Carraway, H.E., 2009. Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies.

polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. *Cancer Res.* 63, 7563-

Structure-function relationships and the therapeutic potential for cancer. *Curr. Med.* 

Jacobsen, S.E., Bestor, T.H., 2006. Methylation of tRNAAsp by the DNA

wide range of computational approaches are being used to assist in the discovery and development of novel DNMT inhibitors. Molecular docking, pharmacophore modelling and molecular dynamics have been used to better understand the mechanism of action of established DNMT inhibitors; *in silico* screening of large compound libraries followed by experimental testing has been successful in identifying non-nucleoside inhibitors with novel chemical scaffolds; structure-based design is being used to guide the optimization of inhibitors such as hydralazine. Homology models of the catalytic domain of DNMT1 has played an important role to conduct the computational approaches that rely on the three dimensional structure of the target. It is expected that the recently published crystal structure of human DNMT1 bound to duplex DNA containing unmethylated CG sites will be the starting point of future structure-based studies with inhibitors of DNA methylation. It is also anticipated that the synergistic combination of computational approaches with combinatorial chemistry, and the systematic *in silico* and experimental screening of natural products will boost the discovery and optimization of inhibitors of DNMT for cancer therapy.

### **5. Acknowledgment**

Discussions with Dr. Fabian López-Vallejo and other members of the group are highly appreciated. Authors are also very grateful to Karen Gottwald for proofreading the chapter. This work was supported by the State of Florida, Executive Office of the Governor's Office of Tourism, Trade, and Economic Development. J.L.M-F. also thanks the Menopause & Women's Health Research Center for funding.

#### **6. References**


wide range of computational approaches are being used to assist in the discovery and development of novel DNMT inhibitors. Molecular docking, pharmacophore modelling and molecular dynamics have been used to better understand the mechanism of action of established DNMT inhibitors; *in silico* screening of large compound libraries followed by experimental testing has been successful in identifying non-nucleoside inhibitors with novel chemical scaffolds; structure-based design is being used to guide the optimization of inhibitors such as hydralazine. Homology models of the catalytic domain of DNMT1 has played an important role to conduct the computational approaches that rely on the three dimensional structure of the target. It is expected that the recently published crystal structure of human DNMT1 bound to duplex DNA containing unmethylated CG sites will be the starting point of future structure-based studies with inhibitors of DNA methylation. It is also anticipated that the synergistic combination of computational approaches with combinatorial chemistry, and the systematic *in silico* and experimental screening of natural products will boost the discovery and optimization of inhibitors of DNMT for cancer

Discussions with Dr. Fabian López-Vallejo and other members of the group are highly appreciated. Authors are also very grateful to Karen Gottwald for proofreading the chapter. This work was supported by the State of Florida, Executive Office of the Governor's Office of Tourism, Trade, and Economic Development. J.L.M-F. also thanks the Menopause &

Bender, A., 2010. Databases compound bioactivities go public. *Nat. Chem. Biol.* 6, 309-

Bender, A., Glen, R.C., 2004. Molecular similarity: A key technique in molecular informatics.

Brown, N., Jacoby, E., 2006. On scaffolds and hopping in medicinal chemistry. *Mini-Rev.* 

Brueckner, B., Boy, R.G., Siedlecki, P., Musch, T., Kliem, H.C., Zielenkiewicz, P., Suhai, S.,

Castellano, S., Kuck, D., Sala, M., Novellino, E., Lyko, F., Sbardella, G., 2008. Constrained

Charifson, P.S., Walters, W.P., 2002. Filtering databases and chemical libraries. *J. Comput.-*

Chen, T.P., Hevi, S., Gay, F., Tsujimoto, N., He, T., Zhang, B.L., Ueda, Y., Li, E., 2007.

Wiessler, M., Lyko, F., 2005. Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. *Cancer Res.* 65,

analogues of procaine as novel small molecule inhibitors of DNA

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer

therapy.

**5. Acknowledgment** 

**6. References** 

309.

6305-6311.

Women's Health Research Center for funding.

*Org. Biomol. Chem.* 2, 3204-3218.

methyltransferase-1. *J. Med. Chem.* 51, 2321-2325.

*Med. Chem.* 6, 1217-1229.

*Aided Mol. Des.* 16, 311-323.

cells. *Nat. Genet.* 39, 391-396.


Discovery and Optimization of Inhibitors

*Inf. Model.* 46, 1535-1535.

15, 293-304.

477-491.

933.

*Biol.* 28, 752-771.

18, 7380-7391.

28, 1057-1068.

*Chem. Comm.* 2, 44-49.

screening. *Drug Discovery Today* 16, 372-376.

*Drug Des.* 4, 322-333.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 19

Lyko, F., Brown, R., 2005. DNA methyltransferase inhibitors and the development of

Maggiora, G.M., 2006. On outliers and activity cliffs-why QSAR often disappoints. *J. Chem.* 

Maggiora, G.M., Shanmugasundaram, V., 2011. Molecular similarity measures, In:

Medina-Franco, J., López-Vallejo, F., Kuck, D., Lyko, F., 2011. Natural products as DNA

Medina-Franco, J.L., Caulfield, T., 2011. Advances in the computational development of DNA methyltransferase inhibitors. *Drug Discovery Today* 16, 418-425. Medina-Franco, J.L., López-Vallejo, F., Castillo, R., 2006. Diseño de fármacos asistido por

Medina-Franco, J.L., Martínez-Mayorga, K., Giulianotti, M.A., Houghten, R.A., Pinilla, C.,

Medina-Franco, J.L., Martínez-Mayorga, K., Bender, A., Marín, R.M., Giulianotti, M.A.,

Miller, C.A., Gavin, C.F., White, J.A., Parrish, R.R., Honasoge, A., Yancey, C.R., Rivera, I.M.,

Muegge, I., 2008. Synergies of virtual screening approaches. *Mini-Rev. Med. Chem.* 8, 927-

Ooms, F., 2000. Molecular modeling and computer aided drug design. Examples of their

Palii, S.S., Van Emburgh, B.O., Sankpal, U.T., Brown, K.D., Robertson, K.D., 2008. DNA

Pérez-Villanueva, J., Santos, R., Hernández-Campos, A., Giulianotti, M.A., Castillo, R.,

Pérez-Villanueva, J., Santos, R., Hernandez-Campos, A., Giulianotti, M.A., Castillo, R.,

Portela, A., Esteller, M., 2010. Epigenetic modifications and human disease. *Nat. Biotechnol.*

Ripphausen, P., Nisius, B., Bajorath, J., 2011. State-of-the-art in ligand-based virtual

applications in medicinal chemistry. *Curr. Med. Chem.* 7, 141-158.

maintains remote memory. *Nat. Neurosci.* 13, 664-666.

*Chemoinformatics and Computational Chemical Biology, Methods in Molecular Biology*, J.

methyltransferase inhibitors: A computer-aided discovery approach. *Mol. Diversity*

2008. Visualization of the chemical space in drug discovery. *Curr. Comput.-Aided* 

Pinilla, C., Houghten, R.A., 2009. Characterization of activity landscapes using 2D and 3D similarity methods: Consensus activity cliffs. *J. Chem. Inf. Model.* 49,

Rubio, M.D., Rumbaugh, G., Sweatt, J.D., 2010. Cortical DNA methylation

methylation inhibitor 5-aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B. *Mol. Cell.* 

Medina-Franco, J.L., 2010. Towards a systematic characterization of the antiprotozoal activity landscape of benzimidazole derivatives. *Bioorg. Med. Chem.*

Medina-Franco, J.L., 2011. Structure-activity relationships of benzimidazole derivatives as antiparasitic agents: Dual activity-difference (DAD) maps. *Med.* 

epigenetic cancer therapies. *J. Natl. Cancer Inst.* 97, 1498-1506.

Bajorath, (Ed.), pp. 39-100, Springer, New York.

computadora. *Educ. Quim.* 17, 452-457.


Issa, J.-P.J., Kantarjian, H.M., 2009. Targeting DNA methylation. *Clin. Cancer Res.* 15, 3938-

Issa, J.P.J., Kantarjian, H.M., Kirkpatrick, P., 2005. Azacitidine. *Nat. Rev. Drug Discovery* 4,

Jagadeesh, S., Sinha, S., Pal, B.C., Bhattacharya, S., Banerjee, P.P., 2007. Mahanine reverses

Jordan, A.M., Roughley, S.D., 2009. Drug discovery chemistry: A primer for the non-

Jurkowska, R.Z., Jurkowski, T.P., Jeltsch, A., 2011. Structure and function of mammalian

Kelly, T.K., De Carvalho, D.D., Jones, P.A., 2010. Epigenetic modifications as therapeutic

Kitchen, D.B., Decornez, H., Furr, J.R., Bajorath, J., 2004. Docking and scoring in virtual

Kuck, D., Singh, N., Lyko, F., Medina-Franco, J.L., 2010a. Novel and selective DNA

Kuck, D., Caulfield, T., Lyko, F., Medina-Franco, J.L., 2010b. Nanaomycin A selectively

Kumar, S., Cheng, X.D., Klimasauskas, S., Mi, S., Posfai, J., Roberts, R.J., Wilson, G.G., 1994. The DNA (cytosine-5) methyltransferases. *Nucleic Acids Res.* 22, 1-10. Lan, J., Hua, S., He, X.N., Zhang, Y., 2010. DNA methyltransferases and methyl-binding

Lee, B.H., Yegnasubramanian, S., Lin, X.H., Nelson, W.G., 2005a. Procainamide is a specific inhibitor of DNA methyltransferase 1. *J. Biol. Chem.* 280, 40749-40756. Lee, W.J., Shim, J.Y., Zhu, B.T., 2005b. Mechanisms for the inhibition of DNA

Li, Y., Tollefsbol, T.O., 2010. Impact on DNA methylation in cancer prevention and therapy

Liu, Z.F., Xie, Z.L., Jones, W., Pavlovicz, R.E., Liu, S.J., Yu, J.H., Li, P.K., Lin, J.Y., Fuchs, J.R.,

López-Vallejo, F., Caulfield, T., Martínez-Mayorga, K., Giulianotti, M.A., Nefzi, A.,

López-Vallejo, F., Nefzi, A., Bender, A., Owen, J.R., Nabney, I.T., Houghten, R.A., Medina-

analysis of bis-diazacyclic libraries. *Chem. Biol. Drug Des.* 77, 328-342.

by bioactive dietary components. *Curr. Med. Chem.* 17, 2141-2151.

hypomethylation agent. *Bioorg. Med. Chem. Lett.* 19, 706-709.

proteins of mammals. *Acta Biochim. Biophys. Sin.* 42, 243-252.

screening for drug discovery: Methods and applications. *Nat. Rev. Drug Discov.* 3,

methyltransferase inhibitors: Docking-based virtual screening and experimental

inhibits DNMT3B and reactivates silenced tumor suppressor genes in human

methyltransferases by tea catechins and bioflavonoids. *Mol. Pharmacol.* 68, 1018-

Marcucci, G., Li, C.L., Chan, K.K., 2009. Curcumin is a potent DNA

Houghten, R.A., Medina-Franco, J.L., 2011. Integrating virtual screening and combinatorial chemistry for accelerated drug discovery. *Comb. Chem. High* 

Franco, J.L., 2011. Increased diversity of libraries from libraries: Chemoinformatic

cancer cells. *Biochem. Biophys. Res. Commun.* 362, 212-217. Jones, P.A., Baylin, S.B., 2007. The epigenomics of cancer. *Cell* 128, 683-692.

specialist. *Drug Discovery Today* 14, 731-744.

targets. *Nat. Biotechnol.* 28, 1069-1078.

evaluation. *Bioorg. Med. Chem.* 18, 822-829.

cancer cells. *Mol. Cancer Ther.* 9, 3015-23.

*Throughput Screening* 14, 475-487.

DNA methyltransferases. *ChemBioChem* 12, 206-222.

an epigenetically silenced tumor suppressor gene RASSF1A in human prostate

3946.

275-276.

935-949.

1030.


Discovery and Optimization of Inhibitors

1010-1024.

Weinheim.

1036-1040.

4989.

11745.

25, 555-567.

of DNA Methyltransferase as Novel Drugs for Cancer Therapy 21

Singh, N., Guha, R., Giulianotti, M.A., Pinilla, C., Houghten, R.A., Medina-Franco, J.L.,

Sippl, W., Jung, M., 2009. DNA methyltransferase inhibitors, In: *Epigenetic Targets in* 

Song, J., Rechkoblit, O., Bestor, T.H., Patel, D.J., 2011. Structure of DNMT1-DNA complex

Sperandio, O., Miteva, M.A., Villoutreix, B.O., 2008. Combining ligand- and structure-based methods in drug design projects. *Curr. Comput.-Aided Drug Des.* 4, 250-258. Stresemann, C., Lyko, F., 2008. Modes of action of the DNA methyltransferase inhibitors

Suzuki, T., Tanaka, R., Hamada, S., Nakagawa, H., Miyata, N., 2010. Design, synthesis,

Tommasi, R., Cornella, I., 2006. Focused libraries: The evolution in strategy from large-

Vilkaitis, G., Merkiene, E., Serva, S., Weinhold, E., Klimasauskas, S., 2001. The mechanism of

Villar-Garea, A., Fraga, M.F., Espada, J., Esteller, M., 2003. Procaine is a DNA-demethylating

Villoutreix, B.O., Eudes, R., Miteva, M.A., 2009. Structure-based virtual ligand screening: Recent success stories. *Comb. Chem. & High Throughput Screening* 12, 1000-1016. Villoutreix, B.O., Renault, N., Lagorce, D., Sperandio, O., Montes, M., Miteva, M.A., 2007.

Wawer, M., Lounkine, E., Wassermann, A.M., Bajorath, J., 2010. Data structures and

Yokochi, T., Robertson, K.D., 2002. Preferential methylation of unmethylated DNA by

Yoo, J., Medina-Franco, J.L., 2011. Homology modeling, docking, and structure-based

Zawia, N.H., Lahiri, D.K., Cardozo-Pelaez, F., 2009. Epigenetics, oxidative stress, and

Alzheimer disease. *Free Radical Biol. Med.* 46, 1241-1249.

azacytidine and decitabine. *Int. J. Cancer* 123, 8-13.

inhibitors. *Bioorg. Med. Chem. Lett.* 20, 1124-1127.

methyltransferase. *J. Biol. Chem.* 276, 20924-20934.

Society of Chemistry, Cambridge.

*Protein Pept. Sci.* 8, 381-411.

sets. *Drug Discovery Today* 15, 630-639.

2009b. Chemoinformatic analysis of combinatorial libraries, drugs, natural products, and molecular libraries small molecule repository. *J. Chem. Inf. Model.* 49,

*DrugDiscovery*, W. Sippl, and M. Jung (Eds.), pp. 163-183, Wiley-VCH,

reveals a role for autoinhibition in maintenance DNA methylation. *Science* 331,

inhibitory activity, and binding mode study of novel DNA methyltransferase 1

diversity libraries to the focused library approach, In: *Exploiting Chemical Diversity for Drug Discovery,* A. Bartlett and M. Entzeroth, (Eds.), pp. 163-183, The Royal

DNA cytosine-5 methylation. Kinetic and mutational dissection of HhaI

agent with growth-inhibitory effects in human cancer cells. *Cancer Res.* 63, 4984-

Free resources to assist structure-based virtual ligand screening experiments. *Curr.* 

computational tools for the extraction of SAR information from large compound

mammalian de novo DNA methyltransferase dnmt3a. *J. Biol. Chem.* 277, 11735-

pharmacophore of inhibitors of DNA methyltransferase. *J. Comp.-Aided Mol. Des.*


Ripphausen, P., Nisius, B., Peltason, L., Bajorath, J.R., 2010. *Quo vadis*, virtual screening?

Robertson, K.D., 2001. DNA methylation, methyltransferases, and cancer. *Oncogene* 20, 3139-

Salam, N.K., Nuti, R., Sherman, W., 2009. Novel method for generating structure-based pharmacophores using energetic analysis. *J. Chem. Inf. Model.* 49, 2356-2368.

Schermelleh, L., Spada, F., Easwaran, H.P., Zolghadr, K., Margot, J.B., Cardoso, M.C.,

Schrump, D.S., Fischette, M.R., Nguyen, D.M., Zhao, M., Li, X.M., Kunst, T.F., Hancox, A.,

involving the lungs, esophagus, or pleura. *Clin. Cancer Res.* 12, 5777-5785. Scior, T., Bernard, P., Medina-Franco, J.L., Maggiora, G.M., 2007. Large compound databases

Scior, T., Medina-Franco, J.L., Do, Q.T., Martinez-Mayorga, K., Yunes Rojas, J.A., Bernard,

Segura-Pacheco, B., Trejo-Becerril, C., Perez-Cardenas, E., Taja-Chayeb, L., Mariscal, I.,

Sheikh, K.D., Banerjee, P.P., Jagadeesh, S., Grindrod, S.C., Zhang, L., Paige, M., Brown, M.L.,

Shelat, A.A., Guy, R.K., 2007. The interdependence between screening methods and

Siedlecki, P., Boy, R.G., Musch, T., Brueckner, B., Suhai, S., Lyko, F., Zielenkiewicz, P., 2006.

Siedlecki, P., Boy, R.G., Comagic, S., Schirrmacher, R., Wiessler, M., Zielenkiewicz, P., Suhai,

Singh, N., Dueñas-González, A., Lyko, F., Medina-Franco, J.L., 2009a. Molecular modeling

Leonhardt, H., 2005. Trapped in action: Direct visualization of DNA

Hong, J.A., Chen, G.A., Pishchik, V., Figg, W.D., Murgo, A.J., Steinberg, S.M., 2006. Phase I study of decitabine-mediated gene expression in patients with cancers

for structure-activity relationships studies in drug discovery. *Mini-Rev. Med. Chem.*

P., 2009. How to recognize and workaround pitfalls in QSAR studies: A critical

Chavez, A., Acuña, C., Salazar, A.M., Lizano, M., Dueñas-Gonzalez, A., 2003. Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy. *Clin. Cancer Res.* 9,

2010. Fluorescent epigenetic small molecule induces expression of the tumor suppressor Ras-association domain family 1a and inhibits human prostate

Discovery of two novel, small-molecule inhibitors of DNA methylation. *J. Med.* 

S., Lyko, F., 2003. Establishment and functional validation of a structural homology model for human DNA methyltransferase 1. *Biochem. Biophys. Res. Commun.* 306,

and dynamics studies of hydralazine with human DNA methyltransferase 1.

Schaefer, M., Lyko, F., 2010. Solving the dnmt2 enigma. *Chromosoma* 119, 35-40.

methyltransferase activity in living cells. *Nat. Methods* 2, 751-756.

8467.

3155.

7, 851-860.

1596-1603.

*Chem.* 49, 678-683.

*ChemMedChem* 4, 792-799.

558-563.

review. *Curr. Med. Chem.* 16, 4297-4313.

xenograft. *J. Med. Chem.* 53, 2376-2382.

screening libraries. *Curr. Opin. Chem. Biol.* 11, 244-251.

Shoichet, B.K., 2004. Virtual screening of chemical libraries. *Nature* 432, 862-865.

A comprehensive survey of prospective applications. *J. Med. Chem.* 53, 8461-


**2** 

**Development of Novel Secondary** 

**Castrate-Resistant Prostate Cancer**

Androgen deprivation therapy (ADT), initially via surgical orchiectomy and more contemporarily with medical castration through the use of luteinizing hormone-releasing hormone (LHRH) agonists, has been the mainstay of treatment for advanced prostate cancer for more than 60 years (Huggins and Hodges, 1941). Though initially effective in decreasing serum PSA, lessening pain from bone metastases, and delaying clinical progression, almost all men develop disease progression despite ADT within 2-3 years. Initially, this disease state was considered hormone-refractory and androgen-independent. However, more recent research has led to the understanding that many prostate cancers continue to depend on androgen receptor (AR) signalling in this state of low but still detectable circulating androgens. Thus, a more appropriate term for this disease state is castrate resistant prostate cancer (CRPC). In this chapter we will discuss the biology behind continued AR signalling in CRPC, traditional non-selective secondary hormonal therapies, and the development of novel secondary hormonal agents which selectively and potently target the AR axis in

**2. Androgen Receptor signalling in Castrate Resistant Prostate Cancer** 

**2.1 Ligand production in Castrate Resistant Prostate Cancer** 

In many cases, CRPC retains the ability to activate the AR to stimulate prostate cancer growth and progression, despite low circulating levels of testosterone induced by medical or surgical castration (i.e. less than 50 ng/dL). Various research efforts have sought to understand the mechanism through which this occurs, both as a means of understanding tumor biology and as a means of developing new targeted therapies exploiting the AR axis in CRPC. Signalling can be conceptually divided into efforts to understand ligand

Current ADT strategies using LHRH agonists suppress gonadal androgen production, resulting in a decrease in circulating serum testosterone to castrate levels (less than 50 ng/dL). Despite gonadal androgen suppression, low levels of circulating androgens persist

**1. Introduction** 

CRPC.

production and AR modification.

**2.1.1 Endocrine ligands** 

**Hormonal Therapies for** 

Rahul Aggarwal and Charles J. Ryan *University of California San Francisco* 

*United States of America* 

Zheng, W., Johnson, S.R., 2008. Compound library design - principles and applications, In: *Chemoinformatics Approaches to Virtual Screening,* A. Varnek and A. Tropsha, (Eds.), pp. 268-294, Royal Society of Chemistry, Cambridge.

## **Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer**

Rahul Aggarwal and Charles J. Ryan *University of California San Francisco United States of America* 

## **1. Introduction**

22 Drug Development – A Case Study Based Insight into Modern Strategies

Zheng, W., Johnson, S.R., 2008. Compound library design - principles and applications, In:

pp. 268-294, Royal Society of Chemistry, Cambridge.

*Chemoinformatics Approaches to Virtual Screening,* A. Varnek and A. Tropsha, (Eds.),

Androgen deprivation therapy (ADT), initially via surgical orchiectomy and more contemporarily with medical castration through the use of luteinizing hormone-releasing hormone (LHRH) agonists, has been the mainstay of treatment for advanced prostate cancer for more than 60 years (Huggins and Hodges, 1941). Though initially effective in decreasing serum PSA, lessening pain from bone metastases, and delaying clinical progression, almost all men develop disease progression despite ADT within 2-3 years. Initially, this disease state was considered hormone-refractory and androgen-independent. However, more recent research has led to the understanding that many prostate cancers continue to depend on androgen receptor (AR) signalling in this state of low but still detectable circulating androgens. Thus, a more appropriate term for this disease state is castrate resistant prostate cancer (CRPC). In this chapter we will discuss the biology behind continued AR signalling in CRPC, traditional non-selective secondary hormonal therapies, and the development of novel secondary hormonal agents which selectively and potently target the AR axis in CRPC.

## **2. Androgen Receptor signalling in Castrate Resistant Prostate Cancer**

In many cases, CRPC retains the ability to activate the AR to stimulate prostate cancer growth and progression, despite low circulating levels of testosterone induced by medical or surgical castration (i.e. less than 50 ng/dL). Various research efforts have sought to understand the mechanism through which this occurs, both as a means of understanding tumor biology and as a means of developing new targeted therapies exploiting the AR axis in CRPC. Signalling can be conceptually divided into efforts to understand ligand production and AR modification.

#### **2.1 Ligand production in Castrate Resistant Prostate Cancer 2.1.1 Endocrine ligands**

Current ADT strategies using LHRH agonists suppress gonadal androgen production, resulting in a decrease in circulating serum testosterone to castrate levels (less than 50 ng/dL). Despite gonadal androgen suppression, low levels of circulating androgens persist

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 25

More recently, research has shown that CRPC tissue has the ability to convert adrenal steroids to androgens, thereby creating an intracrine signalling system capable of converting steroid precursors to testosterone and dihydrotestosterone (DHT) which leads to continued stimulation of the AR and prostate cancer progression. The evidence for this comes from various lines of research. Direct measurements of intra-prostatic androgens including DHT demonstrates that levels of androgens in CRPC tissue is not significantly different compared with normal prostate tissue, despite significantly lower levels of circulating testosterone in the castrate men (Nishiyama T, et al. 2004). This finding implies that CRPC tissue acquires the ability to produce testosterone and DHT in an intracrine fashion, a finding which has been supported by further studies showing up-regulation of many of the steroid enzymes

For example, Stanbrough et al. analysed oligonucleotide microarrays from 33 CRPC bone metastasis samples and compared their gene expression with samples from 22 hormonesensitive primary cancers. The CRPC bone metastases demonstrated up-regulated expression of several enzymes involved in the steroid synthetic pathway: 17-beta hydroxysteroid dehydrogenase which converts androstenedione to testosterone; 3-beta hydroxysteroid dehydrogenase, which converts DHEA to androstenedione, and increased ratio of 5-alpha reductase isoform 1 to 2, which converts testosterone to DHT (Stanbrough

In a follow up study, Montgomery et al. evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. In this study, castrate-resistant tissues displayed increased expression of several key enzymes involved in androgen synthesis, including: CYP17A1 (C17,20 lyase), a key enzyme which converts progesterone and pregnenolone to 17-hydroxyprogesterone and 17-OH pregnenolone, as well as subsequent conversion of these steroids to androstenedione and DHEA respectively; 3-beta hydroxysteroid dehydrogenase as in the prior study. Furthermore, metastatic prostate cancers from CRPC patient samples express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival in a xenograft model (Montgomery R et al, 2008). Finally, in an innovative study by Locke et al., it was demonstrated that tumor explants isolated from CRPC progression are capable of de novo conversion of [14C] acetic acid to dihydrotestosterone and that uptake of [3H] progesterone allows detection of the

This cumulative body of evidence suggests that CRPCs are capable of adapting to lower circulating levels of androgens induced by castration, in which steroid enzymes involved in the synthetic pathway are upregulated, and thereby maintain high levels of intra-tumor androgens capable of stimulating the AR and driving prostate cancer progression. Understanding this mechanism of castration resistance has led to the development of targeted secondary hormonal therapies which specifically inhibit key enzymes of the

In addition to modification in the enzymes involved in steroid hormone production, the AR itself undergoes adaptation in the castrate state, and is implicated in disease progression to

production of six other steroids upstream of dihydrotestosterone.

androgen synthetic pathway, as will be discussed in the later section.

**2.2 Androgen Receptor modification in Castrate Resistant Prostate Cancer** 

**2.1.2 Intracrine ligands** 

M, et al 2006).

involved in androgen synthesis (see figure 1).

in CRPC, often via production of adrenal androgens, such as dihydroepiandrostenedione (DHEA), DHEA-sulfate (DHEA-S), and androstenedione, which are then converted to testosterone in peripheral tissues. Figure 1 below displays the steroid biosynthetic pathway and several secondary hormonal agents which block various steps of steroidogenesis, to be discussed in the following sections.

Fig. 1. Steroid Biosynthetic Pathway. Adapted from Aggarwal R and Ryan C, 2011.

Due to the peripheral conversion of adrenal steroids, low levels of circulating testosterone persists, and may account for levels up to 10% of that of pre-castrate levels (Puche, C et al. 2002). Low levels of circulating testosterone, along with circulating adrenal androgens, are hypothesized to subsequently stimulate CRPC progression through activation of the AR.

## **2.1.2 Intracrine ligands**

24 Drug Development – A Case Study Based Insight into Modern Strategies

in CRPC, often via production of adrenal androgens, such as dihydroepiandrostenedione (DHEA), DHEA-sulfate (DHEA-S), and androstenedione, which are then converted to testosterone in peripheral tissues. Figure 1 below displays the steroid biosynthetic pathway and several secondary hormonal agents which block various steps of steroidogenesis, to be

Fig. 1. Steroid Biosynthetic Pathway. Adapted from Aggarwal R and Ryan C, 2011.

Due to the peripheral conversion of adrenal steroids, low levels of circulating testosterone persists, and may account for levels up to 10% of that of pre-castrate levels (Puche, C et al. 2002). Low levels of circulating testosterone, along with circulating adrenal androgens, are hypothesized to subsequently stimulate CRPC progression through activation of the AR.

discussed in the following sections.

More recently, research has shown that CRPC tissue has the ability to convert adrenal steroids to androgens, thereby creating an intracrine signalling system capable of converting steroid precursors to testosterone and dihydrotestosterone (DHT) which leads to continued stimulation of the AR and prostate cancer progression. The evidence for this comes from various lines of research. Direct measurements of intra-prostatic androgens including DHT demonstrates that levels of androgens in CRPC tissue is not significantly different compared with normal prostate tissue, despite significantly lower levels of circulating testosterone in the castrate men (Nishiyama T, et al. 2004). This finding implies that CRPC tissue acquires the ability to produce testosterone and DHT in an intracrine fashion, a finding which has been supported by further studies showing up-regulation of many of the steroid enzymes involved in androgen synthesis (see figure 1).

For example, Stanbrough et al. analysed oligonucleotide microarrays from 33 CRPC bone metastasis samples and compared their gene expression with samples from 22 hormonesensitive primary cancers. The CRPC bone metastases demonstrated up-regulated expression of several enzymes involved in the steroid synthetic pathway: 17-beta hydroxysteroid dehydrogenase which converts androstenedione to testosterone; 3-beta hydroxysteroid dehydrogenase, which converts DHEA to androstenedione, and increased ratio of 5-alpha reductase isoform 1 to 2, which converts testosterone to DHT (Stanbrough M, et al 2006).

In a follow up study, Montgomery et al. evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. In this study, castrate-resistant tissues displayed increased expression of several key enzymes involved in androgen synthesis, including: CYP17A1 (C17,20 lyase), a key enzyme which converts progesterone and pregnenolone to 17-hydroxyprogesterone and 17-OH pregnenolone, as well as subsequent conversion of these steroids to androstenedione and DHEA respectively; 3-beta hydroxysteroid dehydrogenase as in the prior study. Furthermore, metastatic prostate cancers from CRPC patient samples express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival in a xenograft model (Montgomery R et al, 2008). Finally, in an innovative study by Locke et al., it was demonstrated that tumor explants isolated from CRPC progression are capable of de novo conversion of [14C] acetic acid to dihydrotestosterone and that uptake of [3H] progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone.

This cumulative body of evidence suggests that CRPCs are capable of adapting to lower circulating levels of androgens induced by castration, in which steroid enzymes involved in the synthetic pathway are upregulated, and thereby maintain high levels of intra-tumor androgens capable of stimulating the AR and driving prostate cancer progression. Understanding this mechanism of castration resistance has led to the development of targeted secondary hormonal therapies which specifically inhibit key enzymes of the androgen synthetic pathway, as will be discussed in the later section.

## **2.2 Androgen Receptor modification in Castrate Resistant Prostate Cancer**

In addition to modification in the enzymes involved in steroid hormone production, the AR itself undergoes adaptation in the castrate state, and is implicated in disease progression to

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 27

CW, et al. 2001). This data suggests that AR activation and subsequent AR-mediated gene expression may in part be stimulated in CRPCs by mechanisms to prevent AR degradation and enhance localization to the nucleus. The mechanism of AR stabilization in CRPCs may in part related to increased cyclin-dependent kinase 1, which has been shown to phosphorylate and stabilize the AR and is also upregulated in castrate-resistant cell lines in

Estimating the true frequency of acquired point mutations with functional significance in advanced prostate cancer has been difficult, due to various factors including patient selection, tumor heterogeneity, tissue source (prostate gland v metastases), method of tissue preservation, and molecular methods. They appear to be fairly uncommon in early prostate cancer and more prevalent in advanced prostate cancer. In a correlative analysis of bone marrow samples from patients with CRPC being treated with first generation anti-androgen withdrawal (CALGB study 9663), 10% of the patient samples had an AR point mutation, which was found within the hormone binding domain involved with transcription factor binding (Taplin M, et al. 2003). From a functional standpoint, it appears that certain AR point mutations lead to a more promiscuous AR, capable of being activated by a wider range of ligands. In a prior study of a mutant AR transfected into various cell lines, the adrenal androgen DHEA was capable of inducing greater AR-mediated transcriptional

activity in the mutant AR cell line compared with wild type AR (Tan J, et al. 1997).

**2.2.4 Ligand-independent activation of the Androgen Receptor** 

growth factor pathways, among others (Feldman B & Feldman D, 2001).

**2.2.5 Androgen Receptor splice variants** 

In this way, the increase in AR promiscuity may complement the changes in ligand production as outlined in the previous section, in which point mutations in the AR confer a greater ability for the AR to be activated by alternative ligands in the presence of low circulating testosterone levels, including the adrenal androgens such as DHEA. Mutations in the AR may also lead to partial agonistic activity of the first generation anti-androgens, such as flutamide, nilutamide, and bicalutamide, as will be discussed in the following section.

There is a wide-ranging body of evidence which suggests that for a subset of prostate cancers, ligand-independent activation of the AR, via activation from other signal transduction pathways, can independently activate the AR and lead to disease progression in the absence of hormone binding to the AR. Though not the focus of the current book chapter, the various signaling pathways that have been implicated in such a manner include the insulin-like growth factor pathway, epidermal growth factor receptor, and keratinocyte

Over the past several years a growing body of research implicates the generation of AR splice variants as a potential mechanism of driving disease progression to CRPC. Such AR splice variants have "hidden exons" within introns that are not normally transcribed in the wild type AR. The alternate splicing that incorporates such hidden exons into the variant mRNA transcripts creates pre-mature stop codons prior to the translation of the C-terminal ligand binding domain. Thus, variant AR proteins are created which lack the traditional ligand binding domain (see figure 2 below). In a seminal paper by Hu et al. prostate cancer tissue from primary hormone-sensitive and metastatic CRPC cancer tissue was analyzed by

prior pre-clinical study (Chen S, et al. 2006).

**2.2.3 Androgen Receptor point mutations** 

CRPC. Mechanisms by which the AR adapts to the castrate state have been extensively studied in the past several decades, and include: (1) AR amplification and overexpression (2) heightened AR sensitivity to ligand activation through increased AR stabilization, enhanced nuclear localization, and overexpression of nuclear co-activators (3) increased AR promiscuity through various point mutations (4) ligand-independent activation of the AR through various signal transduction pathways, (5) AR splice variants with constitutive activity. In the following sections we will examine some of the evidence behind these modifications to the AR.

#### **2.2.1 Androgen Receptor gene amplification and overexpression**

In the late 1990s, research was starting to show that AR activation continued to play an important role in prostate cancer progression despite low circulating testosterone levels, and a potential mechanism through which this might occur was AR gene amplification and overexpression. In a study by Koivisto, et al, AR gene amplification was analyzed in 54 patient tumor samples at the time of recurrence after prior therapy, as well as in 26 cases, paired primary tumor samples prior to any therapy. In this study, 28% of the recurrent therapy-resistant tumors, versus none of the primary tumor samples, displayed AR gene amplification. Furthermore, through genomic analysis, the AR gene was wild type in all but one of the 15 AR gene amplified tumor samples. Interestingly, this study went on to show a clinicopathologic correlation between AR gene amplification and prior responsiveness to ADT, as well as improved subsequent prognosis (Koivisto P et al. 1997). In a follow up study by Linja et al., in which real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze AR expression levels in eight benign prostate hyperplasias, 33 untreated and 13 castrate-resistant locally recurrent carcinomas, as well as 10 prostate cancer xenografts. All castrate-resistant tumors showed on average, 6-fold higher expression than androgen-dependent tumors or benign prostate hyperplasias (P < 0.001). Four of 13 (31%) castrate-resistant tumors contained AR gene amplification detected by fluorescence in situ hybridization. Finally, and equally as important, two of the ten prostate cancer xenograft models displayed AR overexpression, thus providing a key model for testing future drugs targeting the AR in the AR-amplified disease state (Linja MJ, et al. 2001).

Early studies such as these provided compelling evidence that AR gene amplification and thus overexpression may represent an important mechanism by which prostate cancers overcome low circulating androgen levels. Given this, a logical therapeutic strategy is the development of potent AR antagonists which would have activity in this AR-amplified disease state, and indeed, there are several novel potent, AR antagonists which are in clinical phase of drug development (see section below).

#### **2.2.2 Androgen Receptor stabilization and heightened activity**

In addition to numerical increase in the number of ARs per cancer cell, increased stabilization and nuclear localization of the AR may also factor into the mechanism of prostate cancer progression in the castrate resistant disease state. In a prior study by Gregory et al, recurrent prostate cancer cell lines had an AR degradation half-life that was 2- 4 times longer than that of androgen-sensitive cancer cell lines. Furthermore, IHC staining showing that AR localization was entirely nuclear in the recurrent cancer cell lines; while localizing to both the cytoplasm and nucleus in the androgen sensitive cell lines (Gregory CW, et al. 2001). This data suggests that AR activation and subsequent AR-mediated gene expression may in part be stimulated in CRPCs by mechanisms to prevent AR degradation and enhance localization to the nucleus. The mechanism of AR stabilization in CRPCs may in part related to increased cyclin-dependent kinase 1, which has been shown to phosphorylate and stabilize the AR and is also upregulated in castrate-resistant cell lines in prior pre-clinical study (Chen S, et al. 2006).

## **2.2.3 Androgen Receptor point mutations**

26 Drug Development – A Case Study Based Insight into Modern Strategies

CRPC. Mechanisms by which the AR adapts to the castrate state have been extensively studied in the past several decades, and include: (1) AR amplification and overexpression (2) heightened AR sensitivity to ligand activation through increased AR stabilization, enhanced nuclear localization, and overexpression of nuclear co-activators (3) increased AR promiscuity through various point mutations (4) ligand-independent activation of the AR through various signal transduction pathways, (5) AR splice variants with constitutive activity. In the following sections we will examine some of the evidence behind these

In the late 1990s, research was starting to show that AR activation continued to play an important role in prostate cancer progression despite low circulating testosterone levels, and a potential mechanism through which this might occur was AR gene amplification and overexpression. In a study by Koivisto, et al, AR gene amplification was analyzed in 54 patient tumor samples at the time of recurrence after prior therapy, as well as in 26 cases, paired primary tumor samples prior to any therapy. In this study, 28% of the recurrent therapy-resistant tumors, versus none of the primary tumor samples, displayed AR gene amplification. Furthermore, through genomic analysis, the AR gene was wild type in all but one of the 15 AR gene amplified tumor samples. Interestingly, this study went on to show a clinicopathologic correlation between AR gene amplification and prior responsiveness to ADT, as well as improved subsequent prognosis (Koivisto P et al. 1997). In a follow up study by Linja et al., in which real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze AR expression levels in eight benign prostate hyperplasias, 33 untreated and 13 castrate-resistant locally recurrent carcinomas, as well as 10 prostate cancer xenografts. All castrate-resistant tumors showed on average, 6-fold higher expression than androgen-dependent tumors or benign prostate hyperplasias (P < 0.001). Four of 13 (31%) castrate-resistant tumors contained AR gene amplification detected by fluorescence in situ hybridization. Finally, and equally as important, two of the ten prostate cancer xenograft models displayed AR overexpression, thus providing a key model for testing future drugs targeting the AR in the AR-amplified disease state (Linja MJ, et al.

Early studies such as these provided compelling evidence that AR gene amplification and thus overexpression may represent an important mechanism by which prostate cancers overcome low circulating androgen levels. Given this, a logical therapeutic strategy is the development of potent AR antagonists which would have activity in this AR-amplified disease state, and indeed, there are several novel potent, AR antagonists which are in

In addition to numerical increase in the number of ARs per cancer cell, increased stabilization and nuclear localization of the AR may also factor into the mechanism of prostate cancer progression in the castrate resistant disease state. In a prior study by Gregory et al, recurrent prostate cancer cell lines had an AR degradation half-life that was 2- 4 times longer than that of androgen-sensitive cancer cell lines. Furthermore, IHC staining showing that AR localization was entirely nuclear in the recurrent cancer cell lines; while localizing to both the cytoplasm and nucleus in the androgen sensitive cell lines (Gregory

clinical phase of drug development (see section below).

**2.2.2 Androgen Receptor stabilization and heightened activity** 

**2.2.1 Androgen Receptor gene amplification and overexpression** 

modifications to the AR.

2001).

Estimating the true frequency of acquired point mutations with functional significance in advanced prostate cancer has been difficult, due to various factors including patient selection, tumor heterogeneity, tissue source (prostate gland v metastases), method of tissue preservation, and molecular methods. They appear to be fairly uncommon in early prostate cancer and more prevalent in advanced prostate cancer. In a correlative analysis of bone marrow samples from patients with CRPC being treated with first generation anti-androgen withdrawal (CALGB study 9663), 10% of the patient samples had an AR point mutation, which was found within the hormone binding domain involved with transcription factor binding (Taplin M, et al. 2003). From a functional standpoint, it appears that certain AR point mutations lead to a more promiscuous AR, capable of being activated by a wider range of ligands. In a prior study of a mutant AR transfected into various cell lines, the adrenal androgen DHEA was capable of inducing greater AR-mediated transcriptional activity in the mutant AR cell line compared with wild type AR (Tan J, et al. 1997).

In this way, the increase in AR promiscuity may complement the changes in ligand production as outlined in the previous section, in which point mutations in the AR confer a greater ability for the AR to be activated by alternative ligands in the presence of low circulating testosterone levels, including the adrenal androgens such as DHEA. Mutations in the AR may also lead to partial agonistic activity of the first generation anti-androgens, such as flutamide, nilutamide, and bicalutamide, as will be discussed in the following section.

### **2.2.4 Ligand-independent activation of the Androgen Receptor**

There is a wide-ranging body of evidence which suggests that for a subset of prostate cancers, ligand-independent activation of the AR, via activation from other signal transduction pathways, can independently activate the AR and lead to disease progression in the absence of hormone binding to the AR. Though not the focus of the current book chapter, the various signaling pathways that have been implicated in such a manner include the insulin-like growth factor pathway, epidermal growth factor receptor, and keratinocyte growth factor pathways, among others (Feldman B & Feldman D, 2001).

### **2.2.5 Androgen Receptor splice variants**

Over the past several years a growing body of research implicates the generation of AR splice variants as a potential mechanism of driving disease progression to CRPC. Such AR splice variants have "hidden exons" within introns that are not normally transcribed in the wild type AR. The alternate splicing that incorporates such hidden exons into the variant mRNA transcripts creates pre-mature stop codons prior to the translation of the C-terminal ligand binding domain. Thus, variant AR proteins are created which lack the traditional ligand binding domain (see figure 2 below). In a seminal paper by Hu et al. prostate cancer tissue from primary hormone-sensitive and metastatic CRPC cancer tissue was analyzed by

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 29

each successive hormonal manipulation. Chemotherapy has traditionally been the mainstay of treatment for CRPC patients who have failed secondary hormonal therapy; however the median increase in overall survival with first line docetaxel chemotherapy is a modest 3 months, and fewer than 20% of patients with CRPC live beyond 3 years (Tannock, et al. 2004; Petyrlak DP, et al. 2004). Clearly, novel therapies are needed which applied together or in succession can lead to meaningful improvement in the quality and quantity of time for patients with CRPC. In the following sections we will first discuss the traditional secondary hormonal agents which have been used to treat CRPC. We will then continue onwards with a discussion of the novel secondary hormonal therapies currently in clinical development, which more selectively and potently inhibit either steroid ligand production or AR

First generation antiandrogens, which competitively inhibit the binding of androgens to the ligand binding doman of the AR, remain in widespread use in the treatment of prostate cancer of various disease stages. The addition of a first generation antiandrogen (i.e. flutamide, nilutamide, or bicalutamide) to medical castration (combined androgen blockade) demonstrates only modest benefits in the hormone-sensitive disease population, with a small absolute survival benefit of less than 5% in most studies and meta-analyses. Similarly, the addition of an anti-androgen after ADT fails has demonstrated only modest benefit in prior clinical studies. In a prior clinical trial of flutamide 250 mg orally three times daily versus prednisone 5 mg orally four times per day, the median time to symptomatic progression on flutamide was only 2.3 months (as compared to 3.4 months with prednisone), and the proportion of patients with a greater than 50% decline in PSA or greater was 23% in the flutamide group vs. 21% in the prednisone group (Fossa SD, et al.

Similar rates of biochemical response were noted in a trial of 232 men who received either flutamide 375 mg/day or bicalutamide 80 mg/day after disease progression on combined androgen blockade. The percentage of men with a greater than 50% decline in PSA was 35.8%; the response duration was a little over 6 months (Suzuki H, et al. 2008). In another small trial of 31 men with CRPC treated with high dose bicalutamide 150 mg/day, only 22.5% of men had a PSA decline of > 50% for more than 2 months, almost all in men without

The modest efficacy and limited duration of response of first generation anti-androgens may in part be due to the fact that these molecules can act as partial agonists of the AR, especially AR which develop point mutations as a mechanism of resistance to these anti-androgens. Clinically, this partial agonist effect is observed with the phenomenon of anti-androgen withdrawal, a therapeutic maneuver in which the anti-androgen is discontinued in a patient who is progressing despite combined androgen blockade. In a prior study of anti-androgen withdrawal, 11% of patients demonstrated a decline of 50% or more in serum PSA after antiandrogen withdrawal (Small E, et al. 2004). Presumably, in these small subsets of patients who respond to antiandrogen withdrawal, the AR may have developed mutations which

Novel second generation antiandrogens which lack any agonist activity against the AR and demonstrate markedly more potent AR inhibition, including MDV-3100, will be discussed

activation.

2001).

**3.1 First generation antiandrogens** 

prior treatment with flutamide (Joyce R, et al. 1998).

confer the ability to be activated by the antiandrogen.

in the upcoming section.

*in silico* DNA sequencing for the presence of AR splice variants. In total, 7 variant AR transcripts were discovered, AR-V1 through AR-V7. The two most abundantly expressed were AR-V1 and AR-V7. On average, there was 20-fold higher expression of these two variant transcripts in CRPC as opposed to hormone-sensitive prostate cancer. Functionally, AR-V7 was found to localize to the nucleus of prostate cancer cell line under androgen depleted conditions, and most importantly, was constitutively active in driving the expression of androgen-responsive genes (Hu, R, et al. 2009).

Fig. 2. Androgen Receptor Transcript and Splice Variants. NTD = N terminal domain. DBD = DNA binding domain. The hatched areas represent "hidden" exons spliced into the DNA biding domain exons (2 and 3), thus creating variant AR transcripts. The hidden exons of the variant AR transcripts encode stop codons, leading to premature termination and exclusion of the C-terminal ligand binding domain (exons 5-8 in green). Figure adapted from Guo, Z & Qiu, Y, 2011.

The exciting discovery of AR splice variants represents another potential mechanism by which cancer cells modify AR processing to adapt to a low circulating testosterone environment, creating AR splice variants which are not dependent on hormone binding to drive gene expression and cancer cell division and metastasis. Targeting the variant AR proteins, perhaps at the more ubiquitous N-terminal domain, represents a potential therapeutic approach to overcome this mechanism of resistance.

## **3. Traditional secondary hormonal therapies for Castrate Resistant Prostate Cancer**

Traditional hormonal manipulations can be of some benefit to patients with CRPC; however significant responses are not seen in the majority of patients, and responses tend to be shortlived. Furthermore, the response duration and magnitude of benefit tend to diminish with each successive hormonal manipulation. Chemotherapy has traditionally been the mainstay of treatment for CRPC patients who have failed secondary hormonal therapy; however the median increase in overall survival with first line docetaxel chemotherapy is a modest 3 months, and fewer than 20% of patients with CRPC live beyond 3 years (Tannock, et al. 2004; Petyrlak DP, et al. 2004). Clearly, novel therapies are needed which applied together or in succession can lead to meaningful improvement in the quality and quantity of time for patients with CRPC. In the following sections we will first discuss the traditional secondary hormonal agents which have been used to treat CRPC. We will then continue onwards with a discussion of the novel secondary hormonal therapies currently in clinical development, which more selectively and potently inhibit either steroid ligand production or AR activation.

#### **3.1 First generation antiandrogens**

28 Drug Development – A Case Study Based Insight into Modern Strategies

*in silico* DNA sequencing for the presence of AR splice variants. In total, 7 variant AR transcripts were discovered, AR-V1 through AR-V7. The two most abundantly expressed were AR-V1 and AR-V7. On average, there was 20-fold higher expression of these two variant transcripts in CRPC as opposed to hormone-sensitive prostate cancer. Functionally, AR-V7 was found to localize to the nucleus of prostate cancer cell line under androgen depleted conditions, and most importantly, was constitutively active in driving the

**Variant AR Proteins Which Lack the Ligand Binding Domain** 

Fig. 2. Androgen Receptor Transcript and Splice Variants. NTD = N terminal domain. DBD = DNA binding domain. The hatched areas represent "hidden" exons spliced into the DNA biding domain exons (2 and 3), thus creating variant AR transcripts. The hidden exons of the variant AR transcripts encode stop codons, leading to premature termination and exclusion of the C-terminal ligand binding domain (exons 5-8 in green). Figure adapted from Guo, Z &

The exciting discovery of AR splice variants represents another potential mechanism by which cancer cells modify AR processing to adapt to a low circulating testosterone environment, creating AR splice variants which are not dependent on hormone binding to drive gene expression and cancer cell division and metastasis. Targeting the variant AR proteins, perhaps at the more ubiquitous N-terminal domain, represents a potential

**3. Traditional secondary hormonal therapies for Castrate Resistant Prostate** 

Traditional hormonal manipulations can be of some benefit to patients with CRPC; however significant responses are not seen in the majority of patients, and responses tend to be shortlived. Furthermore, the response duration and magnitude of benefit tend to diminish with

therapeutic approach to overcome this mechanism of resistance.

expression of androgen-responsive genes (Hu, R, et al. 2009).

Qiu, Y, 2011.

**Cancer** 

First generation antiandrogens, which competitively inhibit the binding of androgens to the ligand binding doman of the AR, remain in widespread use in the treatment of prostate cancer of various disease stages. The addition of a first generation antiandrogen (i.e. flutamide, nilutamide, or bicalutamide) to medical castration (combined androgen blockade) demonstrates only modest benefits in the hormone-sensitive disease population, with a small absolute survival benefit of less than 5% in most studies and meta-analyses. Similarly, the addition of an anti-androgen after ADT fails has demonstrated only modest benefit in prior clinical studies. In a prior clinical trial of flutamide 250 mg orally three times daily versus prednisone 5 mg orally four times per day, the median time to symptomatic progression on flutamide was only 2.3 months (as compared to 3.4 months with prednisone), and the proportion of patients with a greater than 50% decline in PSA or greater was 23% in the flutamide group vs. 21% in the prednisone group (Fossa SD, et al. 2001).

Similar rates of biochemical response were noted in a trial of 232 men who received either flutamide 375 mg/day or bicalutamide 80 mg/day after disease progression on combined androgen blockade. The percentage of men with a greater than 50% decline in PSA was 35.8%; the response duration was a little over 6 months (Suzuki H, et al. 2008). In another small trial of 31 men with CRPC treated with high dose bicalutamide 150 mg/day, only 22.5% of men had a PSA decline of > 50% for more than 2 months, almost all in men without prior treatment with flutamide (Joyce R, et al. 1998).

The modest efficacy and limited duration of response of first generation anti-androgens may in part be due to the fact that these molecules can act as partial agonists of the AR, especially AR which develop point mutations as a mechanism of resistance to these anti-androgens. Clinically, this partial agonist effect is observed with the phenomenon of anti-androgen withdrawal, a therapeutic maneuver in which the anti-androgen is discontinued in a patient who is progressing despite combined androgen blockade. In a prior study of anti-androgen withdrawal, 11% of patients demonstrated a decline of 50% or more in serum PSA after antiandrogen withdrawal (Small E, et al. 2004). Presumably, in these small subsets of patients who respond to antiandrogen withdrawal, the AR may have developed mutations which confer the ability to be activated by the antiandrogen.

Novel second generation antiandrogens which lack any agonist activity against the AR and demonstrate markedly more potent AR inhibition, including MDV-3100, will be discussed in the upcoming section.

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 31

with asymptomatic or minimally symptomatic bone-only metastatic or rising PSA-only

Insights into the mechanisms of continued AR signaling in CRPC, as discussed above, including (1) adrenal and intra-tumoral androgen ligand production, and (2) modifications of the AR, including gene amplification, over-expression, point mutations, ligandindependent activation, and splice variants, have led to the development of novel secondary hormonal therapies for CRPC. These new therapies are more selective and potent than their traditional counterparts. In the following subsections we will discuss the clinical

As displayed in figure 1, CYP17 (17-alpha hydroxylase/C17, 20 lyase) catalyzes two key steps of androgen synthesis within the steroid biosynthetic pathway: the 17-hydroxylation of progesterone and pregnenolone and subsequent conversion to DHEA and androstenedione respectively. Inhibition of this enzyme would divert cholesterol derivatives away from androgen production, and towards mineralocorticoid production (corticosterone and aldosterone). As outlined above, intra-tumoral upregulation of CYP17 has been previously implicated in the progression to CRPC. Logically then, selective inhibition of CYP17 represents an attractive strategy for inhibiting adrenal and intra-tumor androgen

Abiraterone acetate is the prodrug of abiraterone, a potent, highly selective, irreversible inhibitor of CYP17. In pre-clinical in vivo study using WHT mice, this compound was able to markedly decrease the level of serum testosterone to less than 0.1 nanomolar concentration, despite 3-4 fold increase in serum LH concentration (Barrie SE, et al. 1994). In the first phase 1 study of abiraterone, O'Donnell et al. studied various dosing schedules ranging from 10 to 500 mg x 1 dose in castrate resistant men. At a dose level of 500 mg, there was suppression of serum testosterone to less than lower limit of detection (< 0.14 nmol/L) with parallel reduction androstenedione levels, supporting its mechanism of action of CYP17 inhibition. The duration of testosterone suppression after a single dose was variable, but generally ranged from days 2-5 post-dose (O'Donnell A, et al. 2004). In a follow up phase I trial by Attard and colleagues, 21 patients with CRPC and progression through multiple prior traditional secondary hormonal therapies were treated with abiraterone with doses ranging from 250 mg to 2000 mg/day. Pharmacodynamic effects on serum hormone levels showed a plateau at a dose of 1000 mg/day, which was the dose level of an expanded cohort of 9 patients and the subsequent recommended phase II/III dose. There were no treatment-related grade 3 or 4 adverse events from this trial. As expected, increases in levels of ACTH, corticosterone, and 11-deoxycorticosterone were observed, and there were adverse events related to subsequent mineralocorticoid excess, namely hypokalemia and hypertension. This was effectively managed with the use of eplerenone, a mineralocorticoid antagonist. The median baseline serum testosterone level was 7 ng/mL in this study; at all

**4. Novel secondary hormonal therapies for Castrate Resistant Prostate** 

development of several of the new hormonal therapies for CRPC.

production in CRPCs and thereby slowing disease progression.

**4.1 Selective inhibition of CYP17** 

**4.1.1 Abiraterone acetate** 

disease.

**Cancer** 

## **3.2 Estrogens**

Estrogens have long known to have been active in the treatment of prostate cancer; however the exact mechanism of actions remains uncertain. Putative mechanisms include inhibition of LH hormone release from the pituitary gland, inhibition of adrenal androgen production, and a direct cytotoxic effect on prostate cancer cells (Robertson CN, et al. 1996). In a prior phase randomized phase II trial comparing the estrogenic herbal compound PC-SPES with diethylstilbestrol, a greater than 50% decline in baseline PSA was noted in 40% and 24% of patients respectively; median time to progression was 5.5 vs. 2.9 months respectively (Oh W, et al. 2004).

There is clearly a modest degree of activity of estrogenic compounds in the treatment of CRPC; however current use of these agents (i.e. diethylstilbestrol, Premarin, etc.) is limited by the small but not insignificant risk of venous thromboembolic events and possibly increased risk of myocardial infarction and stroke; these particular co-morbidities are especially concerning in a disease population of elderly men. Concomitant prophylactic anticoagulation is recommended when using these agents.

## **3.3 Ketoconazole**

Ketoconazole is a broad, non-specific inhibitor of multiple cytochrome p450 enzymes involved in androgen biosynthesis, including the conversion of cholesterol to pregnenolone, 11-beta hydroxylation, and 17-alpha hydroxylase/C17, 20 lyase (CYP17) activity. In a previously referred to randomized phase II study of 260 men with CRPC, with progressive disease despite combined androgen blockade, randomized to treatment with antiandrogen withdrawal alone or in combination with ketoconazole, 27% of patients assigned to the ketoconazole arm had a 50% or greater decline in serum PSA level, and 20% of patients had an objective response (Small EJ, et al. 2004). Interestingly, at the time of disease progression on ketoconazole, levels of adrenal androgens including DHEA, DHEA-S, and androstenedione had all increased compared to month 1 levels, which suggest that ketoconazole resistance may in part reflect inadequate androgen production suppression. This mechanism of resistance has implications for the development of novel androgen synthetic enzyme inhibitors such as abiraterone acetate. In an intriguing analysis of adrenal androgen hormone levels from the study by Small et al., patients who had higher baseline levels of androstenedione had a higher likelihood of response to treatment with ketoconazole (Ryan CJ, et al. 2007). This suggests that baseline adrenal androgen levels may be used as predictive biomarker for the use of adrenal androgen blockade as a therapeutic maneuver for CRPC; however this hypothesis requires prospective validation in larger studies.

Ketoconazole is a relatively non-specific inhibitor of multiple enzymes involved in the steroid synthetic pathway, and as such, as blocks normal corticosteroid production and causes iatrogenic adrenal insufficiency. Accordingly, side effects of this medication include lethargy, rash, nausea, and liver toxicity. Supplementation with physiologic replacement doses of hydrocortisone (i.e. 20 mg in the morning, 10 mg in the evening) is required while patients are taking ketoconazole. Furthermore, given the relatively non-specific CYPP450 inhibition, ketoconazole interacts with a wide number of other medications. Its oral absorption and bioavailability can be variable, depending on the acidity of the stomach and fed/fasting state and use of acid suppressing medications.

Despite these potential side effects and drug interactions, ketoconazole represents a viable and widely used secondary hormonal agent for CRPC, especially in the patient population with asymptomatic or minimally symptomatic bone-only metastatic or rising PSA-only disease.

## **4. Novel secondary hormonal therapies for Castrate Resistant Prostate Cancer**

Insights into the mechanisms of continued AR signaling in CRPC, as discussed above, including (1) adrenal and intra-tumoral androgen ligand production, and (2) modifications of the AR, including gene amplification, over-expression, point mutations, ligandindependent activation, and splice variants, have led to the development of novel secondary hormonal therapies for CRPC. These new therapies are more selective and potent than their traditional counterparts. In the following subsections we will discuss the clinical development of several of the new hormonal therapies for CRPC.

#### **4.1 Selective inhibition of CYP17**

30 Drug Development – A Case Study Based Insight into Modern Strategies

Estrogens have long known to have been active in the treatment of prostate cancer; however the exact mechanism of actions remains uncertain. Putative mechanisms include inhibition of LH hormone release from the pituitary gland, inhibition of adrenal androgen production, and a direct cytotoxic effect on prostate cancer cells (Robertson CN, et al. 1996). In a prior phase randomized phase II trial comparing the estrogenic herbal compound PC-SPES with diethylstilbestrol, a greater than 50% decline in baseline PSA was noted in 40% and 24% of patients respectively; median time to progression was 5.5 vs. 2.9 months respectively (Oh W,

There is clearly a modest degree of activity of estrogenic compounds in the treatment of CRPC; however current use of these agents (i.e. diethylstilbestrol, Premarin, etc.) is limited by the small but not insignificant risk of venous thromboembolic events and possibly increased risk of myocardial infarction and stroke; these particular co-morbidities are especially concerning in a disease population of elderly men. Concomitant prophylactic

Ketoconazole is a broad, non-specific inhibitor of multiple cytochrome p450 enzymes involved in androgen biosynthesis, including the conversion of cholesterol to pregnenolone, 11-beta hydroxylation, and 17-alpha hydroxylase/C17, 20 lyase (CYP17) activity. In a previously referred to randomized phase II study of 260 men with CRPC, with progressive disease despite combined androgen blockade, randomized to treatment with antiandrogen withdrawal alone or in combination with ketoconazole, 27% of patients assigned to the ketoconazole arm had a 50% or greater decline in serum PSA level, and 20% of patients had an objective response (Small EJ, et al. 2004). Interestingly, at the time of disease progression on ketoconazole, levels of adrenal androgens including DHEA, DHEA-S, and androstenedione had all increased compared to month 1 levels, which suggest that ketoconazole resistance may in part reflect inadequate androgen production suppression. This mechanism of resistance has implications for the development of novel androgen synthetic enzyme inhibitors such as abiraterone acetate. In an intriguing analysis of adrenal androgen hormone levels from the study by Small et al., patients who had higher baseline levels of androstenedione had a higher likelihood of response to treatment with ketoconazole (Ryan CJ, et al. 2007). This suggests that baseline adrenal androgen levels may be used as predictive biomarker for the use of adrenal androgen blockade as a therapeutic maneuver for CRPC; however this hypothesis requires prospective validation in larger

Ketoconazole is a relatively non-specific inhibitor of multiple enzymes involved in the steroid synthetic pathway, and as such, as blocks normal corticosteroid production and causes iatrogenic adrenal insufficiency. Accordingly, side effects of this medication include lethargy, rash, nausea, and liver toxicity. Supplementation with physiologic replacement doses of hydrocortisone (i.e. 20 mg in the morning, 10 mg in the evening) is required while patients are taking ketoconazole. Furthermore, given the relatively non-specific CYPP450 inhibition, ketoconazole interacts with a wide number of other medications. Its oral absorption and bioavailability can be variable, depending on the acidity of the stomach and

Despite these potential side effects and drug interactions, ketoconazole represents a viable and widely used secondary hormonal agent for CRPC, especially in the patient population

anticoagulation is recommended when using these agents.

fed/fasting state and use of acid suppressing medications.

**3.2 Estrogens** 

et al. 2004).

**3.3 Ketoconazole** 

studies.

As displayed in figure 1, CYP17 (17-alpha hydroxylase/C17, 20 lyase) catalyzes two key steps of androgen synthesis within the steroid biosynthetic pathway: the 17-hydroxylation of progesterone and pregnenolone and subsequent conversion to DHEA and androstenedione respectively. Inhibition of this enzyme would divert cholesterol derivatives away from androgen production, and towards mineralocorticoid production (corticosterone and aldosterone). As outlined above, intra-tumoral upregulation of CYP17 has been previously implicated in the progression to CRPC. Logically then, selective inhibition of CYP17 represents an attractive strategy for inhibiting adrenal and intra-tumor androgen production in CRPCs and thereby slowing disease progression.

#### **4.1.1 Abiraterone acetate**

Abiraterone acetate is the prodrug of abiraterone, a potent, highly selective, irreversible inhibitor of CYP17. In pre-clinical in vivo study using WHT mice, this compound was able to markedly decrease the level of serum testosterone to less than 0.1 nanomolar concentration, despite 3-4 fold increase in serum LH concentration (Barrie SE, et al. 1994). In the first phase 1 study of abiraterone, O'Donnell et al. studied various dosing schedules ranging from 10 to 500 mg x 1 dose in castrate resistant men. At a dose level of 500 mg, there was suppression of serum testosterone to less than lower limit of detection (< 0.14 nmol/L) with parallel reduction androstenedione levels, supporting its mechanism of action of CYP17 inhibition. The duration of testosterone suppression after a single dose was variable, but generally ranged from days 2-5 post-dose (O'Donnell A, et al. 2004). In a follow up phase I trial by Attard and colleagues, 21 patients with CRPC and progression through multiple prior traditional secondary hormonal therapies were treated with abiraterone with doses ranging from 250 mg to 2000 mg/day. Pharmacodynamic effects on serum hormone levels showed a plateau at a dose of 1000 mg/day, which was the dose level of an expanded cohort of 9 patients and the subsequent recommended phase II/III dose. There were no treatment-related grade 3 or 4 adverse events from this trial. As expected, increases in levels of ACTH, corticosterone, and 11-deoxycorticosterone were observed, and there were adverse events related to subsequent mineralocorticoid excess, namely hypokalemia and hypertension. This was effectively managed with the use of eplerenone, a mineralocorticoid antagonist. The median baseline serum testosterone level was 7 ng/mL in this study; at all

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 33

endpoints, including progression-free survival, objective response rate, and PSA response rate favored the abiraterone treatment arm. Hypokalemia was noted in 17% of abiraterone group patients, and 10% of patients experienced hypertension of any grade severity. As a result of the overall survival benefit demonstrated in this phase III trial, abiraterone acetate was granted FDA approval on April 28th, 2011 for use in men with metastatic CRPC who had received prior chemotherapy containing docetaxel. An ongoing phase III trial of prednisone with or without abiraterone in men with metastatic CRPC without prior chemotherapy has finished accrual; study results are expected within the next year

The drug development of abiraterone acetate has unfolded rapidly over the past decade, based on a strong scientific rationale, pre-clinical and early clinical phase data indicating potent blockade of CYP17, a rational phase II/III dose selection, and the selection of clinically relevant endpoints for confirmatory phase III trials. Development of this drug remains ongoing, and many questions remain to be answered, including: (1) mechanisms of abiraterone resistance (2) optimal sequencing in the therapy of men with CRPC (e.g. before or after docetaxel?) (3) potential combination with other secondary hormonal agents (4) activity in patients with prior ketoconazole (patients treated with ketoconazole were excluded from the above mentioned phase III trials) (5) population pharmacokinetic analysis, and (6) development of predictive biomarkers that might allow for individualized patient selection for those most likely to benefit from abiraterone. This last issue is likely to become increasingly more relevant in an era of rising medical costs and the choice of multiple new agents for the treatment of CRPC. Preliminary data suggests that patients with higher levels of baseline adrenal androgen levels are more likely to respond to abiraterone, similar to the results obtained with prior studies of ketoconazole

Orteronel (TAK-700) is a selective CYP17 inhibitor which has reached clinical development in CRPC. Preliminary phase 1 data of 26 men with CRPC treated with dose levels ranging from 100 through 600 mg twice daily as well as 400 mg twice daily + low dose prednisone were recently presented (Dreicer R, et al. 2010). No dose limiting toxicities were seen. Fatigue was the most common adverse event, seen in 62% of patients, including 3 patients with grade 3 fatigue at the 600 mg twice daily dose. Other common adverse events included nausea, vomiting, anorexia, and constipation. Doses at or above 300 mg twice daily produced a 50% or greater decline in PSA in 70% of patients, of whom 29% had an impressive > 90% decline in serum PSA. Phase 3 trials of orteronel in men with metastatic CRPC pre and post docetaxel are ongoing (NCT01193244 and

TOK-100, in a pre-clinical model, selectively inhibits CYP17 enzymatic activity and down regulates AR expression. In the LAPC4 xenograft model, TOK-100 combined with castration inhibited tumor growth and down-regulated AR expression, in contrast to treatment with castration or bicalutamide alone, in which AR expression was up-regulated (Vasaitis T, et al. 2008). Phase I/II trials of TOK-001 are underway in CRPC. The potential for down regulation of AR expression in addition to CYP17 inhibition may lead to more potent suppression of AR-mediated disease progression in CRPC, a hypothesis that warrants

testing in current and future clinical trials of this compound.

(NCT00887198).

(Logothetis CJ, et al. 2008).

**4.1.2 TAK-700 and TOK-001** 

NCT01193257 respectively).

dose levels serum testosterone was decreased to < 1 ng/mL within 8 days of treatment initiation.

In a separate phase I dose escalation study of abiraterone acetate in 33 men, including 19 with prior ketoconazole treatment, daily dosing from 250 mg to 1000 mg was well tolerated with no dose-limiting toxicities (DLTs) (Ryan CJ, et al. 2010). Hypertension and hyperkalemia, signs of mineralocorticoid excess, as might be expected by the mechanism of action, were the most common serious toxicities (grade 3 or higher 12% and 9% respectively), which responded to medical management including low dose corticosteroids or mineralocorticoid receptor antagonists such as eplerenone. Spironolactone was avoided given its potential androgenic properties. Overall, 55% of patients in this study had a confirmed 50% or greater decline in serum PSA level at 12 weeks. In the subset of 19 patients with prior ketoconazole exposure, 46% had a greater than or equal to 50% decline in serum PSA at 12 weeks. Importantly, this data suggests that CRPCs which are resistant to ketoconazole may still be sensitive to the effects of abiraterone, which is a more potent and selective inhibitor of androgen synthesis compared to ketoconazole. In contrast to prior studies of ketoconazole in CRPC, in which adrenal androgens levels rose at the time of disease progression, serum hormone levels including testosterone and DHEA-S did not rise at the time of disease progression on abiraterone. This data suggests the mechanism of resistance to abiraterone may be unrelated to a rise in androgen levels. The phase II portion of this study included added prednisone 5 mg orally twice daily, and excluded patients with prior chemotherapy or ketoconazole (Ryan CJ, et al. 2009). Preliminary results indicated a 50% or greater decrease in PSA in 88% of patients; median time to PSA progression was 337 days.

Subsequent various phase II studies have evaluated abiraterone as monotherapy and combined with low dose prednisone in men with CRPC and prior docetaxel chemotherapy. In a two stage phase II trial by Reid and colleagues of 47 men with CRPC and previous treatment with docetaxel, treated with abiraterone 1000 mg/day monotherapy, 51% of patients demonstrated a 50% or greater decline in serum PSA level. Furthermore, the median time to PSA progression was 169 days; the objective response rate was 28% among men with measurable disease at baseline. 8 patients had prior ketoconazole treatment; all but one had prior treatment with a first generation antiandrogen. Adverse events were as expected due to secondary mineralocorticoid excess, including 55% with hypokalemia, 17% with hypertension, and 15% with fluid retention. In a phase II trial of abiraterone 1000 mg/day + prednisone 5 mg twice daily in 58 men with CRPC and prior docetaxel treatment, a confirmed ≥ 50% decline in PSA was observed in 36% of patients, including 27% of patients with prior ketoconazole treatment (Danila DC, et al. 2010). The median time to PSA progression was 169 days. The addition of prednisone decrease the incidence of clinical mineralocorticoid excess, and no patients required treatment with eplerenone while on study.

Results of the follow up confirmatory randomized phase III trial of abiraterone in the postdocetaxel CRPC population were recently reported (de Bono JS, et al. 2011). In this trial, 1195 patients with CRPC and prior docetaxel were randomized in a 2:1 fashion to receive either the combination of abiraterone 1000 mg/day + prednisone 5 mg twice daily versus placebo + prednisone 5 mg twice daily. After a median follow up of 12.8 months, overall survival was longer in the abiraterone group vs. the placebo group (median overall survival of 14.8 vs. 10.9 months; HR = 0.65, p < 0.0001). The data was unblinded at the time of interim analysis, as the results exceeded the pre-planned stopping rule for efficacy. All secondary endpoints, including progression-free survival, objective response rate, and PSA response rate favored the abiraterone treatment arm. Hypokalemia was noted in 17% of abiraterone group patients, and 10% of patients experienced hypertension of any grade severity. As a result of the overall survival benefit demonstrated in this phase III trial, abiraterone acetate was granted FDA approval on April 28th, 2011 for use in men with metastatic CRPC who had received prior chemotherapy containing docetaxel. An ongoing phase III trial of prednisone with or without abiraterone in men with metastatic CRPC without prior chemotherapy has finished accrual; study results are expected within the next year (NCT00887198).

The drug development of abiraterone acetate has unfolded rapidly over the past decade, based on a strong scientific rationale, pre-clinical and early clinical phase data indicating potent blockade of CYP17, a rational phase II/III dose selection, and the selection of clinically relevant endpoints for confirmatory phase III trials. Development of this drug remains ongoing, and many questions remain to be answered, including: (1) mechanisms of abiraterone resistance (2) optimal sequencing in the therapy of men with CRPC (e.g. before or after docetaxel?) (3) potential combination with other secondary hormonal agents (4) activity in patients with prior ketoconazole (patients treated with ketoconazole were excluded from the above mentioned phase III trials) (5) population pharmacokinetic analysis, and (6) development of predictive biomarkers that might allow for individualized patient selection for those most likely to benefit from abiraterone. This last issue is likely to become increasingly more relevant in an era of rising medical costs and the choice of multiple new agents for the treatment of CRPC. Preliminary data suggests that patients with higher levels of baseline adrenal androgen levels are more likely to respond to abiraterone, similar to the results obtained with prior studies of ketoconazole (Logothetis CJ, et al. 2008).

#### **4.1.2 TAK-700 and TOK-001**

32 Drug Development – A Case Study Based Insight into Modern Strategies

dose levels serum testosterone was decreased to < 1 ng/mL within 8 days of treatment

In a separate phase I dose escalation study of abiraterone acetate in 33 men, including 19 with prior ketoconazole treatment, daily dosing from 250 mg to 1000 mg was well tolerated with no dose-limiting toxicities (DLTs) (Ryan CJ, et al. 2010). Hypertension and hyperkalemia, signs of mineralocorticoid excess, as might be expected by the mechanism of action, were the most common serious toxicities (grade 3 or higher 12% and 9% respectively), which responded to medical management including low dose corticosteroids or mineralocorticoid receptor antagonists such as eplerenone. Spironolactone was avoided given its potential androgenic properties. Overall, 55% of patients in this study had a confirmed 50% or greater decline in serum PSA level at 12 weeks. In the subset of 19 patients with prior ketoconazole exposure, 46% had a greater than or equal to 50% decline in serum PSA at 12 weeks. Importantly, this data suggests that CRPCs which are resistant to ketoconazole may still be sensitive to the effects of abiraterone, which is a more potent and selective inhibitor of androgen synthesis compared to ketoconazole. In contrast to prior studies of ketoconazole in CRPC, in which adrenal androgens levels rose at the time of disease progression, serum hormone levels including testosterone and DHEA-S did not rise at the time of disease progression on abiraterone. This data suggests the mechanism of resistance to abiraterone may be unrelated to a rise in androgen levels. The phase II portion of this study included added prednisone 5 mg orally twice daily, and excluded patients with prior chemotherapy or ketoconazole (Ryan CJ, et al. 2009). Preliminary results indicated a 50% or greater decrease in PSA in 88% of patients; median time to PSA progression was 337

Subsequent various phase II studies have evaluated abiraterone as monotherapy and combined with low dose prednisone in men with CRPC and prior docetaxel chemotherapy. In a two stage phase II trial by Reid and colleagues of 47 men with CRPC and previous treatment with docetaxel, treated with abiraterone 1000 mg/day monotherapy, 51% of patients demonstrated a 50% or greater decline in serum PSA level. Furthermore, the median time to PSA progression was 169 days; the objective response rate was 28% among men with measurable disease at baseline. 8 patients had prior ketoconazole treatment; all but one had prior treatment with a first generation antiandrogen. Adverse events were as expected due to secondary mineralocorticoid excess, including 55% with hypokalemia, 17% with hypertension, and 15% with fluid retention. In a phase II trial of abiraterone 1000 mg/day + prednisone 5 mg twice daily in 58 men with CRPC and prior docetaxel treatment, a confirmed ≥ 50% decline in PSA was observed in 36% of patients, including 27% of patients with prior ketoconazole treatment (Danila DC, et al. 2010). The median time to PSA progression was 169 days. The addition of prednisone decrease the incidence of clinical mineralocorticoid excess, and no patients required treatment with eplerenone while on

Results of the follow up confirmatory randomized phase III trial of abiraterone in the postdocetaxel CRPC population were recently reported (de Bono JS, et al. 2011). In this trial, 1195 patients with CRPC and prior docetaxel were randomized in a 2:1 fashion to receive either the combination of abiraterone 1000 mg/day + prednisone 5 mg twice daily versus placebo + prednisone 5 mg twice daily. After a median follow up of 12.8 months, overall survival was longer in the abiraterone group vs. the placebo group (median overall survival of 14.8 vs. 10.9 months; HR = 0.65, p < 0.0001). The data was unblinded at the time of interim analysis, as the results exceeded the pre-planned stopping rule for efficacy. All secondary

initiation.

days.

study.

Orteronel (TAK-700) is a selective CYP17 inhibitor which has reached clinical development in CRPC. Preliminary phase 1 data of 26 men with CRPC treated with dose levels ranging from 100 through 600 mg twice daily as well as 400 mg twice daily + low dose prednisone were recently presented (Dreicer R, et al. 2010). No dose limiting toxicities were seen. Fatigue was the most common adverse event, seen in 62% of patients, including 3 patients with grade 3 fatigue at the 600 mg twice daily dose. Other common adverse events included nausea, vomiting, anorexia, and constipation. Doses at or above 300 mg twice daily produced a 50% or greater decline in PSA in 70% of patients, of whom 29% had an impressive > 90% decline in serum PSA. Phase 3 trials of orteronel in men with metastatic CRPC pre and post docetaxel are ongoing (NCT01193244 and NCT01193257 respectively).

TOK-100, in a pre-clinical model, selectively inhibits CYP17 enzymatic activity and down regulates AR expression. In the LAPC4 xenograft model, TOK-100 combined with castration inhibited tumor growth and down-regulated AR expression, in contrast to treatment with castration or bicalutamide alone, in which AR expression was up-regulated (Vasaitis T, et al. 2008). Phase I/II trials of TOK-001 are underway in CRPC. The potential for down regulation of AR expression in addition to CYP17 inhibition may lead to more potent suppression of AR-mediated disease progression in CRPC, a hypothesis that warrants testing in current and future clinical trials of this compound.

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 35

AR crystal structure. In pre-clinical study, this AR antagonist showed a > 1 log increase in potency of AR inhibition compared with bicalutamide, both in regards to AR binding and inhibition of AR-mediated gene expression (Attar RM, et al. 2009). Furthermore, in a human xenograft model, BMS-6419888 displayed greater growth inhibition compared with bicalutamide. Based on the encouraging pre-clinical data, this compound was subsequently tested in a phase I dose escalation study (Rathkopf D, et al. 2010). In this trial, doses of BMS-6419888 were escalated from 5 mg to 150 mg. In total, 61 men were treated. One patient experienced an epileptic seizure at a dose of 60 mg twice daily. Antitumor activity was limited to one partial response, and partial agonism was seen as evidenced by a decrease in serum PSA upon drug withdrawal. Based the limited anti-tumor activity despite achieving target therapeutic levels, as well as the epileptic seizure, the study was closed prematurely

ARN-509 is a potent AR antagonist in the early phases of clinical development. It inhibits AR nuclear translocation and DNA binding, thereby modulating expression of genes which drive prostate cancer growth. It is currently being tested in a phase I/II clinical trial of men with metastatic CRPC with up to two prior chemotherapy regimens (NCT01171898). The primary endpoint is maximum tolerated dose; secondary endpoints include change in PSA, number of new bone lesions, and objective response by RECIST criteria. Enrollment began in July of 2010 and results are expected in 2012. Likely due to the several seizure events during prior clinical trials of MDV3100 and BMS-6419888, patients with a history of seizures or potentially lower seizure threshold are excluded from this phase I/II trial of

The clinical activity of the novel secondary hormonal therapies which attack the AR axis continues to lend credence to the now widely held hypothesis that continued activation of the AR plays an important role in the progression of disease to CRPC and ultimately to prostate cancer death. Much progress has been made over the past several decades in the drug development of secondary hormonal therapies for CRPC. However, there are many questions that remain yet to be answered, including: (1) optimal timing and sequence of hormonal therapies in relation to chemotherapy and each other (2) relative risks and benefits of combination versus sequential hormonal monotherapy (3) mechanisms of resistance (4) patterns of disease progression on these novel therapies (5) potential predictive biomarkers to help individualize patient therapy, including the molecular characterization of circulating tumor cells (6) pharmacokinetic studies across various study populations and ethnicities (7) pharmacogenomics analysis of potential associations between germ line mutations and response (8) long term safety data, and (9) optimal phase II/III clinical trial endpoints to assess efficacy of these agents, including the potential use of

Furthermore, there are new treatment strategies which target the AR axis that are in the infancy of drug discovery and development. Among them is EPI-001, a compound which inhibits transactivation of the N-terminal domain of the AR, without interacting with the AR ligand-binding doman, and thus may serve as a potential inhibitor of the AR splice variants that are hypothesized to play a role in the resistance to androgen ablation therapy (Andersen et al, 2010). Additionally, inhibitors of heat shock proteins, which act to stabilize

and further clinical development of this compound discontinued.

surrogate markers such as change in number of circulating tumor cells.

the AR among other proteins, are also in clinical development.

ARN-509.

**5. Future directions** 

## **4.2 Selective and potent inhibition of the Androgen Receptor**

AR gene amplification and over-expression appears to be a relatively common phenomenon as tumors adapt to a low circulating testosterone environment, and may lead to progression to CRPC. First generation AR antagonists such as flutamide or bicalutamide inhibit ligand binding to the AR and thereby decrease nuclear localization and activation of AR-mediated gene expression. However, in the AR-amplified state, the first generation antiandrogens may not block the AR in a potent enough manner to block ligand-mediated AR activation. Furthermore, acquired point mutations in the AR may cause first generation antiandrogens to exhibit partial agonistic activity towards the AR, as supported by the clinical phenomenon of response to antiandrogen withdrawal. Pre-clinically, first generation antiandrogens exhibit partial agonist activity towards the AR in prostate cancer cell lines engineered to expression higher amounts of AR. More potent AR antagonists, which are capable of inhibiting the AR even in cells with AR overexpression, and do not possess any agonistic activity towards the AR, would be highly desirable as a hormonal therapy in CRPC, a potentially AR-amplified disease state.

## **4.2.1 MDV3100**

MDV3100 was developed pre-clinically in an iterative screening process of various compounds that retain AR antagonistic activity in an AR-overexpressed cell line. MDV3100 binds to AR with 5-8 fold greater affinity compared to the first generation antiandrogen bicalutamide (Tran C, et al. 2009), impairs AR nuclear translocation, and inhibits AR binding to DNA, and blocks binding of AR to co-activators to a greater degree than bicalutamide. In tumor xenograft models known to overexpress AR, treatment with MDV3100 led to substantial tumor shrinkage.

MDV3100 was studied in a phase I/II clinical trial of 140 men with CRPC, including 45% of patients with prior ketoconazole and 54% with prior chemotherapy, in doses ranging from 30 mg to 600 mg/day. The maximum tolerated dose for ≥ 28 days was 240 mg/day. At doses of 360 mg/day and higher, 13% of patients discontinued treatment due to an adverse event, including three patients with seizures and one patient with a myocardial infarction. In contrast, at doses of 240 mg/day or lower, 1% of patients (1 out of 87 patients) discontinued treatment due to an adverse event. The most common grade 3 or higher doselimiting toxicity was fatigue, which generally resolved with dose reduction. Anti-tumor activity was noted at all dose levels. In total, 56% of patients showed a 50% or greater reduction in serum PSA level; 22% of patients had an objective radiographic response among those with measurable disease at baseline, and conversion from unfavorable to favorable circulating tumor cell (CTC) count in 49% of patients. Similar PSA response rates were seen in patients with and without prior chemotherapy, though among patients with prior ketoconazole exposure, there was a lower percentage of patients with a 50% or greater decline in serum PSA (37% vs. 71% for those with and without prior ketoconazole respectively). The median time to radiographic progression was 47 weeks. Based on the encouraging results of this phase I/II clinical trial, ongoing phase III trials of MDV3100 vs. placebo, at a dose of 160 mg/day, are ongoing in patients with metastatic CRPC with and without prior docetaxel treatment (NCT00974311 and NCT01212991 respectively).

#### **4.2.2 Other Androgen Receptor antagonists**

Several other second generation, highly potent, pure AR antagonists have reached clinical development in CRPC. BMS-641988 is a highly potent AR inhibitor was designed based on AR crystal structure. In pre-clinical study, this AR antagonist showed a > 1 log increase in potency of AR inhibition compared with bicalutamide, both in regards to AR binding and inhibition of AR-mediated gene expression (Attar RM, et al. 2009). Furthermore, in a human xenograft model, BMS-6419888 displayed greater growth inhibition compared with bicalutamide. Based on the encouraging pre-clinical data, this compound was subsequently tested in a phase I dose escalation study (Rathkopf D, et al. 2010). In this trial, doses of BMS-6419888 were escalated from 5 mg to 150 mg. In total, 61 men were treated. One patient experienced an epileptic seizure at a dose of 60 mg twice daily. Antitumor activity was limited to one partial response, and partial agonism was seen as evidenced by a decrease in serum PSA upon drug withdrawal. Based the limited anti-tumor activity despite achieving target therapeutic levels, as well as the epileptic seizure, the study was closed prematurely and further clinical development of this compound discontinued.

ARN-509 is a potent AR antagonist in the early phases of clinical development. It inhibits AR nuclear translocation and DNA binding, thereby modulating expression of genes which drive prostate cancer growth. It is currently being tested in a phase I/II clinical trial of men with metastatic CRPC with up to two prior chemotherapy regimens (NCT01171898). The primary endpoint is maximum tolerated dose; secondary endpoints include change in PSA, number of new bone lesions, and objective response by RECIST criteria. Enrollment began in July of 2010 and results are expected in 2012. Likely due to the several seizure events during prior clinical trials of MDV3100 and BMS-6419888, patients with a history of seizures or potentially lower seizure threshold are excluded from this phase I/II trial of ARN-509.

#### **5. Future directions**

34 Drug Development – A Case Study Based Insight into Modern Strategies

AR gene amplification and over-expression appears to be a relatively common phenomenon as tumors adapt to a low circulating testosterone environment, and may lead to progression to CRPC. First generation AR antagonists such as flutamide or bicalutamide inhibit ligand binding to the AR and thereby decrease nuclear localization and activation of AR-mediated gene expression. However, in the AR-amplified state, the first generation antiandrogens may not block the AR in a potent enough manner to block ligand-mediated AR activation. Furthermore, acquired point mutations in the AR may cause first generation antiandrogens to exhibit partial agonistic activity towards the AR, as supported by the clinical phenomenon of response to antiandrogen withdrawal. Pre-clinically, first generation antiandrogens exhibit partial agonist activity towards the AR in prostate cancer cell lines engineered to expression higher amounts of AR. More potent AR antagonists, which are capable of inhibiting the AR even in cells with AR overexpression, and do not possess any agonistic activity towards the AR, would be highly desirable as a hormonal therapy in

MDV3100 was developed pre-clinically in an iterative screening process of various compounds that retain AR antagonistic activity in an AR-overexpressed cell line. MDV3100 binds to AR with 5-8 fold greater affinity compared to the first generation antiandrogen bicalutamide (Tran C, et al. 2009), impairs AR nuclear translocation, and inhibits AR binding to DNA, and blocks binding of AR to co-activators to a greater degree than bicalutamide. In tumor xenograft models known to overexpress AR, treatment with MDV3100 led to

MDV3100 was studied in a phase I/II clinical trial of 140 men with CRPC, including 45% of patients with prior ketoconazole and 54% with prior chemotherapy, in doses ranging from 30 mg to 600 mg/day. The maximum tolerated dose for ≥ 28 days was 240 mg/day. At doses of 360 mg/day and higher, 13% of patients discontinued treatment due to an adverse event, including three patients with seizures and one patient with a myocardial infarction. In contrast, at doses of 240 mg/day or lower, 1% of patients (1 out of 87 patients) discontinued treatment due to an adverse event. The most common grade 3 or higher doselimiting toxicity was fatigue, which generally resolved with dose reduction. Anti-tumor activity was noted at all dose levels. In total, 56% of patients showed a 50% or greater reduction in serum PSA level; 22% of patients had an objective radiographic response among those with measurable disease at baseline, and conversion from unfavorable to favorable circulating tumor cell (CTC) count in 49% of patients. Similar PSA response rates were seen in patients with and without prior chemotherapy, though among patients with prior ketoconazole exposure, there was a lower percentage of patients with a 50% or greater decline in serum PSA (37% vs. 71% for those with and without prior ketoconazole respectively). The median time to radiographic progression was 47 weeks. Based on the encouraging results of this phase I/II clinical trial, ongoing phase III trials of MDV3100 vs. placebo, at a dose of 160 mg/day, are ongoing in patients with metastatic CRPC with and

without prior docetaxel treatment (NCT00974311 and NCT01212991 respectively).

Several other second generation, highly potent, pure AR antagonists have reached clinical development in CRPC. BMS-641988 is a highly potent AR inhibitor was designed based on

**4.2 Selective and potent inhibition of the Androgen Receptor** 

CRPC, a potentially AR-amplified disease state.

**4.2.2 Other Androgen Receptor antagonists** 

**4.2.1 MDV3100** 

substantial tumor shrinkage.

The clinical activity of the novel secondary hormonal therapies which attack the AR axis continues to lend credence to the now widely held hypothesis that continued activation of the AR plays an important role in the progression of disease to CRPC and ultimately to prostate cancer death. Much progress has been made over the past several decades in the drug development of secondary hormonal therapies for CRPC. However, there are many questions that remain yet to be answered, including: (1) optimal timing and sequence of hormonal therapies in relation to chemotherapy and each other (2) relative risks and benefits of combination versus sequential hormonal monotherapy (3) mechanisms of resistance (4) patterns of disease progression on these novel therapies (5) potential predictive biomarkers to help individualize patient therapy, including the molecular characterization of circulating tumor cells (6) pharmacokinetic studies across various study populations and ethnicities (7) pharmacogenomics analysis of potential associations between germ line mutations and response (8) long term safety data, and (9) optimal phase II/III clinical trial endpoints to assess efficacy of these agents, including the potential use of surrogate markers such as change in number of circulating tumor cells.

Furthermore, there are new treatment strategies which target the AR axis that are in the infancy of drug discovery and development. Among them is EPI-001, a compound which inhibits transactivation of the N-terminal domain of the AR, without interacting with the AR ligand-binding doman, and thus may serve as a potential inhibitor of the AR splice variants that are hypothesized to play a role in the resistance to androgen ablation therapy (Andersen et al, 2010). Additionally, inhibitors of heat shock proteins, which act to stabilize the AR among other proteins, are also in clinical development.

Development of Novel Secondary Hormonal Therapies for Castrate-Resistant Prostate Cancer 37

Fossa, SD; Slee, PH; Brausi, M; et al. (2001). Flutamide versus prednisone in patients with

Gregory, CW; Johnson, RT; Mohler, JL; et al. (2006). Androgen receptor stabilization in

Guo, Z & Qiu, Y. A New Trick of an Old Molecule: Androgen Receptor Splice Variants

Hu, R; Dunn, TA; Wei, S; et al. (2009). Ligand-independent androgen receptor variants

Huggins, C; Stevens Jr, RE; & Hodges, CV. (1941). Studies on prostatic cancer: The effect of

Joyce, R; Fenton, MA; Rode, P; et al. High dose bicalutamide for androgen independent

Koivisto, P; Kononen, J; Palmberg, C; et al. (1997). Androgen receptor gene amplification: a

Linja, MJ; Savinainen, KJ; Saramaki, OR; et al. (2001). Amplification and overexpression of

Logothetis, CJ; Wen, S; Molina, A; et al. Identification of an androgen withdrawal responsive

acetate. *Journal of Clinical Oncology*, Vol. 26, supplement, abstract 5017. Montgomery, RB; Mostaghel, EA; Vessella R, et al. (2008). Maintenance of intratumoral

tumor growth. *Cancer Research*, Vol. 68, No. 11 (June 2008), pp. 4447-4454. Nishiyama, T; Hashimoto, Y & Takahashi, K. (2004). The influence of androgen deprivation

prostate cancer. *British Journal of Cancer*, No. 90 (May 2004), pp. 2317-2325. Oh, WK; Kantoff, PW; Weinberg, V; et al. (2004). Prospective, multicenter, randomized

Petyrlak, DP; Tangen, CM; Hussain, MH; et al. (2004). Docetaxel and estramustine

prostate cancer. *Clinical Cancer Research*, Vol. 10 (2004), pp. 7121-7126. O'Donnell, A; Judson, I; Dowsett, M; et al. (2004). Hormonal impact of the 17 alpha-

*Cancer Research*, Vol. 61 (April 2001), pp. 2892-2898.

*Cancer Research*, Vol. 69, No. 1 (January 2009), pp. 16-22.

cancer. *Cancer Research*, Vol. 57 (January 1997), pp. 314-319.

2001), pp. 62-71.

2011), pp. 815-822.

(1941), pp. 209-223.

(January 1998), pp. 149-153

Vol. 61 (May 2001), pp. 3550-3555.

No. 18 (September 2004), pp. 3705-3712.

prostate cancer symptomatically progressing after androgen-ablative therapy: a phase III study of the European Organization for Research and Treatment of Cancer Genitourinary Group. *Journal of Clinical Oncology*, Vol. 19, No. 1 (January

recurrent prostate cancer is associated with hypersensitivity to low androgen.

Taking the Stage?!. *International Journal of Biological Sciences*, Vol. 7, No. 6 (July

derived from splicing of cryptic exons signify hormone-refractory prostate cancer.

castration on advanced carcinoma of the prostate gland. *Archives of Surgery,*Vol. 43

prostate cancer: effect of prior hormonal therapy. *Journal of Urology*, Vol. 159, No. 1

possible molecular mechanism for androgen deprivation therapy failure in prostate

androgen receptor gene in hormone-refractory prostate cancer. *Cancer Research*,

phenotype in castrate resistant prostate cancer patients treated with abiraterone

androgens in metastatic prostate cancer: a mechanism for castration-resistant

therapy in dihydrotestosterone levels in the prostate tissue of patients with

hydroxylase/C(17,20) lyase inhibitor abiraterone acetate (CB7630) in patients with

phase II trial of the herbal supplement, PC-SPES, and diethylstilbestrol in patients with androgen-independent prostate cancer. *Journal of Clinical Oncology*, Vol. 22,

compared with mitoxantrone and prednisone for advanced refractory prostate cancer. *New England Journal of Medicine*, Vol. 351 (October 2004), pp. 1513-1520.

## **6. Conclusions**

AR activation continues to play a role in the progression of CRPC, despite low circulating serum testosterone levels in this disease state. This is accomplished through endocrine ligand production via adrenal androgen synthesis, intracrine ligand formation via upregulation of the enzymes involved in androgen synthesis, including CYP17, AR overexpression and point mutations which confer receptor promiscuity and promote agonistic activity of traditional antiandrogen therapy, ligand-independent AR activation, and generation of constitutively active AR splice variants, among others. Pre-clinical drug discovery and development targeting specific steps in these mechanisms has led to the clinical development of numerous secondary hormonal agents which specifically and potently target the AR axis. Ongoing research is directed at optimizing and personalizing the use of the current novel secondary hormonal therapies as well as developing new therapeutic strategies to overcome treatment resistance in CRPC.

## **7. References**


AR activation continues to play a role in the progression of CRPC, despite low circulating serum testosterone levels in this disease state. This is accomplished through endocrine ligand production via adrenal androgen synthesis, intracrine ligand formation via upregulation of the enzymes involved in androgen synthesis, including CYP17, AR overexpression and point mutations which confer receptor promiscuity and promote agonistic activity of traditional antiandrogen therapy, ligand-independent AR activation, and generation of constitutively active AR splice variants, among others. Pre-clinical drug discovery and development targeting specific steps in these mechanisms has led to the clinical development of numerous secondary hormonal agents which specifically and potently target the AR axis. Ongoing research is directed at optimizing and personalizing the use of the current novel secondary hormonal therapies as well as developing new

Aggarwal, R & Ryan, CJ. (2011). Castration-resistant prostate cancer: targeted therapies and individualized treatment. *The Oncologist*, Vol. 16, No. 3 (March 2011), pp. 264-275. Andersen, RJ; Mawji, NR; Wang, J; et al. Regression of castrate-recurrent prostate cancer by

Attar, RM; Jure-Kunkel, M; Balog, A; et al. Discovery of BMS-641988: a novel and potent

Attard, G; Reid, AHM; Yap, TA; et al. Phase I clinical trial of a selective inhibitor of CYP17,

Barrie, SE; Potter, GA; Goddard, PM; et al. (1994). Pharmacology of novel steroidal

Danila, DC; Morris, MJ; de Bono, J; et al. Phase II multicenter study of abiraterone acetate

Dreicer, R; Agus, DB; MacVicar, GR; et al. Safety, pharmacokinetics, and efficacy of TAK-700

Feldman, BJ & Feldman D. (2001). The development of androgen independent prostate

a small-molecule inhibitor of the amino-terminus domain of the androgen receptor.

inhibitor of androgen receptor signaling for the treatment of prostate cancer. *Cancer* 

abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. *Journal of Clinical Oncology*, Vol. 28, No. 28 (October 2008),

inhibitors of cytochrome P450 (17) alpha (17 alpha-hydroxylase/C17-20 lyase). *Journal of Steroid Biochemical Molecular Biology*, Vol. 50, No. 5/6 (2004), pp. 267-273. Chen, S; Xu, Y; Yuan, X; et al. Androgen receptor phosphorylation and stabilization in

prostate cancer by cyclin-dependent kinase 1. *Proceedings of the National Academy of* 

plus prednisone in patients with docetaxel-treated castration-resistant prostate cancer. *Journal of Clinical Oncology*, Vol. 28, No. 9 (March 2010), pp. 1496-1501. de Bono, JS; Logothetis, CJ; Molina, A; et al. Abiraterone and increased survival in

metastatic prostate cancer. *New England Journal of Medicine*, Vol. 364, No. 21 (May

in castration-resistant metastatic prostate cancer: a phase I/II open label study.

therapeutic strategies to overcome treatment resistance in CRPC.

*Cancer Cell*, Vol. 17 (June 2010), pp. 535-546.

*Research*, Vol. 69, No. 16 (August 2009), pp. 6522-6530.

*Sciences*, Vol. 103, No. 43 (October 2006), pp. 15969-15974.

ASCO Genitourinary Cancer Symposium 2010, abstract 103.

cancer. *Nature Reviews: Cancer*, Vol. 1 (October 2001), pp. 34-45.

**6. Conclusions** 

**7. References** 

pp. 4563-4571.

2011), pp. 1995-2005.


**3** 

*Poland* 

**Inhibitors of Proteinases** 

Karolina Gluza and Paweł Kafarski

*Wrocław University of Technology, Wrocław* 

**as Potential Anti-Cancer Agents** 

*Department of Bioorganic Chemistry, Faculty of Chemistry* 

Cancer is a collection of over 100 devastating diseases that share a number of characteristics, a primary hallmark of which is out-of-control growth. However, in reality there are significant differences among these diseases, a fact that underlies the difficulties in the past few decades in their chemotherapeutic intervention. It is becoming evident that there are multiple routes to development of cancer, in part because so many distinct metabolic and

There is a positive correlation between the aggressiveness of a tumor and the secretion of various proteinases. Using bioinformatic analysis approximately 600 proteinases have been determined in human and mouse genomes (2-4% of the genome), many of which are orthologous (Puente et al., 2003). Only some of them are involved in tumor progression and growth, both at the primary and metastatic sites. As tumor progresses towards increased malignancy, it passes through several important stages that require the action of proteinases. First, the induction of angiogenesis requires degradation of the vascular basement membrane and the release of matrix-bound proangiogenic growth factors. Second, invasion of cancer cells into the surrounding tissue involves the dissolution of cell-cell junctions, degradation of the epithelial basement membrane and remodeling of extracellular matrix to allow cancer cells to be released from the primary tumor mass. Third, at least two key steps in metastasis require proteolysis: intravasation of cancer cells into the blood or lymphatic circulation at the primary site and then extravasation at the secondary site, where proteinases can play a part in promoting the colonization and growth of cancer. Proteinases may co-operatively mediate these steps with individual ones having distinct roles. Therefore, inhibition of their activity might be one of the means to combat the development of cancer. Despite of the described facts, recent findings have revealed that the functions of proteinases in tumors are significantly more complex and varied. For example, they are now seen as extremely important signaling molecules that are involved in numerous vital processes. Proteinase signaling pathways are strictly regulated, and the deregulation of their activities can lead to various pathologies, including cancer. Thus, construction of the inhibitor, which should have an impact on tumor progression and metastasis, cannot be done without placing certain proteinase in the proper metabolic context. Inhibitor therapy design is further complicated because different types of cancers utilize diverse proteinases at

biochemical steps can be altered to give rise to uncontrolled cell growth.

**1. Introduction** 

varying stages of cancer development.


## **Inhibitors of Proteinases as Potential Anti-Cancer Agents**

Karolina Gluza and Paweł Kafarski

*Department of Bioorganic Chemistry, Faculty of Chemistry Wrocław University of Technology, Wrocław Poland* 

#### **1. Introduction**

38 Drug Development – A Case Study Based Insight into Modern Strategies

Puche, C; Jose, M; Cabero, A; et al. (2002). Expression and enzymatic activity of the P450c17

Reid, AH; Attard, D; Danila, D; et al. Significant and sustained antitumor activity in post-

Ryan, CJ; Halabi, S; Ou, S; et al. (2007). Adrenal androgen levels as predictors of outcome in

Ryan, CJ; Smith, MR; Fong, L; et al. Phase I clinical trial of the CYP17 inhibitor abiraterone

Ryan, CJ; Efstathiou, E; Smith, M; et al. Phase II multicenter study of chemotherapy

Small, EJ; Halabi, S; Dawson, NA; et al. (2004). Antiandrogen withdrawal alone or in

Suzuki, H; Okihara, K; Miyake, H; et al. (2008). Alternative nonsteroidal antiandrogen

blockade. *Journal of Urology*, Vol. 180, No. 3 (September 2008), pp. 921-927. Tan, J; Sharief, Y; Hamil, KG; et al. (1997) Dehydroepiandrosterone activates mutant

Tannock, IF; de Wit, R; Berry, WR; et al. (2004) Docetaxel plus prednisone or mitoxantrone

Taplin, M; Rajeshkumar, B; Halabi, S; et al. Androgen receptor mutations in androgen-

Tran, C; Ouk, S; Clegg, NJ; et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. *Science*, Vol. 324 (May 2009), pp. 787-790. Vasaitis, T; Belosay, A; Schayowitz, A; et al. Androgen receptor inactivation contributes to

*Journal of Clinical Oncology*, Vol. 22, No. 6 (March 2004), pp. 1025-1033. Stanbrough, M; Bubley, GJ; Ross K, et al. (2006). Increased expression of genes converting

*Cancer Institute*, Vol. 88, No. 13 (April 1996), pp. 908-917.

*Oncology*, Vol. 27, No. 15s (June 2009), abstract 5046.

*Research*, Vol. 66, No. 5 (March 2006), pp. 2815-2825.

*Clinical Oncology*, Vol. 21 (July 2003), pp. 2673-2678.

*Therapeutics*, Vol. 7, No. 8 (August 2008), pp. 2348-2357.

13, No. 7 (April 2007), pp. 2030-2037.

(March 2010), pp. 1481-1488.

1997), pp. 450-459.

351 (October 2004), pp. 1502-1512.

2002), pp. 223-229.

gene in human adipose tissue. *European Journal of Endocrinology*, Vol. 146 (February

docetaxel, castration-resistant prostate cancer with the CYP17 inhibitor abiraterone acetate. *Journal of Clinical Oncology*, Vol. 28, No. 9 (March 2010), pp. 1489-1495. Robertson, CN; Roberson, KM; Padilla, GM; et al. (1996). Induction of apoptosis by

diethylstilbestrol in hormone-insensitive prostate cancer cells. *Journal of the National* 

prostate cancer patients treated with ketoconazole plus antiandrogen withdrawal: results from a Cancer and Leukemia Group B Study. *Clinical Cancer Research*, Vol.

acetate demonstrating clinical activity in patients with castration-resistant prostate cancer who received prior ketoconazole. *Journal of Clinical Oncology*, Vol. 28, No. 9

(chemo)-naïve castration resistant prostate cancer (CRPC) not exposed to ketoconazole, treated with abiraterone acetate plus prednisone. *Journal of Clinical* 

combination with ketoconazole in androgen-independent prostate cancer patients.

adrenal androgens to testosterone in androgen-independent prostate cancer. *Cancer* 

therapy for advanced prostate cancer that relapsed after initial maximum androgen

androgen receptors expressed in the androgen- dependent human prostate cancer xenograft CWR22 and LNCaP cells. *Molecular Endocrinology*, Vol. 11, No. 4 (April

plus prednisone for advanced prostate cancer. *New England Journal of Medicine*, Vol.

independent prostate cancer: Cancer and Leukemia Group B Study 9663. *Journal of* 

antitumor efficacy of 17-alpha hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17- (1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer. *Molecular Cancer*  Cancer is a collection of over 100 devastating diseases that share a number of characteristics, a primary hallmark of which is out-of-control growth. However, in reality there are significant differences among these diseases, a fact that underlies the difficulties in the past few decades in their chemotherapeutic intervention. It is becoming evident that there are multiple routes to development of cancer, in part because so many distinct metabolic and biochemical steps can be altered to give rise to uncontrolled cell growth.

There is a positive correlation between the aggressiveness of a tumor and the secretion of various proteinases. Using bioinformatic analysis approximately 600 proteinases have been determined in human and mouse genomes (2-4% of the genome), many of which are orthologous (Puente et al., 2003). Only some of them are involved in tumor progression and growth, both at the primary and metastatic sites. As tumor progresses towards increased malignancy, it passes through several important stages that require the action of proteinases. First, the induction of angiogenesis requires degradation of the vascular basement membrane and the release of matrix-bound proangiogenic growth factors. Second, invasion of cancer cells into the surrounding tissue involves the dissolution of cell-cell junctions, degradation of the epithelial basement membrane and remodeling of extracellular matrix to allow cancer cells to be released from the primary tumor mass. Third, at least two key steps in metastasis require proteolysis: intravasation of cancer cells into the blood or lymphatic circulation at the primary site and then extravasation at the secondary site, where proteinases can play a part in promoting the colonization and growth of cancer. Proteinases may co-operatively mediate these steps with individual ones having distinct roles. Therefore, inhibition of their activity might be one of the means to combat the development of cancer. Despite of the described facts, recent findings have revealed that the functions of proteinases in tumors are significantly more complex and varied. For example, they are now seen as extremely important signaling molecules that are involved in numerous vital processes. Proteinase signaling pathways are strictly regulated, and the deregulation of their activities can lead to various pathologies, including cancer. Thus, construction of the inhibitor, which should have an impact on tumor progression and metastasis, cannot be done without placing certain proteinase in the proper metabolic context. Inhibitor therapy design is further complicated because different types of cancers utilize diverse proteinases at varying stages of cancer development.

Inhibitors of Proteinases as Potential Anti-Cancer Agents 41

localization in the tumor microenvironment. Some of its best-known substrates are proteins of extracellular matrix, as well as several important proteinases and their inhibitors (Skrzydlewska et al., 2005; Mason & Joyce, 2011). This complicated pattern of activity emphasizes the central role of cathepsin B in tumor progression simultaneously showing

Cystatins are a superfamily of endogenous inhibitors of proteinases of papain family. So far, 25 representatives of these proteins have been determined. Their main function is to ensure protection of cells and tissue against the proteolytic activity of lysosomal peptidases that are released during normal cell death, or intentionally by proliferating cancer cells or by invading organisms, such as parasites. They exhibit low specificity towards their target proteases, meaning that one cystatin can inhibit several cathepsins. This is because they have apparently similar three-dimensional structure. In some types of cancers, the changes in cysteine cathepsin epression or activity have diagnostic or prognostic value with imbalance between cathepsins and cystatins being associated with tumor phenotype. Since the latter ones are able to inhibit cathepsins tumor-associated activity many studies have indicated their potential use in therapeutic approaches (Keppler, 2006; Kopitz et al, 2005; Palermo & Joyce, 2007). Indeed, one of these inhibitors, cystatin C (mostly the one isolated from egg white) has been used in preclinical research studies for more than 20 years, however, it has been introduced into clinical practice quite scarcely. Despite some isolated promising results (Saleh at al., 2006) this approach is also highly criticized (Keppler, 2006; Mussap & Plebani, 2004) with the greatest problems being high cost of the inhibitor (140 \$ USA per milligram), its low bioavailability and short circulation time, and general

Despite the fact that cystatins of different families posses different biochemical properties their inhibitory properties are rather common. They are tight and reversible inhibitors of cathepsins and interact with the active sites of these proteinases via their inhibitory reactive site, made up of the juxtaposition of three regions of the molecule, which form a wedgeshaped edge that is highly complementary to the active site of papain family of proteinases

that design of its inhibitors as anticancer agents is a difficult task.

**2.1 Cystatins** 

skepticism amongst clinicians.

Fig. 1. Stefin A (violet) complexed with cathepsinB.

(Fig. 1).

Through the evolution, proteinases have adapted to the wide range of conditions found in human organism (variations in pH, reductive environment and so on) and they use several catalytic mechanisms for substrate hydrolysis. Basing on the chemical mechanism of their action human proteinases may be classified as: cysteine, serine, threonine, aspartic acid and metallo proteinases. In most cases specific inhibitors for each class of these enzymes are being designed.

A number of reviews on various aspects of the use of proteinase inhibitors as a mean to combat cancer have been published recently (Castro-Guillen et el., 2010; Lee et al., 2004; Magdolen et al., 2002; Pandey et al., 2007; Puxbaum & Mach, 2009; Turk, 2006). Therefore, in this review the current trends in designing of such inhibitors will be presented. Special emphasis will be put on rational design using the techniques which are based either on the knowledge of detailed mechanism of enzymatic catalysis or on three-dimensional structure of active sites of chosen enzymes. Indeed, several small-molecule drugs targeting proteinases obtained in that manner are already on the market and many more are in development.

## **2. Cysteine proteinases**

Despite mounting evidence in the last 30 years showing that expression, localization and activation of lysosomal cysteine proteinases are aberrant in tumor cells, when compared to normal cells, this class of proteases has received little attention. Studies on increased expression, elevated activity and mislocalization of certain enzymes have indicated that members of the cysteine proteinases have been implicated in cancer progression. In mammalian cells, cysteine proteinases are localized mainly in the cytoplasm (calpain and caspase families) and lysosomal compartments (cathepsin and legumain families). Cathepsins are the most directly involved in tumor progression. There are 11 human cathepsins: B, C, F, H, K, L, O, S, W, V and X. These enzymes alongside with aspartic proteinases - cathepsins D and E are mainly involved in intracellular proteolysis within lysosomes. Their increased expression correlates with more aggressive tumors and poorer prognoses for patients (Berdowska, 2004). Cathepsins B and L expression is increased in many human cancers and these enzymes have been investigated most intensively (Bell-McGuinn, et al., 2007; Koblinski et al, 2000). In addition, the predominant expression of cathepsin K in osteoclasts has rendered this enzyme as a major target for the development of novel drugs against bone tumors (Lindeman et al., 2004).

The common belief is that cathepsin-mediated degradation of he extracellular matrix is primarily extracellular at the invasive front of tumor cells. This proteolytic process is associated both with early tumor development, affecting tumor cell proliferation and angiogenesis, and with dissemination of malignant cells from primary tumors (Turk et al 2004). Therefore inhibitors of cathepsins are most intensively studied.

Recent evidence reveals that tumor-promoting proteinases act as part of an extensive multidirectional network of proteolytic interactions. These networks involve various constituents of the tumor microenvironment, with cathepsin B being one of the best examples. An aspartic enzyme - cathepsin D converts pro-cathepsin B into cathepsin B. Cathepsin B can be also activated by a series of other proteinases with cathepsins C and G, urokinase-type plasminogen activator and tissue-type plasminogen activator being the most active ones. Finally, cathepsin B may undergo auto-activation under certain conditions. Activated cathepsin B cleaves a wide variety of targets depending on its subcellular localization in the tumor microenvironment. Some of its best-known substrates are proteins of extracellular matrix, as well as several important proteinases and their inhibitors (Skrzydlewska et al., 2005; Mason & Joyce, 2011). This complicated pattern of activity emphasizes the central role of cathepsin B in tumor progression simultaneously showing that design of its inhibitors as anticancer agents is a difficult task.

## **2.1 Cystatins**

40 Drug Development – A Case Study Based Insight into Modern Strategies

Through the evolution, proteinases have adapted to the wide range of conditions found in human organism (variations in pH, reductive environment and so on) and they use several catalytic mechanisms for substrate hydrolysis. Basing on the chemical mechanism of their action human proteinases may be classified as: cysteine, serine, threonine, aspartic acid and metallo proteinases. In most cases specific inhibitors for each class of these enzymes are

A number of reviews on various aspects of the use of proteinase inhibitors as a mean to combat cancer have been published recently (Castro-Guillen et el., 2010; Lee et al., 2004; Magdolen et al., 2002; Pandey et al., 2007; Puxbaum & Mach, 2009; Turk, 2006). Therefore, in this review the current trends in designing of such inhibitors will be presented. Special emphasis will be put on rational design using the techniques which are based either on the knowledge of detailed mechanism of enzymatic catalysis or on three-dimensional structure of active sites of chosen enzymes. Indeed, several small-molecule drugs targeting proteinases obtained in that manner are already on the market and many more are in

Despite mounting evidence in the last 30 years showing that expression, localization and activation of lysosomal cysteine proteinases are aberrant in tumor cells, when compared to normal cells, this class of proteases has received little attention. Studies on increased expression, elevated activity and mislocalization of certain enzymes have indicated that members of the cysteine proteinases have been implicated in cancer progression. In mammalian cells, cysteine proteinases are localized mainly in the cytoplasm (calpain and caspase families) and lysosomal compartments (cathepsin and legumain families). Cathepsins are the most directly involved in tumor progression. There are 11 human cathepsins: B, C, F, H, K, L, O, S, W, V and X. These enzymes alongside with aspartic proteinases - cathepsins D and E are mainly involved in intracellular proteolysis within lysosomes. Their increased expression correlates with more aggressive tumors and poorer prognoses for patients (Berdowska, 2004). Cathepsins B and L expression is increased in many human cancers and these enzymes have been investigated most intensively (Bell-McGuinn, et al., 2007; Koblinski et al, 2000). In addition, the predominant expression of cathepsin K in osteoclasts has rendered this enzyme as a major target for the development of

The common belief is that cathepsin-mediated degradation of he extracellular matrix is primarily extracellular at the invasive front of tumor cells. This proteolytic process is associated both with early tumor development, affecting tumor cell proliferation and angiogenesis, and with dissemination of malignant cells from primary tumors (Turk et al

Recent evidence reveals that tumor-promoting proteinases act as part of an extensive multidirectional network of proteolytic interactions. These networks involve various constituents of the tumor microenvironment, with cathepsin B being one of the best examples. An aspartic enzyme - cathepsin D converts pro-cathepsin B into cathepsin B. Cathepsin B can be also activated by a series of other proteinases with cathepsins C and G, urokinase-type plasminogen activator and tissue-type plasminogen activator being the most active ones. Finally, cathepsin B may undergo auto-activation under certain conditions. Activated cathepsin B cleaves a wide variety of targets depending on its subcellular

being designed.

development.

**2. Cysteine proteinases** 

novel drugs against bone tumors (Lindeman et al., 2004).

2004). Therefore inhibitors of cathepsins are most intensively studied.

Cystatins are a superfamily of endogenous inhibitors of proteinases of papain family. So far, 25 representatives of these proteins have been determined. Their main function is to ensure protection of cells and tissue against the proteolytic activity of lysosomal peptidases that are released during normal cell death, or intentionally by proliferating cancer cells or by invading organisms, such as parasites. They exhibit low specificity towards their target proteases, meaning that one cystatin can inhibit several cathepsins. This is because they have apparently similar three-dimensional structure. In some types of cancers, the changes in cysteine cathepsin epression or activity have diagnostic or prognostic value with imbalance between cathepsins and cystatins being associated with tumor phenotype. Since the latter ones are able to inhibit cathepsins tumor-associated activity many studies have indicated their potential use in therapeutic approaches (Keppler, 2006; Kopitz et al, 2005; Palermo & Joyce, 2007). Indeed, one of these inhibitors, cystatin C (mostly the one isolated from egg white) has been used in preclinical research studies for more than 20 years, however, it has been introduced into clinical practice quite scarcely. Despite some isolated promising results (Saleh at al., 2006) this approach is also highly criticized (Keppler, 2006; Mussap & Plebani, 2004) with the greatest problems being high cost of the inhibitor (140 \$ USA per milligram), its low bioavailability and short circulation time, and general skepticism amongst clinicians.

Despite the fact that cystatins of different families posses different biochemical properties their inhibitory properties are rather common. They are tight and reversible inhibitors of cathepsins and interact with the active sites of these proteinases via their inhibitory reactive site, made up of the juxtaposition of three regions of the molecule, which form a wedgeshaped edge that is highly complementary to the active site of papain family of proteinases (Fig. 1).

Fig. 1. Stefin A (violet) complexed with cathepsinB.

Inhibitors of Proteinases as Potential Anti-Cancer Agents 43

peptide metabolites from *Streptomyces* NCIM 2081 (Fig. 4) exhibited potent inhibitory action against papain and significantly inhibited tumor cell migration at subcytotoxic concentrations, indicating its remarkable potential to be developed as antimetastatic drug

> O HOOC <sup>N</sup>

H

O

H

E-64

<sup>N</sup> <sup>N</sup>

H

NH2 NH

O

N H

O

N H

O

COOH R = H lub H2C OH

O

<sup>N</sup> <sup>N</sup>

O COOH

Me2N

O

H

cathepsin B inhibitor

H <sup>N</sup> <sup>N</sup> H

Fig. 3. Specific inhibitors of cathepsins B and L built up on the frame of E-64.

O

O

N H N

O

cathepsin L inhibitor

O

(Singh et al, 2010).

O

O O

> N H

**2.3 Irreversible inhibitors** 

O

H N O

N H <sup>S</sup> <sup>O</sup>

O

N H

cathepsin B inhibitor

H3COOC

O

N

COOH

O

H N

Fig. 4. Anticancer peptides produced by *Streptomyces* NCIM 2081.

NH

R O

N H

The majority of synthetic cysteine proteinase inhibitors contain a peptide segment for recognition by the chosen enzyme and an electrophilic functionality that is able to react with the thiolate moiety of active site cysteine. In most cases this results in covalent modification of the enzyme and irreversible inhibition. A wide variety of such reactive groups have been employed, including: azomethyl- or halomethyl ketone, acyloxymethyl ketone,

O

Thus, mimicking the segment of cystatin interacting with the cathepsin active site (Fig. 1) seems to be the method of choice. This approach is well represented by highly active inhibitor (N-1845, Fig. 2) of cathepsin B (Ki value of 0.088nM) containing an azaglycine residue in place of evolutionary conserved glycine residue in the N terminal part of cystatin (Wieczerzak et al., 2002). Further modification of this molecule, enforced by the use of molecular dynamic and NMR, afforded next potent and selective inhibitor of cathepsin B (Ki of 0.48 nM, Fig. 2) (Wieczerzak et al.; 2007).

Fig. 2. Two potent azapeptide inhibitors of cathepsin B.

#### **2.2 Inhibitors from natural sources**

General strategy employed for discovery of a new drug relays on random screening of libraries of newly available compounds and selection of these, which exhibit desired activity at micromolar range. The leads are then being modified in order to obtain significantly more potent and selective inhibitors, which might be further introduced as drugs. Nature is strongly exploited as a source of lead substances. Isolated in 1978 from *Aspergillus japonicus*, non-specific, irreversible inhibitor of cysteine proteinases, E-64 can serve as a good example (Hanada et al, 1978). The epoxysucciante fragment of this molecule reacts with active-site cysteine and binds covalently to the enzyme. By using this inhibitor as a frame and applying X-ray crystal structures of cathepsins B and L, specific inhibitors of these enzymes were designed (Fig. 3), prepared and shown to have promising anticancer activity in animal studies (Katunuma, 2011).

Traditionally, secondary metabolites from streptomyces show a wide range of diversity with respect to their biological activity and chemical nature. Therefore it is not surprising that their secondary metabolites appear to be interesting lead compounds. A mixture of two

Thus, mimicking the segment of cystatin interacting with the cathepsin active site (Fig. 1) seems to be the method of choice. This approach is well represented by highly active inhibitor (N-1845, Fig. 2) of cathepsin B (Ki value of 0.088nM) containing an azaglycine residue in place of evolutionary conserved glycine residue in the N terminal part of cystatin (Wieczerzak et al., 2002). Further modification of this molecule, enforced by the use of molecular dynamic and NMR, afforded next potent and selective inhibitor of cathepsin B (Ki

of 0.48 nM, Fig. 2) (Wieczerzak et al.; 2007).

O N H

O N H

Fig. 2. Two potent azapeptide inhibitors of cathepsin B.

**2.2 Inhibitors from natural sources** 

studies (Katunuma, 2011).

O

O

NH

HN NH2

H N

NH

HN NH2

H N O

N H

O

General strategy employed for discovery of a new drug relays on random screening of libraries of newly available compounds and selection of these, which exhibit desired activity at micromolar range. The leads are then being modified in order to obtain significantly more potent and selective inhibitors, which might be further introduced as drugs. Nature is strongly exploited as a source of lead substances. Isolated in 1978 from *Aspergillus japonicus*, non-specific, irreversible inhibitor of cysteine proteinases, E-64 can serve as a good example (Hanada et al, 1978). The epoxysucciante fragment of this molecule reacts with active-site cysteine and binds covalently to the enzyme. By using this inhibitor as a frame and applying X-ray crystal structures of cathepsins B and L, specific inhibitors of these enzymes were designed (Fig. 3), prepared and shown to have promising anticancer activity in animal

Traditionally, secondary metabolites from streptomyces show a wide range of diversity with respect to their biological activity and chemical nature. Therefore it is not surprising that their secondary metabolites appear to be interesting lead compounds. A mixture of two

H <sup>N</sup> <sup>N</sup> H

NH HN NH2

O

N <sup>H</sup> <sup>O</sup> H

N COOCH3

O

O

N H

O

N-1845

H <sup>N</sup> <sup>N</sup> H O

N <sup>H</sup> <sup>O</sup> H

N COOCH3

O

peptide metabolites from *Streptomyces* NCIM 2081 (Fig. 4) exhibited potent inhibitory action against papain and significantly inhibited tumor cell migration at subcytotoxic concentrations, indicating its remarkable potential to be developed as antimetastatic drug (Singh et al, 2010).

cathepsin B inhibitor

Fig. 3. Specific inhibitors of cathepsins B and L built up on the frame of E-64.

Fig. 4. Anticancer peptides produced by *Streptomyces* NCIM 2081.

## **2.3 Irreversible inhibitors**

The majority of synthetic cysteine proteinase inhibitors contain a peptide segment for recognition by the chosen enzyme and an electrophilic functionality that is able to react with the thiolate moiety of active site cysteine. In most cases this results in covalent modification of the enzyme and irreversible inhibition. A wide variety of such reactive groups have been employed, including: azomethyl- or halomethyl ketone, acyloxymethyl ketone,

Inhibitors of Proteinases as Potential Anti-Cancer Agents 45

with other cysteine proteinases, thus causing toxic side effects or generating immunogenic

Rational design of the peptidyl or peptidomimetic part of inhibitor requires X-ray determination of either cysteine proteinase alone or complexed with already known inhibitors. This provides the detailed insight into the active site and binding pockets of certain enzyme and makes the design process viable. The knowledge of the architecture of the active site of cathepsin B and molecular docking studies were used to design the mechanism-based inhibitor of this enzyme with dual action (Lim et el., 2004)). First, active site Cys-29 is acylated by the inhibitor, which is followed by transfer of acetyloxy moiety of the inhibitor catalyzed by His-199. Thus, two vital active site amino acids are blocked

adducts (Joyce et al., 2004).

irreversibly (Fig. 6).

F

N H

O

H <sup>N</sup> <sup>O</sup> O

+

**Cys**

SH

O O

N <sup>O</sup> <sup>O</sup> O

**His**

O

O S

H

N <sup>O</sup> <sup>O</sup> O

**His**

N NH

N NH

**Cys**

The strategy in design of reversible inhibitors of cysteine proteinases is commonly the same as in the case of irreversible ones with the exception that the reaction between electrophilic warhead of the inhibitor and the enzyme is reversible. An aldehyde, a metyl ketone, a αketoamide or a nitrile groups usually act as the reactive electrophiles. Representative examples of such inhibitors are shown in Figure 7. Some of them are currently being profiled in animal models to further delineate the role of these enzymes in cancer disease

A wide variety of these inhibitors were obtained using computer-aided design. For example, high-resolution X-ray crystallographic data and molecular modeling studies were used to find out one of the most potent inhibitors of cathepsin B (Ki=7nM) - dipeptide nitrile shown

O

F

N H

O

H <sup>N</sup> <sup>O</sup> O

Fig. 6. Inhibition of cathepsin B by mechanism-base dual inhibitor.

O

F

F

**2.4 Reversible inhibitors** 

processes.

acylhydroxamate, vinyl sulfone and chloromethyl sufoxide functions. It is also worth to mention that epoxysuccinates, described earlier, also fall within this class of inhibitors. Representative examples of structurally variable inhibitors are shown in Figure 5.

Fig. 5. Representative examples of irreversible inhibitors of cysteine proteinases. The curved arrows indicate possible sites of nucleophilic attack by the active site –SH of active site cysteine.

The reactivity of the electrophilic group greatly determines the selectivity and reaction rate of the formation of the covalent enzyme-inhibitor complex. With this respect halomethylketones are known to react not only with cysteine but also with serine proteinases, thus being non-selective. Although irreversible inhibitors possess high potency and selectivity, they are not considered to be viable drug candidates for treating diseases like cancer, osteoporosis or arthritis. This is because such inhibitors often react over time

acylhydroxamate, vinyl sulfone and chloromethyl sufoxide functions. It is also worth to mention that epoxysuccinates, described earlier, also fall within this class of inhibitors.

Representative examples of structurally variable inhibitors are shown in Figure 5.

Cl

H N

COOH

N H

<sup>O</sup> <sup>H</sup>

O

Fig. 5. Representative examples of irreversible inhibitors of cysteine proteinases. The curved arrows indicate possible sites of nucleophilic attack by the active site –SH of active site

The reactivity of the electrophilic group greatly determines the selectivity and reaction rate of the formation of the covalent enzyme-inhibitor complex. With this respect halomethylketones are known to react not only with cysteine but also with serine proteinases, thus being non-selective. Although irreversible inhibitors possess high potency and selectivity, they are not considered to be viable drug candidates for treating diseases like cancer, osteoporosis or arthritis. This is because such inhibitors often react over time

N O

OH

O N H H N H <sup>N</sup> <sup>O</sup> O

N N

O

<sup>H</sup> <sup>O</sup>

H N

O N <sup>H</sup> <sup>O</sup>

HN

H N

O N H

O

COOH

acyloxymethyl ketone caspase 9 (Berger et. al., 2006)

O O

S O

Cl

chlorometyhyl sulfoxide papain (Brouwer at al., 2007)

O

<sup>S</sup> <sup>S</sup>

OCH3

disufide cathepsin S (Chang et al. 2007)

O

CONH2

O O N <sup>H</sup> <sup>O</sup>

H N

chloromethyl ketone legumain (Niestroj, et.el, 2002)

HO

O2N

N H

O

H N

*O*-acylo hydroxyurea cathepsins B and L (Verhelst, 2006)

> S O O

vinylsulfone cathepsin S (Chang et al. 2007)

NH2

O

O

cysteine.

N N

O

<sup>H</sup> <sup>O</sup>

H N O N H N O

> O N <sup>H</sup> <sup>O</sup>

with other cysteine proteinases, thus causing toxic side effects or generating immunogenic adducts (Joyce et al., 2004).

Rational design of the peptidyl or peptidomimetic part of inhibitor requires X-ray determination of either cysteine proteinase alone or complexed with already known inhibitors. This provides the detailed insight into the active site and binding pockets of certain enzyme and makes the design process viable. The knowledge of the architecture of the active site of cathepsin B and molecular docking studies were used to design the mechanism-based inhibitor of this enzyme with dual action (Lim et el., 2004)). First, active site Cys-29 is acylated by the inhibitor, which is followed by transfer of acetyloxy moiety of the inhibitor catalyzed by His-199. Thus, two vital active site amino acids are blocked irreversibly (Fig. 6).

Fig. 6. Inhibition of cathepsin B by mechanism-base dual inhibitor.

#### **2.4 Reversible inhibitors**

The strategy in design of reversible inhibitors of cysteine proteinases is commonly the same as in the case of irreversible ones with the exception that the reaction between electrophilic warhead of the inhibitor and the enzyme is reversible. An aldehyde, a metyl ketone, a αketoamide or a nitrile groups usually act as the reactive electrophiles. Representative examples of such inhibitors are shown in Figure 7. Some of them are currently being profiled in animal models to further delineate the role of these enzymes in cancer disease processes.

A wide variety of these inhibitors were obtained using computer-aided design. For example, high-resolution X-ray crystallographic data and molecular modeling studies were used to find out one of the most potent inhibitors of cathepsin B (Ki=7nM) - dipeptide nitrile shown

Inhibitors of Proteinases as Potential Anti-Cancer Agents 47

The field of metallodrugs is dominated by compounds, which interact with DNA and cause its direct damage. In recent years, however, it was well established that some of them exert cytotoxic activity affecting certain enzymes. Rhutenium (II)–arene derivatives exhibit remarkable selectivity towards solid tumors, most likely by inhibiting two vital enzymes for cancer development – thioredoxin reductase and cathepsin B. The most active inhibitor of cathepsin B is reversibly bound to the active site of the enzyme (Casini et al., 2008). Docking studies revealed that the most important interactions responsible for its activity are those

Fig. 9. The most active organorhutenium inhibitor of cathepsin B and its mode of binding in

Quite contrary, newly synthesized series of organotelluranes appeared to be potent, irreversible inhibitors of cathepsins V and S (Piovan et al., 2011). Tellurium atom is an electrophilic center, which undergoes nucleophilic attack of cysteine thiol at the active site of the enzyme. In this reaction tellurium-halogen bond is broken and new tellurium sulfur bond is formed (Fig. 10). Considering the electrophilicity of the chalcogen, it is known that tellurium is less electronegative than selenium and, due to its greater capacity to stabilize the negative charge, bromide is a better leaving group than the chloride, we can explain the

OCH3

Te Br S

**Cys**

highest reactivity of the dibromo-organotelluranes toward cysteine cathepsins.

Fig. 10. Mechanism of irreversible inhibition of cathepsins by organotellurane.

the active site of the enzyme as modeled by docking approach.

OCH3

HS **Cys**

Te Br Br

**2.5 Metalloinhibitors** 

with the residues flanking the active site (Fig. 9).

in Figure 8 (Greenspan et al., 2001). In the Figure 8 also the mechanism of reversible binding of this inhibitors was outlined.

Fig. 7. Representative examples of reversible inhibitors of cysteine proteinases.

Fig. 8. Mechanism of inhibition of cathepsin B by dipeptidyl nitrile.

#### **2.5 Metalloinhibitors**

46 Drug Development – A Case Study Based Insight into Modern Strategies

in Figure 8 (Greenspan et al., 2001). In the Figure 8 also the mechanism of reversible binding

of this inhibitors was outlined.

O

F

O N H

<sup>O</sup> <sup>H</sup>

N

<sup>O</sup> <sup>O</sup>

N N H

> N H

O


> H N

O


**Cys**

H N

CF3

O N H

O

O S O O

nitrile cathepsin S (Ward et al., 2002)

H N CN

O

O

<sup>O</sup> <sup>H</sup> N O

H N O

H O

Fig. 7. Representative examples of reversible inhibitors of cysteine proteinases.

COOH

Fig. 8. Mechanism of inhibition of cathepsin B by dipeptidyl nitrile.

SH N

O

O


H N O

O

N N

Br F

N H

O

N N NH2

<sup>N</sup> NH

thiosemicarbazone cathepsin L (Kumar et al., 2010)

> H N O

> > O N H

<sup>O</sup> <sup>H</sup>

N

COOH

**Cys**

NH

<sup>O</sup> <sup>O</sup>

azepanone cathepsin K (Stroup et al., 2001)

O

N S

<sup>O</sup> <sup>O</sup> N

O NH2

N H

O

HN O

OCH3

H N O

aldehyde cathepsin S (Katanuma, 2011)

CHO

O

O

The field of metallodrugs is dominated by compounds, which interact with DNA and cause its direct damage. In recent years, however, it was well established that some of them exert cytotoxic activity affecting certain enzymes. Rhutenium (II)–arene derivatives exhibit remarkable selectivity towards solid tumors, most likely by inhibiting two vital enzymes for cancer development – thioredoxin reductase and cathepsin B. The most active inhibitor of cathepsin B is reversibly bound to the active site of the enzyme (Casini et al., 2008). Docking studies revealed that the most important interactions responsible for its activity are those with the residues flanking the active site (Fig. 9).

Fig. 9. The most active organorhutenium inhibitor of cathepsin B and its mode of binding in the active site of the enzyme as modeled by docking approach.

Quite contrary, newly synthesized series of organotelluranes appeared to be potent, irreversible inhibitors of cathepsins V and S (Piovan et al., 2011). Tellurium atom is an electrophilic center, which undergoes nucleophilic attack of cysteine thiol at the active site of the enzyme. In this reaction tellurium-halogen bond is broken and new tellurium sulfur bond is formed (Fig. 10). Considering the electrophilicity of the chalcogen, it is known that tellurium is less electronegative than selenium and, due to its greater capacity to stabilize the negative charge, bromide is a better leaving group than the chloride, we can explain the highest reactivity of the dibromo-organotelluranes toward cysteine cathepsins.

Fig. 10. Mechanism of irreversible inhibition of cathepsins by organotellurane.

Inhibitors of Proteinases as Potential Anti-Cancer Agents 49

Typically serine proteinases have active site clefts that are relatively exposed to solvent. This permits the access to polypeptide loops of substrates and endogenous inhibitors. By forming strong proteinase-inhibitor complexes the latter ones regulate the activity of proteolytic enzymes and play important physiological roles in all organisms. Therefore, it is not surprising that they are considered as potential anticancer drugs and are already being

Proteinous serine proteinase inhibitors were the first used against cancer and are so far the most intensively studied (Castro-Guillén et al., 2010; Otlewski et al., 2005). A small metalloprotein, Birk-Bowman inhibitor, isolated from soybeans as far as in 1946, is 8kDa polypeptide of the documented activity in a variety of tumors. Other members of this family have also proved their anti cancer activity, with field bean protease inhibitor being strongly active against skin and lung tumors, and tepary bean inhibitor affecting proliferation and metastasis of fibroblast (Castro-Guillén et al., 2010; Joanitti et al., 2010; Sakuhari et al., 2008). Another classes of similar inhibitors of serine proteinases also exhibit promising anti-cancer properties, to mention only: Kunitz-type inhibitors (Sierko et el., 2007; Wang et al., 2010), serpins (Catanzaro et al., 2011; Li et al., 2006), antileukoprotease (Xuan et el., 2008), nexin

Paradoxically, the action of proteinase inhibitors in some cases results in poorer prognosis and promotion of the cancer development (Fayard et al, 2009; Ozaki et al., 2009). This is contrary to what would be expected from proteinase inhibitor and shows that more detailed studies are required in order to understand their action. These observations also indicate the need for development of inhibitors of different types. Examination of crystal structures of inhibitors bound by various proteinases is a useful tool to study architecture and requirements of serine proteinase binding sites. This is because 3-5 amino acid residues of proteinaceus inhibitor, properly spatially located with respect to each other, interact with small binding region of the enzyme. The binding modes of Bowman-Birk inhibitor from *Vigna unguinocula* with β-chymotrypsin (Barbosa et. al., 2007), and structure of textilinin-1 from the venom of Australian *Pseudonaja textilis* snake complexed with trypsin (Millers et

Fig. 11. The binding modes of Bowman-Birk inhibitor with β-chymotrypsin (left-hand side)

Mutation of the already known protein inhibitors is one of the means to construct highly specific inhibitors of chosen proteinase. Such strategy was applied to obtain specific and potent inhibitors of human kallikrein 14. A human serpin, named α-1-antichymotrypsin,

(Candia et el., 2006) and lunasin (Dia & de Meija, 2010; Hsieh et al., 2010).

al., 2009) are shown in Figure 11 as representative examples.

**3.1 Proteinous inhibitors** 

and textilinin with trypsin

tested in clinics.

## **3. Serine proteinases**

Serine proteinases emerged during evolution as the most abundant and functionally diverse group of proteolytic enzymes - over one third of them belong to this class. They typically contain a catalytic triad of serine, histidine and aspartic acid residues in their catalytic active sites, which are commonly referred to as the charge relay system. This implies common mechanism of peptide bond hydrolysis. It goes through two-step hydrolytic process, which allows acylation of the serine residue by peptide substrate followed by hydrolysis of this adduct and regeneration of the enzyme.

Several serine proteinases have been implicated as important regulators of cancer development. This family includes enzymes involved in mediating of plasminogen (urokinase-type and tissue-type plasminogen activators), as well as serine proteinases stored in secretory lysosomes of various leukocytes, namely mast cell chymase, mast cell tryptase, and neutrophil elastase. Although most secreted serine proteinases emanate from host stromal cells, recent studies implicate a superfamily of cell-surface associated serine proteases, also known as Type II Transmembrane Serine Proteinases (TTSP), such as matriptase and hepsin, as important regulators of cancer development.

Plasmin proteolytic cascade is functionally contributing to neoplastic progression, including acquisition of a migratory and invasive phenotype by tumor cells, as well as remodeling of extracellular matrix components via activation of matrix metalloproteinases. Urokinase-type and tissue-type plasminogen activators (uPA and tPA respectively) regulate enzymatic activity of plasmin. uPA plays a crucial role in tissue remodeling, while tPA is important in vascular fibrinolysis (Naffara et al., 2009).

Mast cell-derived chymases and tryptases are stored in secretory granules. Their release into the extracellular milieu triggers a proinflammatory response as well as induces a cascade of protease activations, culminating in activation of matrix metalloproteinase 9. As a result neoplastic progression is observed (Fiorucci & Ascoli, 2004; Takai et el., 2004).

Neutrophil elastase, a serine protease abundantly present in neutrophil azurophilic (primary) granules, is transcriptionally activated during early myeloid development. Little is known about the role of this proteinase in cancer progression, however, it has the ability to cleave almost every protein contained within the extracellular matrix including, but not limited to: elastin, collagen, fibronectin, laminin, and proteoglycans. Interest in neutrophil elastase during neoplastic processes stems from recent clinical reports that correlate elevated expression of this enzyme with poor survival rates in patients with primary breast cancer and non-small cell lung cancer. It also has recently been found to initiate development of acute promyelocytic leukemia (Naffra et al., 2009; Sato et al., 2006)

Most serine proteinases are expressed by supporting tumor stromal cells, whereas membrane-anchored serine protease appear to be largely expressed by tumor cells at the cell surface and are thus ideally located to regulate cell–cell and cell–matrix interactions. Increasing evidence demonstrates that aberrant expression of enzymes such as matriptase and hepsin is a hallmark of several cancers and recent studies have defined molecular mechanisms underlying TTSP-promoted tumorigenesis, a processes causing carcinomas of skin, breast, and prostate (Choi et al., 2009). Similar association with cancer has led to great interest in kallikreins (Di Cera, 2009), a large family better known for its role in regulation of blood pressure through the kinin system. Prostate-specific antigen (PSA), a serine protease also belonging to the human kallikrein family, is best known as a prostate cancer biomarker since its expression is highly restricted to normal and malignant prostate epithelial cells.

## **3.1 Proteinous inhibitors**

48 Drug Development – A Case Study Based Insight into Modern Strategies

Serine proteinases emerged during evolution as the most abundant and functionally diverse group of proteolytic enzymes - over one third of them belong to this class. They typically contain a catalytic triad of serine, histidine and aspartic acid residues in their catalytic active sites, which are commonly referred to as the charge relay system. This implies common mechanism of peptide bond hydrolysis. It goes through two-step hydrolytic process, which allows acylation of the serine residue by peptide substrate followed by hydrolysis of this

Several serine proteinases have been implicated as important regulators of cancer development. This family includes enzymes involved in mediating of plasminogen (urokinase-type and tissue-type plasminogen activators), as well as serine proteinases stored in secretory lysosomes of various leukocytes, namely mast cell chymase, mast cell tryptase, and neutrophil elastase. Although most secreted serine proteinases emanate from host stromal cells, recent studies implicate a superfamily of cell-surface associated serine proteases, also known as Type II Transmembrane Serine Proteinases (TTSP), such as

Plasmin proteolytic cascade is functionally contributing to neoplastic progression, including acquisition of a migratory and invasive phenotype by tumor cells, as well as remodeling of extracellular matrix components via activation of matrix metalloproteinases. Urokinase-type and tissue-type plasminogen activators (uPA and tPA respectively) regulate enzymatic activity of plasmin. uPA plays a crucial role in tissue remodeling, while tPA is important in

Mast cell-derived chymases and tryptases are stored in secretory granules. Their release into the extracellular milieu triggers a proinflammatory response as well as induces a cascade of protease activations, culminating in activation of matrix metalloproteinase 9. As a result

Neutrophil elastase, a serine protease abundantly present in neutrophil azurophilic (primary) granules, is transcriptionally activated during early myeloid development. Little is known about the role of this proteinase in cancer progression, however, it has the ability to cleave almost every protein contained within the extracellular matrix including, but not limited to: elastin, collagen, fibronectin, laminin, and proteoglycans. Interest in neutrophil elastase during neoplastic processes stems from recent clinical reports that correlate elevated expression of this enzyme with poor survival rates in patients with primary breast cancer and non-small cell lung cancer. It also has recently been found to initiate development of

Most serine proteinases are expressed by supporting tumor stromal cells, whereas membrane-anchored serine protease appear to be largely expressed by tumor cells at the cell surface and are thus ideally located to regulate cell–cell and cell–matrix interactions. Increasing evidence demonstrates that aberrant expression of enzymes such as matriptase and hepsin is a hallmark of several cancers and recent studies have defined molecular mechanisms underlying TTSP-promoted tumorigenesis, a processes causing carcinomas of skin, breast, and prostate (Choi et al., 2009). Similar association with cancer has led to great interest in kallikreins (Di Cera, 2009), a large family better known for its role in regulation of blood pressure through the kinin system. Prostate-specific antigen (PSA), a serine protease also belonging to the human kallikrein family, is best known as a prostate cancer biomarker since its expression is highly restricted to normal and malignant prostate epithelial cells.

matriptase and hepsin, as important regulators of cancer development.

neoplastic progression is observed (Fiorucci & Ascoli, 2004; Takai et el., 2004).

acute promyelocytic leukemia (Naffra et al., 2009; Sato et al., 2006)

**3. Serine proteinases** 

adduct and regeneration of the enzyme.

vascular fibrinolysis (Naffara et al., 2009).

Typically serine proteinases have active site clefts that are relatively exposed to solvent. This permits the access to polypeptide loops of substrates and endogenous inhibitors. By forming strong proteinase-inhibitor complexes the latter ones regulate the activity of proteolytic enzymes and play important physiological roles in all organisms. Therefore, it is not surprising that they are considered as potential anticancer drugs and are already being tested in clinics.

Proteinous serine proteinase inhibitors were the first used against cancer and are so far the most intensively studied (Castro-Guillén et al., 2010; Otlewski et al., 2005). A small metalloprotein, Birk-Bowman inhibitor, isolated from soybeans as far as in 1946, is 8kDa polypeptide of the documented activity in a variety of tumors. Other members of this family have also proved their anti cancer activity, with field bean protease inhibitor being strongly active against skin and lung tumors, and tepary bean inhibitor affecting proliferation and metastasis of fibroblast (Castro-Guillén et al., 2010; Joanitti et al., 2010; Sakuhari et al., 2008). Another classes of similar inhibitors of serine proteinases also exhibit promising anti-cancer properties, to mention only: Kunitz-type inhibitors (Sierko et el., 2007; Wang et al., 2010), serpins (Catanzaro et al., 2011; Li et al., 2006), antileukoprotease (Xuan et el., 2008), nexin (Candia et el., 2006) and lunasin (Dia & de Meija, 2010; Hsieh et al., 2010).

Paradoxically, the action of proteinase inhibitors in some cases results in poorer prognosis and promotion of the cancer development (Fayard et al, 2009; Ozaki et al., 2009). This is contrary to what would be expected from proteinase inhibitor and shows that more detailed studies are required in order to understand their action. These observations also indicate the need for development of inhibitors of different types. Examination of crystal structures of inhibitors bound by various proteinases is a useful tool to study architecture and requirements of serine proteinase binding sites. This is because 3-5 amino acid residues of proteinaceus inhibitor, properly spatially located with respect to each other, interact with small binding region of the enzyme. The binding modes of Bowman-Birk inhibitor from *Vigna unguinocula* with β-chymotrypsin (Barbosa et. al., 2007), and structure of textilinin-1 from the venom of Australian *Pseudonaja textilis* snake complexed with trypsin (Millers et al., 2009) are shown in Figure 11 as representative examples.

Fig. 11. The binding modes of Bowman-Birk inhibitor with β-chymotrypsin (left-hand side) and textilinin with trypsin

Mutation of the already known protein inhibitors is one of the means to construct highly specific inhibitors of chosen proteinase. Such strategy was applied to obtain specific and potent inhibitors of human kallikrein 14. A human serpin, named α-1-antichymotrypsin,

Inhibitors of Proteinases as Potential Anti-Cancer Agents 51

Similar cyclic peptides, inhibitors of various proteinases, were also isolated from natural sources. For example, out of more than 100 compounds of this class isolated from cyanobacteria about half have been reported to inhibit trypsin or chymotrypsin. Recently isolated Symplocamine A (Figure 13), molecule of strong serine protease inhibitory activity, appeared to exert high level of cytotoxicity against variety of cancer cells *in vitro* thus being

Similarly as in the case of cysteine proteinases irreversible inhibitors of serine proteinases are prominent class of their inactivators. They usually bind covalently to one of the nucleophilic moieties of amino acids present in an active site of the enzyme (most likely hydroxylic group of serine) using an electrophilic warhead. Although there are many classes of irreversible inhibitors of serine proteinases available today (Powers, et al., 2002) only limited examples entered clinical studies as anticancer agents. Therefore, new lowmolecular inhibitors of enzymes involved in cancer development and metastasis are still strongly desirable. Recent studies are concentrated on the synthesis of inhibitors containing

a potential agent against cancers (Linington et el., 2008).

non-typical warheads (representative examples are shown in Figure 14).

Fig. 14. Representative examples of irreversible inhibitors of serine proteinases.

Diphenyl phosphonates seem to be the most promising and general group of these inhibitors. They may be also classified as competitive transition-state analogues. On a molecular level they phosphonylate specifically active-site serine residue thus blocking the catalytic triad of serine, histidine and aspartic acids responsible for the formation of enzyme-substrate acyl

**3.2 Irreversible inhibitors** 

was used to change its specificity by modifying five amino acids of its reactive center loop, a region involved in inhibitor–protease interaction. This region was replaced by two pentapeptides, previously selected by kallikrein 14 using phage-display technology. In this manner inhibitors with high reactivity towards the enzyme were generated (Fleber et al., 2006).

Sensing the binding site of chosen proteinase by studying structure of bound regions of its inhibitors and substrates is a classical tool for the design of new inhibitors of these enzymes. This concept is well illustrated by the discovery of cyclic peptides mimicking binding fragment of plasminogen activator (uPA) to its cell surface associated receptor (uPAR). The minimal portion of uPa able to bind effectively to uPAR was selected by systematic deletions of peptidyl fragments from the N- and C-terminus of the starting protein inhibitor. Cyclization of the minimal effective structure results in introduction of constrains that limit the conformational freedom of the molecule and ensure proper spatial arrangement of the amino acid residues interacting with the receptor. In that manner cyclic peptides, mimetics of uPA, (the most effective one is shown in Figure 12) were synthesized and found to effectively compete with uPA binding (Schmiedeberg et al., 2002).

Fig. 12. Fragment of uPA selected as a scaffold for the preparation of cyclic peptidomimetics and the structure of the most effective of them

Fig. 13. Structure of Symplocamine A

Similar cyclic peptides, inhibitors of various proteinases, were also isolated from natural sources. For example, out of more than 100 compounds of this class isolated from cyanobacteria about half have been reported to inhibit trypsin or chymotrypsin. Recently isolated Symplocamine A (Figure 13), molecule of strong serine protease inhibitory activity, appeared to exert high level of cytotoxicity against variety of cancer cells *in vitro* thus being a potential agent against cancers (Linington et el., 2008).

#### **3.2 Irreversible inhibitors**

50 Drug Development – A Case Study Based Insight into Modern Strategies

was used to change its specificity by modifying five amino acids of its reactive center loop, a region involved in inhibitor–protease interaction. This region was replaced by two pentapeptides, previously selected by kallikrein 14 using phage-display technology. In this manner inhibitors with high reactivity towards the enzyme were generated (Fleber et al.,

Sensing the binding site of chosen proteinase by studying structure of bound regions of its inhibitors and substrates is a classical tool for the design of new inhibitors of these enzymes. This concept is well illustrated by the discovery of cyclic peptides mimicking binding fragment of plasminogen activator (uPA) to its cell surface associated receptor (uPAR). The minimal portion of uPa able to bind effectively to uPAR was selected by systematic deletions of peptidyl fragments from the N- and C-terminus of the starting protein inhibitor. Cyclization of the minimal effective structure results in introduction of constrains that limit the conformational freedom of the molecule and ensure proper spatial arrangement of the amino acid residues interacting with the receptor. In that manner cyclic peptides, mimetics of uPA, (the most effective one is shown in Figure 12) were synthesized and found to

N H

O

N H NH2

<sup>O</sup> <sup>H</sup> N

O

CONH2

Fig. 12. Fragment of uPA selected as a scaffold for the preparation of cyclic peptidomimetics

O

HN <sup>O</sup> <sup>H</sup>

O

N H

N

O

N

N

<sup>O</sup> <sup>O</sup>

<sup>O</sup> HO

OCH3

N H

NH O

NH2 S S

O

NH

HN

O

H2NOC

HO

H <sup>N</sup> NH

HO

O

HN

NH

O

HN

HN COOH O

O

CONH2 H2N

O O

effectively compete with uPA binding (Schmiedeberg et al., 2002).

and the structure of the most effective of them

Br

Fig. 13. Structure of Symplocamine A

2006).

Similarly as in the case of cysteine proteinases irreversible inhibitors of serine proteinases are prominent class of their inactivators. They usually bind covalently to one of the nucleophilic moieties of amino acids present in an active site of the enzyme (most likely hydroxylic group of serine) using an electrophilic warhead. Although there are many classes of irreversible inhibitors of serine proteinases available today (Powers, et al., 2002) only limited examples entered clinical studies as anticancer agents. Therefore, new lowmolecular inhibitors of enzymes involved in cancer development and metastasis are still strongly desirable. Recent studies are concentrated on the synthesis of inhibitors containing non-typical warheads (representative examples are shown in Figure 14).

Fig. 14. Representative examples of irreversible inhibitors of serine proteinases.

Diphenyl phosphonates seem to be the most promising and general group of these inhibitors. They may be also classified as competitive transition-state analogues. On a molecular level they phosphonylate specifically active-site serine residue thus blocking the catalytic triad of serine, histidine and aspartic acids responsible for the formation of enzyme-substrate acyl

Inhibitors of Proteinases as Potential Anti-Cancer Agents 53

H N O

> <sup>N</sup> <sup>N</sup> H

O

<sup>S</sup> <sup>N</sup> H

H2N NH

O N

matriptase (Steinmetzer et al., 2006)

OH

N H NH2

NH

O O

CONH2

H N B(OH)2

N H O H

O

O

H2N

O N H

O N H

inhibitors is currently under intensive investigations.

<sup>O</sup> <sup>H</sup>

O HO

Fig. 16. Representative examples of reversible inhibitors of serine proteinases.

tumor cells to radiation or chemotherapy (Adams, 2002; Goldberg, 2007).

PSA (LeBeauet.al. 2009)

H2N

HO

<sup>O</sup> <sup>H</sup>

O HO

<sup>N</sup> <sup>N</sup> H

The sequencing of human genome revealed that threonine proteinases account only for about 5% of the whole pool of proteinases. From these proteinases, only proteasome is considered as a target for potential anticancer agents. Since tightly ordered proteasomal degradation of proteins plays crucial role in the cell cycle control potential of proteasome

The proteasome is a highly conserved intracellular nonlysosomal multicatalytic protease complex, degrading proteins usually tagged with a polyubiquitin chain. The 26S proteasome is a 2,000 kDa multisubunit cylindrical protein comprised of a 20S core catalytic component (the 20S proteasome) capped at one or both ends by 19S regulatory components (Figure 17). Proteasome 20S has three major sites of different activities designed as "chymotrypsin-like", "trypsin-like" and "caspase-like". These three activities are responsible for the cleavage of protein after hydrophobic, basic, and acidic amino acid residues, respectively. Analysis of the proteasome catalytic mechanism has revealed the importance of the N-terminal threonine as catalytic nucleophile. Thus, proteolytic machinery of the proteasome is an important target for the design of anticancer drugs (Abbenante & Fairlie, 2005; Delcros et al., 2003; Goldberg, 2007). A wide variety of inhibitors of proteasome were developed and evaluated (Delcros et al., 2003). This process culminated in discovery of bortezomib (*Velcade*, Figure 17), which decreases proliferation, induces apoptosis and enhances sensitivity of

PSA (LeBeauet.al. 2009)

> <sup>O</sup> <sup>H</sup> N O

HO

H2N

NH

N O O

UK 122

**4. Threonine proteinases** 

intermediate and its further hydrolysis (Fig. 15). Anyway, the mode of action of phosphonates towards serine proteinases is not yet fully elucidated and minor variations were observed, depending on the targeted enzyme and conditions (Grzywa et al. 2007; Joossens et al., 2006; Sieńczyk et al., 2011; Sieńczyk & Oleksyszyn 2006; Sieńczyk & Oleksyszyn, 2009).

Fig. 15. Schematic illustration of the mechanism of action of the diphenyl αaminophosphonate inhibitors of serine proteinases

#### **3.3 Reversible inhibitors**

Inhibitors of urokinase (also called urokinase-type plasminogen activator, uPA) are the biggest family of reversible serine protease inhibitors. Development of small molecule uPA inhibitors has began with aryl guanidines, aryl amidines, and acyl guanidines, molecules that contain positively charged guanidine, amidine, or simple amines as anchors able to interact with the negatively charged site chain of Asp189 (Lee et el., 2004). Although they exhibited moderate potency and poor selectivity they constituted a good starting point for the development of new effective generations of uPA inhibitors. Intensive studies using various approaches resulted in many inhibitors, which quite frequently revealed *in vitro* anticancer properties. Determination of three-dimensional structure of this enzyme either in native state or complexed with various inhibitors is vital for the design of new effectors of urokinase (Huai et al. 2008; Klinghofer et al. 2001; Sperl et. al. 2000).

For, example, an extremely simple inhibitor UK 122 (Fig. 16) was designed in a stepwise process. The first step was a selection of moderate inhibitors of uPA by screening a library of 16,000 synthetic compounds. This resulted in four promising inhibitors of the enzyme sharing very similar chemical structures. They were further optimized by using crystal structure of the enzyme-Amiloride complex and by applying molecular modeling methods. As a result UK 122 was found (Zhu et al., 2007). This compound significantly inhibited the migration and invasion of pancreatic cancer cell line.

Another example may be the use of three-dimensional quantitative structure-activity relationship (3D QSAR) studies to elucidate structural features required for uPA inhibition and to obtain predictive three-dimensional template for the design of new inhibitors. 3D QSAR was performed on five reported classes of the urokinase inhibitors by employing widely used CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Shape Indices Analysis) methods (Bhongade & Gadad 2006). As a result the significance of various structural elements bound at different urokinase subsites was identified. These subsites may be combined to improve overall activity of newly designed inhibitors.

Inhibitors of other serine proteinases were studied as anticancer agents quite scarcely. Most of the obtained inhibitors were designed to affect with prostate specific antigen (PSA) and matriptase by adopting the procedures used for designing of other serine proteinase inhibitors. Some of them exhibit promising anticancer properties in cell culture systems. Representative examples of these inhibitors are shown in Figure 16.

intermediate and its further hydrolysis (Fig. 15). Anyway, the mode of action of phosphonates towards serine proteinases is not yet fully elucidated and minor variations were observed, depending on the targeted enzyme and conditions (Grzywa et al. 2007; Joossens et al., 2006;

O O

O

Inhibitors of urokinase (also called urokinase-type plasminogen activator, uPA) are the biggest family of reversible serine protease inhibitors. Development of small molecule uPA inhibitors has began with aryl guanidines, aryl amidines, and acyl guanidines, molecules that contain positively charged guanidine, amidine, or simple amines as anchors able to interact with the negatively charged site chain of Asp189 (Lee et el., 2004). Although they exhibited moderate potency and poor selectivity they constituted a good starting point for the development of new effective generations of uPA inhibitors. Intensive studies using various approaches resulted in many inhibitors, which quite frequently revealed *in vitro* anticancer properties. Determination of three-dimensional structure of this enzyme either in native state or complexed with various inhibitors is vital for the design of new effectors of

For, example, an extremely simple inhibitor UK 122 (Fig. 16) was designed in a stepwise process. The first step was a selection of moderate inhibitors of uPA by screening a library of 16,000 synthetic compounds. This resulted in four promising inhibitors of the enzyme sharing very similar chemical structures. They were further optimized by using crystal structure of the enzyme-Amiloride complex and by applying molecular modeling methods. As a result UK 122 was found (Zhu et al., 2007). This compound significantly inhibited the

Another example may be the use of three-dimensional quantitative structure-activity relationship (3D QSAR) studies to elucidate structural features required for uPA inhibition and to obtain predictive three-dimensional template for the design of new inhibitors. 3D QSAR was performed on five reported classes of the urokinase inhibitors by employing widely used CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Shape Indices Analysis) methods (Bhongade & Gadad 2006). As a result the significance of various structural elements bound at different urokinase subsites was identified. These subsites may

Inhibitors of other serine proteinases were studied as anticancer agents quite scarcely. Most of the obtained inhibitors were designed to affect with prostate specific antigen (PSA) and matriptase by adopting the procedures used for designing of other serine proteinase inhibitors. Some of them exhibit promising anticancer properties in cell culture systems.

HO **Ser Ser**

H N P R

H2O

O O

OH

Sieńczyk et al., 2011; Sieńczyk & Oleksyszyn 2006; Sieńczyk & Oleksyszyn, 2009).

H N P R

Fig. 15. Schematic illustration of the mechanism of action of the diphenyl α-

urokinase (Huai et al. 2008; Klinghofer et al. 2001; Sperl et. al. 2000).

be combined to improve overall activity of newly designed inhibitors.

Representative examples of these inhibitors are shown in Figure 16.

migration and invasion of pancreatic cancer cell line.

**Ser**

aminophosphonate inhibitors of serine proteinases

H N P R

O O

O

**3.3 Reversible inhibitors** 

Fig. 16. Representative examples of reversible inhibitors of serine proteinases.

### **4. Threonine proteinases**

The sequencing of human genome revealed that threonine proteinases account only for about 5% of the whole pool of proteinases. From these proteinases, only proteasome is considered as a target for potential anticancer agents. Since tightly ordered proteasomal degradation of proteins plays crucial role in the cell cycle control potential of proteasome inhibitors is currently under intensive investigations.

The proteasome is a highly conserved intracellular nonlysosomal multicatalytic protease complex, degrading proteins usually tagged with a polyubiquitin chain. The 26S proteasome is a 2,000 kDa multisubunit cylindrical protein comprised of a 20S core catalytic component (the 20S proteasome) capped at one or both ends by 19S regulatory components (Figure 17). Proteasome 20S has three major sites of different activities designed as "chymotrypsin-like", "trypsin-like" and "caspase-like". These three activities are responsible for the cleavage of protein after hydrophobic, basic, and acidic amino acid residues, respectively. Analysis of the proteasome catalytic mechanism has revealed the importance of the N-terminal threonine as catalytic nucleophile. Thus, proteolytic machinery of the proteasome is an important target for the design of anticancer drugs (Abbenante & Fairlie, 2005; Delcros et al., 2003; Goldberg, 2007). A wide variety of inhibitors of proteasome were developed and evaluated (Delcros et al., 2003). This process culminated in discovery of bortezomib (*Velcade*, Figure 17), which decreases proliferation, induces apoptosis and enhances sensitivity of tumor cells to radiation or chemotherapy (Adams, 2002; Goldberg, 2007).

Inhibitors of Proteinases as Potential Anti-Cancer Agents 55

stimulated studies on their analogues (representative structure is shown in Fig. 18). This resulted in several potent inhibitors, which display non-typical mechanism of action (Elofsson et e., 1999; Zhou et el., 2009). A hemiacetal is first formed between the ketone portion of the inhibitor and threonine hydroxyl, followed by epoxide ring opening by the

OH <sup>H</sup> <sup>O</sup> <sup>N</sup>

N H

<sup>N</sup> <sup>S</sup>

<sup>O</sup> <sup>O</sup>

H <sup>N</sup> <sup>N</sup> H

<sup>O</sup> <sup>O</sup>

Zhou et. el., 2009

NH H2NOC O

NH

N <sup>H</sup> <sup>O</sup>

Clerc et el., 2009

<sup>O</sup> <sup>O</sup> NH

ROOC

Basse et al., 2007

HN

O

Fig. 18. Natural inhibitors of proteasome activity as scaffolds for synthesis improved ones. Next example considers syringolines, reversible inhibitors of proteasome produced by *Pseudomonas siringae* (Coleman, et al. 2006). Elucidation of the crystal structure of syringolin B complexed with proteasome gave an insight into the structural requirements of good inhibitor. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be one of the most potent

A limiting factor in the efficiency of peptidic inhibitors is that they are unstable in living organism because they are easily degraded by endogenous proteinases. This explains growing interest in non-peptidic inhibitors. Nature is an acknowledged source of such compounds and many inhibitors of proteasome were isolated and identified. These include such structurally diverse compounds as: ajoene isolated from garlic (Hassan, 2004), gliotoxin produced by *Aspergillus fumigatus* (Pahl et al., 1996), or triterpene-celastrol isolated from the

N H O H <sup>N</sup> <sup>N</sup> <sup>H</sup> <sup>N</sup> <sup>H</sup> <sup>O</sup> O

O O OH

O

O O

free amine of the N-terminal threonine to give a stable morpholino adduct.

<sup>H</sup> <sup>O</sup> <sup>N</sup>

O O

lactacystin MLN-519

Omuralide

<sup>H</sup> <sup>O</sup> <sup>N</sup> <sup>S</sup>

O

<sup>N</sup> <sup>N</sup> H

O

OHOH

HN COOH O

> H <sup>N</sup> <sup>N</sup>

H <sup>N</sup> <sup>N</sup> <sup>H</sup> <sup>O</sup> OH

> N H

O

O

proteasome inhibitors described so far (Clerc et el., 2009).

root bark of medicinal plant *Tripterigium wolfordii* (Yang et al., 2006).

CONH2

HO HO

N <sup>H</sup> NHO

HO OH <sup>O</sup> N H O

O

TMC-95A

O H <sup>N</sup> <sup>N</sup> <sup>H</sup> <sup>N</sup> <sup>H</sup> COOH

O

syringolin A

HN

O

N H <sup>O</sup> OH

epoxomicin

eponemycin

NH O

O

O

O O

O O

<sup>H</sup> <sup>O</sup>

The most significant step in development of proteasome inhibitors was the decision by A. L. Goldberg and colleagues to create in 1993 the company *MyoGenics.* The goal was to synthesize proteasome inhibitors that could prevent muscle atrophy that occur in various disease states, such as cancer cachexia. This led to the production of a series of inhibitors that were freely distributed to academic laboratories and contributed to the enormous leap forward in understanding the multiple roles of the proteasome in cells.

#### Fig. 17. Schematic structure of proteasome with indication of the binding site of bortezomib

## **4.1 Inhibitors from natural sources**

The 20S proteasome is a tubular molecule with the proteolytic active sites on the inner surface. Thus, substrate molecules have to be translocated through the internal cavity to the catalytic sites. The X-ray crystallographic analysis has shown that the translocation channel is too narrow to allow passage of folded proteins. Protein substrates should be firstly unfolded and then degraded. Quite surprisingly, classical protein inhibitor of serine proteinases, bovine pancreatic trypsin inhibitor (BPTI) appeared to exert similar activity against proteaseome *in vitro* and *ex vivo* (Yabe & Koide, 2009). The molar ratio of BPTI to the proteasome 20S in the complex was estimated as approximately six to one, suggesting that two out of three proteinase activities of this complex were inhibited. This interesting finding has opened a new front in proteasome inhibition studies.

The majority of proteasome inhibitors have a structure of small cyclic and linear peptides built on scaffolds provided by natural substances. Lactacystin (Figure 18), produced by *Strepromyces* (Omura et. al., 1991), rearranges in neutral pH to highly reactive lactone-Omuralide, which irreversibly acylates proteasome active site threonine. Minute modification of the latter one led to the more potent inhibitor MNL-519 (Abbenante & Fairlie, 2005). Isolation of *Actinomycete* products – epoxomycin and eponemycin, and evaluation of their inhibitory activity (Hanada, et. al, 1992; Sugawara et al, 1990) has

The most significant step in development of proteasome inhibitors was the decision by A. L. Goldberg and colleagues to create in 1993 the company *MyoGenics.* The goal was to synthesize proteasome inhibitors that could prevent muscle atrophy that occur in various disease states, such as cancer cachexia. This led to the production of a series of inhibitors that were freely distributed to academic laboratories and contributed to the enormous leap

Fig. 17. Schematic structure of proteasome with indication of the binding site of bortezomib

The 20S proteasome is a tubular molecule with the proteolytic active sites on the inner surface. Thus, substrate molecules have to be translocated through the internal cavity to the catalytic sites. The X-ray crystallographic analysis has shown that the translocation channel is too narrow to allow passage of folded proteins. Protein substrates should be firstly unfolded and then degraded. Quite surprisingly, classical protein inhibitor of serine proteinases, bovine pancreatic trypsin inhibitor (BPTI) appeared to exert similar activity against proteaseome *in vitro* and *ex vivo* (Yabe & Koide, 2009). The molar ratio of BPTI to the proteasome 20S in the complex was estimated as approximately six to one, suggesting that two out of three proteinase activities of this complex were inhibited. This interesting finding

The majority of proteasome inhibitors have a structure of small cyclic and linear peptides built on scaffolds provided by natural substances. Lactacystin (Figure 18), produced by *Strepromyces* (Omura et. al., 1991), rearranges in neutral pH to highly reactive lactone-Omuralide, which irreversibly acylates proteasome active site threonine. Minute modification of the latter one led to the more potent inhibitor MNL-519 (Abbenante & Fairlie, 2005). Isolation of *Actinomycete* products – epoxomycin and eponemycin, and evaluation of their inhibitory activity (Hanada, et. al, 1992; Sugawara et al, 1990) has

**4.1 Inhibitors from natural sources** 

has opened a new front in proteasome inhibition studies.

forward in understanding the multiple roles of the proteasome in cells.

stimulated studies on their analogues (representative structure is shown in Fig. 18). This resulted in several potent inhibitors, which display non-typical mechanism of action (Elofsson et e., 1999; Zhou et el., 2009). A hemiacetal is first formed between the ketone portion of the inhibitor and threonine hydroxyl, followed by epoxide ring opening by the free amine of the N-terminal threonine to give a stable morpholino adduct.

Fig. 18. Natural inhibitors of proteasome activity as scaffolds for synthesis improved ones.

Next example considers syringolines, reversible inhibitors of proteasome produced by *Pseudomonas siringae* (Coleman, et al. 2006). Elucidation of the crystal structure of syringolin B complexed with proteasome gave an insight into the structural requirements of good inhibitor. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be one of the most potent proteasome inhibitors described so far (Clerc et el., 2009).

A limiting factor in the efficiency of peptidic inhibitors is that they are unstable in living organism because they are easily degraded by endogenous proteinases. This explains growing interest in non-peptidic inhibitors. Nature is an acknowledged source of such compounds and many inhibitors of proteasome were isolated and identified. These include such structurally diverse compounds as: ajoene isolated from garlic (Hassan, 2004), gliotoxin produced by *Aspergillus fumigatus* (Pahl et al., 1996), or triterpene-celastrol isolated from the root bark of medicinal plant *Tripterigium wolfordii* (Yang et al., 2006).

Inhibitors of Proteinases as Potential Anti-Cancer Agents 57

Searching for a new class of 20S proteasome inhibitors is a hot subject and to date a plethora of molecules that target the proteasome have been identified or designed (de Bettignies and Coux, 2010). Synthetic inhibitors possess a homogeneous structural profile - they are generally peptide-based compounds with a C-terminal pharmacophore function required for primary interaction with catalytic threonine of the enzyme. The peptide component seems to be important for determining specificity of the interactions with the enzymatic pockets. Essentially, most of these inhibitors act on the chymotrypsin-like activity of the

Protection of the aldehyde moiety in a form of semicarbazone provides compounds that are more stable than counterpart aldehydes. They do not form adducts with cellular proteins and are irreversible inhibitors of proteasome requiring the action of this enzymatic complex to release inhibiting aldehyde. Thus, they may be classified as suicidal inhibitors. Recently two peptide semicarbazones, S-2209 and SC68896, were found to exert anti-melanoma and anti-glioma activities in preclinical studies (Baumann et el., 2009: Leban et al., 2008; Roth et el., 2009). For the latter one company was given an approval to start phase I/II clinical

Structurally related *N*-acylpyrrole peptidyl derivatives were designed as irreversible inhibitors of proteasome. They appeared to possess unique biological profile and interact reversibly with β1 catalytic site of the proteasome also displaying good pharmacological properties (Baldisserotto et al., 2010). Molecular docking of the *N*-acylpyrrole molecule

The vinyl sulfone group is less reactive than the aldehyde group but also binds irreversibly to the active sites. The advantage of vinylsulfone inhibitors is that they are easy to prepare. One of the most potent inhibitor - Ada(Ahx)3-LLL-VFS, specifically and irreversibly inhibits both the constitutive and the induced proteasome by binding to their three active sites with

The screening of huge libraries of structurally variable compounds is a method for the identification of new cell-active inhibitors with novel chemical scaffolds. Such a procedure was also used in order to obtain new inhibitors of proteasome. Thus, a high-throughput screen of the Millennium Pharmaceuticals Inc. library (approximately 352,500 compounds) afforded 3015 hits, which were further optimized by applying X-ray crystallography and molecular modeling. In such manner 16 various structures were selected. They appear to exhibit high potency and selectivity towards β5 subunit of 20S proteasome. The crystal structures determined for the most active compounds (Fig. 20) enabled to determine the structural requirements of the inhibited subunit. Similar screening done on National Cancer Institute Diversity Set library composed of 1,992 compounds resulted in selection of four promising inhibitors of proteasome, with organocopper NCS 321206 (Fig.20) being the most

Different approaches to the selection of new inhibitors of proteasome relayed on the use of computational tools, namely multistep structure-based virtual ligand screening strategy. First scoring engines were standardized using known inhibitors in order to obtain results similar to those found from crystallographic studies. It appeared that none of the presently developed scoring functions are fully reliable nor they fully correlate with experimental affinities. Therefore three protocols were used simultaneously - FRED, LigandFit and Surflex, to dock 300,000 compound collection (Chembridge). This enabled to select 200 molecules for further experimental testing, using MG-132 as a standard. Twenty of these molecules appeared to act as potent proteasome inhibitors showing variable profiles of

proteasome although two remaining activities are also addressed.

shown in Figure 20 enabled to rationalize the mode of their binding.

approximately equal efficiencies (Kessler et al., 2001).

active one (Lavelin et al., 2009).

studies in 2011.

#### **4.2 Synthetic inhibitors**

The first inhibitors of proteasome were identified among the commercially available reversible tripeptide inhibitors of serine and cysteine proteinases. The easy access to the peptide aldehydes had lead to the development of a wide variety of inhibitors with an improved potency and selectivity. MG-132 (for its chemical structure see Figure 20) was one of the first synthetic inhibitors to be described and used in cell culture system (Adams & Stein, 1996). It exerts both, direct antiproliferative and cytotoxic effects towards tumor cells, and increases apoptosis induced by other agents. Recent studies have demonstrated the influence of absolute configuration of this tripeptide aldehyde on its cytotoxicity, with (*L,D,L*) isomer being the most active (Mroczkiewicz et al., 2010). Since a great number of tripeptide aldehydes contain side chains of non-coded amino acids but they usually correspond to natural *L-*amino acids this finding shed a new light on the importance of peptidyl absolute configuration.

Structurally related α-ketoaldehydes exert their action via mechanism similar to this described earlier for epoxyketones (Gräwert et al., 2011). This is a cyclization mechanism, which proceeds through formation of hemiketal with threonine hydroxyl followed by Schiff base formation between the nucleophilic *N*-terminal threonine and aldehyde moiety, which finally results in the reversible formation of a 5,6-dihydro-2*H*-1,4-oxazine ring (Figure 19). The examination of the binding mode of these inhibitors serves as a new lead for the development of anticancer drugs (Fig. 19).

Fig. 19. Molecular mechanism of action of α–ketoaldehyde inhibitor of proteasome and the mode of its binding in the active site.

The first inhibitors of proteasome were identified among the commercially available reversible tripeptide inhibitors of serine and cysteine proteinases. The easy access to the peptide aldehydes had lead to the development of a wide variety of inhibitors with an improved potency and selectivity. MG-132 (for its chemical structure see Figure 20) was one of the first synthetic inhibitors to be described and used in cell culture system (Adams & Stein, 1996). It exerts both, direct antiproliferative and cytotoxic effects towards tumor cells, and increases apoptosis induced by other agents. Recent studies have demonstrated the influence of absolute configuration of this tripeptide aldehyde on its cytotoxicity, with (*L,D,L*) isomer being the most active (Mroczkiewicz et al., 2010). Since a great number of tripeptide aldehydes contain side chains of non-coded amino acids but they usually correspond to natural *L-*amino acids this

Structurally related α-ketoaldehydes exert their action via mechanism similar to this described earlier for epoxyketones (Gräwert et al., 2011). This is a cyclization mechanism, which proceeds through formation of hemiketal with threonine hydroxyl followed by Schiff base formation between the nucleophilic *N*-terminal threonine and aldehyde moiety, which finally results in the reversible formation of a 5,6-dihydro-2*H*-1,4-oxazine ring (Figure 19). The examination of the binding mode of these inhibitors serves as a new lead for the

> H N

H2N O

Fig. 19. Molecular mechanism of action of α–ketoaldehyde inhibitor of proteasome and the

N <sup>H</sup> <sup>O</sup> O

N H H

H O

<sup>O</sup> OH OH

O

finding shed a new light on the importance of peptidyl absolute configuration.

O O N H

H O

**4.2 Synthetic inhibitors** 

development of anticancer drugs (Fig. 19).

O

H2N HO

mode of its binding in the active site.

H O

Searching for a new class of 20S proteasome inhibitors is a hot subject and to date a plethora of molecules that target the proteasome have been identified or designed (de Bettignies and Coux, 2010). Synthetic inhibitors possess a homogeneous structural profile - they are generally peptide-based compounds with a C-terminal pharmacophore function required for primary interaction with catalytic threonine of the enzyme. The peptide component seems to be important for determining specificity of the interactions with the enzymatic pockets. Essentially, most of these inhibitors act on the chymotrypsin-like activity of the proteasome although two remaining activities are also addressed.

Protection of the aldehyde moiety in a form of semicarbazone provides compounds that are more stable than counterpart aldehydes. They do not form adducts with cellular proteins and are irreversible inhibitors of proteasome requiring the action of this enzymatic complex to release inhibiting aldehyde. Thus, they may be classified as suicidal inhibitors. Recently two peptide semicarbazones, S-2209 and SC68896, were found to exert anti-melanoma and anti-glioma activities in preclinical studies (Baumann et el., 2009: Leban et al., 2008; Roth et el., 2009). For the latter one company was given an approval to start phase I/II clinical studies in 2011.

Structurally related *N*-acylpyrrole peptidyl derivatives were designed as irreversible inhibitors of proteasome. They appeared to possess unique biological profile and interact reversibly with β1 catalytic site of the proteasome also displaying good pharmacological properties (Baldisserotto et al., 2010). Molecular docking of the *N*-acylpyrrole molecule shown in Figure 20 enabled to rationalize the mode of their binding.

The vinyl sulfone group is less reactive than the aldehyde group but also binds irreversibly to the active sites. The advantage of vinylsulfone inhibitors is that they are easy to prepare. One of the most potent inhibitor - Ada(Ahx)3-LLL-VFS, specifically and irreversibly inhibits both the constitutive and the induced proteasome by binding to their three active sites with approximately equal efficiencies (Kessler et al., 2001).

The screening of huge libraries of structurally variable compounds is a method for the identification of new cell-active inhibitors with novel chemical scaffolds. Such a procedure was also used in order to obtain new inhibitors of proteasome. Thus, a high-throughput screen of the Millennium Pharmaceuticals Inc. library (approximately 352,500 compounds) afforded 3015 hits, which were further optimized by applying X-ray crystallography and molecular modeling. In such manner 16 various structures were selected. They appear to exhibit high potency and selectivity towards β5 subunit of 20S proteasome. The crystal structures determined for the most active compounds (Fig. 20) enabled to determine the structural requirements of the inhibited subunit. Similar screening done on National Cancer Institute Diversity Set library composed of 1,992 compounds resulted in selection of four promising inhibitors of proteasome, with organocopper NCS 321206 (Fig.20) being the most active one (Lavelin et al., 2009).

Different approaches to the selection of new inhibitors of proteasome relayed on the use of computational tools, namely multistep structure-based virtual ligand screening strategy. First scoring engines were standardized using known inhibitors in order to obtain results similar to those found from crystallographic studies. It appeared that none of the presently developed scoring functions are fully reliable nor they fully correlate with experimental affinities. Therefore three protocols were used simultaneously - FRED, LigandFit and Surflex, to dock 300,000 compound collection (Chembridge). This enabled to select 200 molecules for further experimental testing, using MG-132 as a standard. Twenty of these molecules appeared to act as potent proteasome inhibitors showing variable profiles of

Inhibitors of Proteinases as Potential Anti-Cancer Agents 59

cancer. Interestingly, recent studies have indicated that this drug is a multiple inhibitor and affects also serine proteinases in cell lysates (Arastu-Kapur et al., 2011). This finding may explain better the clinical profile of this drug. Alongside with physiologic studies synthesis and evaluation of inhibitory activity of its analogues have been carried out. Although in some cases inhibitors of similar potency were obtained (Aubin et al., 2005; Vivier et al., 2005;

This is the smallest family of proteinases, which accounts for only 3% of them and includes several physiologically important enzymes such as pepsin, chymosin, renin, gastricsin, cathepsin D and cathepsin E. Some members of this family, in particular cathepsins D and E, have been implicated in cancer progression. High cathepsin D expression is associated with shorter disease-free and overall survival in patients with breast cancer, whereas in patients with ovarian or endometrial cancer, cathepsin E expression has been reported to be

Quite surprisingly, the aspartic protease napsin A, expressed in lung cells, where it is involved in the processing of surfactant protein B, suppressed tumor growth in HEK293 cells in a manner independent of its catalytic activity (Ueno et al., 2008). Further insight into

The most extensively investigated aspartic proteinase in the context of cancer is cathepsin D, with a particular emphasis on its role in breast cancer (Benes et al, 2008). In these studies several inhibitors of this enzyme are most commonly used including peptidomimetic pepstatin (Umezawa et al., 1970) and protein inhibitors from potato and tomato (Carter et al., 2002). Search for new inhibitors of this enzyme is practically limited to peptides containing non-typical amino acid – statine. Inhibitors of this type were obtained from both natural sources as well as were synthesized basing on the crystal structure of pepstatin A (Fig. 21) complexed by this enzyme. Statine, which is a component of pepstatin A, may be considered as an analogue of tetrahedral intermediate (or transition-state) of the enzymatic hydrolysis of *L-*leucylglycine (Fig. 21). Therefore it is not surprising that most of cathepsin D inhibitors contain this amino acid or its analogue within peptidic chain (Bi et al., 2000; McConnell et al., 2003). Of special interest are grassystatins (Fig. 21) isolated from cyanobacterium *Lyngbya* cf. *confervoides*. These peptidomimetics are equally active against

The new approach to the identification of inhibitors is appropriate selection of DNA aptamers strongly interacting with chosen enzyme. This methodology was used to identify the aptamer SF-6-3, which selectively and very strongly binds cathepsin E (Naimuddin et

Metalloproteinases are the largest class of proteinases in human genome. They are a range of enzymes possessing metal ions in their active sites. Most of them are dependent on zinc ions, which play catalytic functions. Understanding their mechanism of action is of key importance to rational design of potent and specific inhibitors of these enzymes and, consequently, to obtain drugs of improved properties. Therefore, a substantial effort has been made to study the mode of binding of their substrates and inhibitors, as well as to elucidate the three dimensional structure of these enzymes and to define the detailed

Zhu et al, 2010) none of them was found to be better than bortezomib.

mechanism involved may help in producing new drugs for renal cancer.

**5. Aspartic proteinases** 

associated with tumor aggressiveness.

cathepsins D and E (Kwan et al., 2009).

al., 2007).

**6. Metalloproteinases** 

activity. Thus six of them inhibited all three activities of proteasome, eleven of them inhibited two types of enzymatic activities, whereas three inhibited only one type of activity (Basse et al., 2010). The most active and selective inhibitors against chymotrypsin-like and trypsin-like activities are shown in Figure 20.

Fig. 20. Structurally diverse, synthetic inhibitors of proteasome.

The discovery of bortezomib was followed by intensive preclinical and clinical studies on many cancer models and cancer patients. This drug was approved in 2003 for treatment of multiple myeloma as a second line of the therapy. Today it is taken by approximately 50,000 patients worldwide (Goldberg, 2007) and is still being tested clinically against other forms of cancer. Interestingly, recent studies have indicated that this drug is a multiple inhibitor and affects also serine proteinases in cell lysates (Arastu-Kapur et al., 2011). This finding may explain better the clinical profile of this drug. Alongside with physiologic studies synthesis and evaluation of inhibitory activity of its analogues have been carried out. Although in some cases inhibitors of similar potency were obtained (Aubin et al., 2005; Vivier et al., 2005; Zhu et al, 2010) none of them was found to be better than bortezomib.

## **5. Aspartic proteinases**

58 Drug Development – A Case Study Based Insight into Modern Strategies

activity. Thus six of them inhibited all three activities of proteasome, eleven of them inhibited two types of enzymatic activities, whereas three inhibited only one type of activity (Basse et al., 2010). The most active and selective inhibitors against chymotrypsin-like and

trypsin-like activities are shown in Figure 20.

Fig. 20. Structurally diverse, synthetic inhibitors of proteasome.

The discovery of bortezomib was followed by intensive preclinical and clinical studies on many cancer models and cancer patients. This drug was approved in 2003 for treatment of multiple myeloma as a second line of the therapy. Today it is taken by approximately 50,000 patients worldwide (Goldberg, 2007) and is still being tested clinically against other forms of This is the smallest family of proteinases, which accounts for only 3% of them and includes several physiologically important enzymes such as pepsin, chymosin, renin, gastricsin, cathepsin D and cathepsin E. Some members of this family, in particular cathepsins D and E, have been implicated in cancer progression. High cathepsin D expression is associated with shorter disease-free and overall survival in patients with breast cancer, whereas in patients with ovarian or endometrial cancer, cathepsin E expression has been reported to be associated with tumor aggressiveness.

Quite surprisingly, the aspartic protease napsin A, expressed in lung cells, where it is involved in the processing of surfactant protein B, suppressed tumor growth in HEK293 cells in a manner independent of its catalytic activity (Ueno et al., 2008). Further insight into mechanism involved may help in producing new drugs for renal cancer.

The most extensively investigated aspartic proteinase in the context of cancer is cathepsin D, with a particular emphasis on its role in breast cancer (Benes et al, 2008). In these studies several inhibitors of this enzyme are most commonly used including peptidomimetic pepstatin (Umezawa et al., 1970) and protein inhibitors from potato and tomato (Carter et al., 2002). Search for new inhibitors of this enzyme is practically limited to peptides containing non-typical amino acid – statine. Inhibitors of this type were obtained from both natural sources as well as were synthesized basing on the crystal structure of pepstatin A (Fig. 21) complexed by this enzyme. Statine, which is a component of pepstatin A, may be considered as an analogue of tetrahedral intermediate (or transition-state) of the enzymatic hydrolysis of *L-*leucylglycine (Fig. 21). Therefore it is not surprising that most of cathepsin D inhibitors contain this amino acid or its analogue within peptidic chain (Bi et al., 2000; McConnell et al., 2003). Of special interest are grassystatins (Fig. 21) isolated from cyanobacterium *Lyngbya* cf. *confervoides*. These peptidomimetics are equally active against cathepsins D and E (Kwan et al., 2009).

The new approach to the identification of inhibitors is appropriate selection of DNA aptamers strongly interacting with chosen enzyme. This methodology was used to identify the aptamer SF-6-3, which selectively and very strongly binds cathepsin E (Naimuddin et al., 2007).

## **6. Metalloproteinases**

Metalloproteinases are the largest class of proteinases in human genome. They are a range of enzymes possessing metal ions in their active sites. Most of them are dependent on zinc ions, which play catalytic functions. Understanding their mechanism of action is of key importance to rational design of potent and specific inhibitors of these enzymes and, consequently, to obtain drugs of improved properties. Therefore, a substantial effort has been made to study the mode of binding of their substrates and inhibitors, as well as to elucidate the three dimensional structure of these enzymes and to define the detailed

Inhibitors of Proteinases as Potential Anti-Cancer Agents 61

HO <sup>N</sup> H O

N

O

tanomastat (*Bayer*) Fig. 22. Matrix metalloproteinase inhibitors, which failed in clinical studies. In parentheses

Clinical studies indicated that timeframe of targeting MMPs differs, depending on the stage of cancer, because the expression profile, as well as the activity of these enzymes, is not the same in the early stage compared to advanced cancer disease. As a consequence, the use of broad-spectrum inhibitors raises concerns that certain MMPs that exert anticancer effects are inhibited, which in turn may result in promotion of the disease (Gialeli et al., 2011). Thus, pharmacological targeting of cancer by the development of a new generation of effective and selective inhibitors to individual matrix metalloproteinases is an emerging and promising area of research (Devel et al., 2010; Manello, 2006). However, despite intense efforts, very few highly selective inhibitors of these metalloproteinases have been discovered up to now. This is because MMPs have catalytic domains composed of 160–170 amino acid residues that share a marked sequence similarity, with the percentage of identical residues being in the range of 33% to 90%. The three dimensional structure of the catalytic domains of 12 out of 23 human MMPs has been solved either by X-ray crystallography or NMR (Maskos, 2005), and the results supported that they are of significant similarity. The other cause of low specificity of most of MMP inhibitors is that their action relays on strong complexation of zinc ion present in the active sites of these enzymes. This is especially true in the case of hydoxamic acid-based inhibitors (Yiotakis & Dive, 2008), which are the most intensively studied so far (Attolino et al., 2010; Fisher &

Among different zinc-binding groups, the phosphoryl moiety was thought to be the weakest binder. Indeed, it turns out that numerous peptide analogues with a phosphorus-containing moiety replacing the scissile amide bond have been found to regulate the activity of

O

COOH S

O SH

N

H <sup>N</sup> <sup>S</sup>

O

O

prinomastat (*Aguron*)

H N

rebimastat (*Bristol-Meyers Squibb*)

O

O

N H

O

O

H N

O

N

S O

HO <sup>N</sup> H

HO <sup>N</sup> H O

Mobashery, 2006; Nuti et al., 2010).

O

S S H <sup>N</sup> <sup>N</sup> H

O

O

batimastat (*British Biotech*)

OH O

marimastat (*British Biotech*)

H <sup>N</sup> <sup>N</sup> H

O

Cl

companies, which introduced these compounds are given.

mechanisms of catalyzed reactions. Despite extensive experimental and theoretical studies the mechanism by which the catalytic center of metalloproteinases functions is still the subject of debate and several mechanism have been proposed (Mucha et al., 2010).

Fig. 21. Pepstatin A and grassystatin as transition-state analogues of peptide hydrolysis.

Matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs, adamalysins) and tissue inhibitors of metalloproteinases (TIMPs) together comprise an important set of proteins that are regulatory in matrix turnover and regulate growth factor bioavailability. There are 23 MMP, 32 ADAM and 4 TIMP proteins present in humans. This shows how complex system is involved in tumorigenesis and its regulation. For example, four tissue inhibitors of metalloproteinases (TIMP1, TIMP2, TIMP3 and TIMP4) are the main endogenous inhibitors for all the metallo-endopeptidases, of which there are more than 180.

#### **6.1 Matrix metalloproteinases**

Matrix metalloproteinases (MMPs) consist of a multigene family of zinc dependent extracellular endopeptidases implicated in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of extracellular matrix, alteration of the cell–cell and cell–matrix interactions, cell migration and angiogenesis (Gialeli et al., 2011). These structurally and functionally related endoproteinases share common functional domains and activation mechanisms. The MMPs were the first proteinase targets seriously considered for combating cancer. After encouraging preclinical results in various cancer models several of the MMP inhibitors were tested in advanced clinical trials but all failed because of severe side effects or no major clinical benefit (Turk et el., 2006). These include: hydroxamate inhibitors batimastat, marimastat, and prinomastat and the non-hydroxamate ones such as neovastat (an extract from shark cartilage of a molecular mass up to 500kDa introduced by Aeterna) rebimastat and tanomastat (Fig. 22).

mechanisms of catalyzed reactions. Despite extensive experimental and theoretical studies the mechanism by which the catalytic center of metalloproteinases functions is still the

> N H

pepstatin A

N H

> N H

CONH2

grassystatin

O

OH <sup>O</sup>

O

H

H N O O

OH O

N COOH

OH

H N

OH O

N H

N O

O N

COOH

O

subject of debate and several mechanism have been proposed (Mucha et al., 2010).

H N

O

<sup>N</sup> <sup>N</sup> <sup>H</sup> <sup>H</sup>

H N

N H

introduced by Aeterna) rebimastat and tanomastat (Fig. 22).

O N

<sup>H</sup> HO OH

N H

O

H N

O

Fig. 21. Pepstatin A and grassystatin as transition-state analogues of peptide hydrolysis.

Matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs, adamalysins) and tissue inhibitors of metalloproteinases (TIMPs) together comprise an important set of proteins that are regulatory in matrix turnover and regulate growth factor bioavailability. There are 23 MMP, 32 ADAM and 4 TIMP proteins present in humans. This shows how complex system is involved in tumorigenesis and its regulation. For example, four tissue inhibitors of metalloproteinases (TIMP1, TIMP2, TIMP3 and TIMP4) are the main endogenous inhibitors for all the metallo-endopeptidases, of which there are more than 180.

Matrix metalloproteinases (MMPs) consist of a multigene family of zinc dependent extracellular endopeptidases implicated in tumor growth and the multistep processes of invasion and metastasis, including proteolytic degradation of extracellular matrix, alteration of the cell–cell and cell–matrix interactions, cell migration and angiogenesis (Gialeli et al., 2011). These structurally and functionally related endoproteinases share common functional domains and activation mechanisms. The MMPs were the first proteinase targets seriously considered for combating cancer. After encouraging preclinical results in various cancer models several of the MMP inhibitors were tested in advanced clinical trials but all failed because of severe side effects or no major clinical benefit (Turk et el., 2006). These include: hydroxamate inhibitors batimastat, marimastat, and prinomastat and the non-hydroxamate ones such as neovastat (an extract from shark cartilage of a molecular mass up to 500kDa

N H

O

H N O N

O

**6.1 Matrix metalloproteinases** 

O

Fig. 22. Matrix metalloproteinase inhibitors, which failed in clinical studies. In parentheses companies, which introduced these compounds are given.

tanomastat (*Bayer*)

Clinical studies indicated that timeframe of targeting MMPs differs, depending on the stage of cancer, because the expression profile, as well as the activity of these enzymes, is not the same in the early stage compared to advanced cancer disease. As a consequence, the use of broad-spectrum inhibitors raises concerns that certain MMPs that exert anticancer effects are inhibited, which in turn may result in promotion of the disease (Gialeli et al., 2011). Thus, pharmacological targeting of cancer by the development of a new generation of effective and selective inhibitors to individual matrix metalloproteinases is an emerging and promising area of research (Devel et al., 2010; Manello, 2006). However, despite intense efforts, very few highly selective inhibitors of these metalloproteinases have been discovered up to now. This is because MMPs have catalytic domains composed of 160–170 amino acid residues that share a marked sequence similarity, with the percentage of identical residues being in the range of 33% to 90%. The three dimensional structure of the catalytic domains of 12 out of 23 human MMPs has been solved either by X-ray crystallography or NMR (Maskos, 2005), and the results supported that they are of significant similarity. The other cause of low specificity of most of MMP inhibitors is that their action relays on strong complexation of zinc ion present in the active sites of these enzymes. This is especially true in the case of hydoxamic acid-based inhibitors (Yiotakis & Dive, 2008), which are the most intensively studied so far (Attolino et al., 2010; Fisher & Mobashery, 2006; Nuti et al., 2010).

Among different zinc-binding groups, the phosphoryl moiety was thought to be the weakest binder. Indeed, it turns out that numerous peptide analogues with a phosphorus-containing moiety replacing the scissile amide bond have been found to regulate the activity of

Inhibitors of Proteinases as Potential Anti-Cancer Agents 63

information about the role of carboxypeptidases in tumorigenesis. However, some of them were proposed as markers of individual tumors (Kemik et al., 2011; Lee et al., 2011). This indicates that they also might be considered as targets in anticancer therapy. Indeed, there are two reports on antitumor activity of two endogenous protein inhibitors of carboxypeptidases – latexin (Pallares et al., 2005) and retinoic acid-induced tumor

> O N H

selective towards MMP-9

N N <sup>N</sup> <sup>N</sup>

HOOC

H

OH

O

bestatin

Fig. 24. Bestatin and tosedostat – general inhibitors of aminopeptidases and promising

N COOH

<sup>O</sup> <sup>H</sup>

N P O HO O O

N

O

selective towards MMP-13

H N

COOH <sup>N</sup>

OH O

H

HO

O

CHR-79888

NH

H N COOH

F

NH

suppressor retinoic acid receptor responder 1 (RARRES 1) (Sahab et al., 2011).

N CONH2

H

COOH N

Cl

H2N

O

O

H N

OH O

O

O

N N HN NH O O

selective towards MMP-13

Fig. 23. Selective MMP inhibitors.

N H

HO

anticancer drugs.

O

tosedostat, CHR-2797

F F

COOH

P N H

HO O

selective towards MMP-12

Br <sup>O</sup>

metalloproteinases (Collinsová & Jiraček, 2000). The intense optimization of the phosphinic inhibitor structures, using parallel or combinatorial chemistry, is generally required to identify nanomolar inhibitors and to get selectivity (Dive et al., 2004). Without selective inhibitors, which are indispensable tools for studying the structure and the role of individual enzymes at different stages of complex tumorigenesis, anticancer strategies based on MMP inhibition are unlikely to provide important therapeutic benefits. Two representative inhibitors of this class of inhibitors are shown in Figure 23.

High-throughput screening of chemical libraries has also led to the discovery of unusual MMP inhibitors, selective against MMP-13. Among these, a new class of MMP inhibitors that do not possess a zinc-binding group and thus do not interact directly with the zinc active site ion is of special interest (Fig. 23) (Chen et al., 2000).

## **6.2 Aminopeptidases**

Aminopeptidases are proteolytic enzymes that hydrolyze peptide bonds from the amino termini of polypeptide chains with the release of a single amino acid residue from polypeptide substrates. Although their involvement in tumorigenesis was well established the studies on their anticancer properties are far less developed than studies on MMPs. This may also result from the fact that physiologic role of these enzymes is far more complex.

A plethora of inhibitors of aminopeptidases have been synthesized and tested clinically against various pathological disorders, including cancers (Bauvois & Dauzonne, 2006; Mucha et al., 2010; Selvakumar et al., 2006; Wickström et al., 2011). Bestatin, a general inhibitor of aminopeptidases and aspartyl proteinases, has been the most intensively studied (Fig. 24). It was originally isolated from *Streptomyces olivoreticuli* more than 30 years ago (Umezawa et al., 1976). Bestatin studies in biological systems both *in vitro* and *in vivo*, resulted in discovery of several interesting properties of this compound such as ability to induce apoptosis in cancer cells, and anti-angiogenic, anti-malarial or immunomodulatory effects. Presently, bestatin (Ubenimex®) is on Japanese market where it is applied for treatment of cancer and bacterial infections. Examples of successful inhibition of aminopeptidases by bestatin include aminopeptidase N (CD13), leucine aminopeptidase (LAP) and aminopeptidase B. These aminopeptidases, as well as methionine aminopeptidase 2 are the most exploited targets to obtain new anticancer agents.

In contrast to MMPs selectivity of the inhibitor is not a required feature and in most cases general inhibitors of aminopeptidases are used in clinical studies. Such an example is tosedostat (Fig. 24) (Krige et. al, 2008; Moore et al., 2009), a hydroxamic acid inhibitor of M1 family of aminopeptidases (especially leucine aminopeptidase), which is now being introduced to the market by Chroma Therapeutics. In clinical studies tosedostat was well tolerated, given orally once a day, and it has produced encouraging response rates in difficult to treat patients with acute leukemia and a variety of blood related cancers. Tosedostat (CHR-2797) is a prodrug and exposure of cancer cells to this drug results in the generation of the active metabolite CHR-79888 (Fig. 24), which is poorly membranepermeable, what limits its pharmacological activity. The use of prodrug results in intracellular accumulation of CHR-79888 and desirable physiological effect.

#### **6.3 Carboxypeptidases**

Carboxypeptidases cleave the peptide bond of amino acid residue at the carboxylic terminus of protein or peptide. Humans contain several types of carboxypeptidases, which have diverse functions ranging from catabolism to protein maturation. There is practically lack of

metalloproteinases (Collinsová & Jiraček, 2000). The intense optimization of the phosphinic inhibitor structures, using parallel or combinatorial chemistry, is generally required to identify nanomolar inhibitors and to get selectivity (Dive et al., 2004). Without selective inhibitors, which are indispensable tools for studying the structure and the role of individual enzymes at different stages of complex tumorigenesis, anticancer strategies based on MMP inhibition are unlikely to provide important therapeutic benefits. Two

High-throughput screening of chemical libraries has also led to the discovery of unusual MMP inhibitors, selective against MMP-13. Among these, a new class of MMP inhibitors that do not possess a zinc-binding group and thus do not interact directly with the zinc

Aminopeptidases are proteolytic enzymes that hydrolyze peptide bonds from the amino termini of polypeptide chains with the release of a single amino acid residue from polypeptide substrates. Although their involvement in tumorigenesis was well established the studies on their anticancer properties are far less developed than studies on MMPs. This may also result from the fact that physiologic role of these enzymes is far more complex. A plethora of inhibitors of aminopeptidases have been synthesized and tested clinically against various pathological disorders, including cancers (Bauvois & Dauzonne, 2006; Mucha et al., 2010; Selvakumar et al., 2006; Wickström et al., 2011). Bestatin, a general inhibitor of aminopeptidases and aspartyl proteinases, has been the most intensively studied (Fig. 24). It was originally isolated from *Streptomyces olivoreticuli* more than 30 years ago (Umezawa et al., 1976). Bestatin studies in biological systems both *in vitro* and *in vivo*, resulted in discovery of several interesting properties of this compound such as ability to induce apoptosis in cancer cells, and anti-angiogenic, anti-malarial or immunomodulatory effects. Presently, bestatin (Ubenimex®) is on Japanese market where it is applied for treatment of cancer and bacterial infections. Examples of successful inhibition of aminopeptidases by bestatin include aminopeptidase N (CD13), leucine aminopeptidase (LAP) and aminopeptidase B. These aminopeptidases, as well as methionine

aminopeptidase 2 are the most exploited targets to obtain new anticancer agents.

intracellular accumulation of CHR-79888 and desirable physiological effect.

In contrast to MMPs selectivity of the inhibitor is not a required feature and in most cases general inhibitors of aminopeptidases are used in clinical studies. Such an example is tosedostat (Fig. 24) (Krige et. al, 2008; Moore et al., 2009), a hydroxamic acid inhibitor of M1 family of aminopeptidases (especially leucine aminopeptidase), which is now being introduced to the market by Chroma Therapeutics. In clinical studies tosedostat was well tolerated, given orally once a day, and it has produced encouraging response rates in difficult to treat patients with acute leukemia and a variety of blood related cancers. Tosedostat (CHR-2797) is a prodrug and exposure of cancer cells to this drug results in the generation of the active metabolite CHR-79888 (Fig. 24), which is poorly membranepermeable, what limits its pharmacological activity. The use of prodrug results in

Carboxypeptidases cleave the peptide bond of amino acid residue at the carboxylic terminus of protein or peptide. Humans contain several types of carboxypeptidases, which have diverse functions ranging from catabolism to protein maturation. There is practically lack of

representative inhibitors of this class of inhibitors are shown in Figure 23.

active site ion is of special interest (Fig. 23) (Chen et al., 2000).

**6.2 Aminopeptidases** 

**6.3 Carboxypeptidases** 

information about the role of carboxypeptidases in tumorigenesis. However, some of them were proposed as markers of individual tumors (Kemik et al., 2011; Lee et al., 2011). This indicates that they also might be considered as targets in anticancer therapy. Indeed, there are two reports on antitumor activity of two endogenous protein inhibitors of carboxypeptidases – latexin (Pallares et al., 2005) and retinoic acid-induced tumor suppressor retinoic acid receptor responder 1 (RARRES 1) (Sahab et al., 2011).

selective towards MMP-13

Fig. 23. Selective MMP inhibitors.

Fig. 24. Bestatin and tosedostat – general inhibitors of aminopeptidases and promising anticancer drugs.

Inhibitors of Proteinases as Potential Anti-Cancer Agents 65

Attolino, E.; Calderone, V.; Dragoni, E.; Fragai, M.; Richichi, B.; Luchinat, C. & Nativi, C.

Aubin, S.; Martin, B.; Delcros, J.-G.; Arlot-Bonnemains, J & Baudy-Floc'h, M. (2005) Retro

Baldisserotto, A.; Ferretta, V.; Destro, F.; Franceschini, C.; Marastoni, M.; Gavioli, R. &

Basse, N.; Piguel, S.; Papapostolu, D.; Ferrier-Berthelot, A.; Richy, N.; Pagano, M.; Sarthou,

Baumann, P.; Müller, K.; Mandl-Weber, S.; Leban, J.; Doblhofer, R.; Ammendola, A.;

Bauvois, B. & Dauzonne, D. (2006) Aminopeptidase N/CD13 (EC 3.4.11.2) Inhibitors:

Bell-McGuinn, K. M.; Garfall, A. M.; Bogyo, M.; Hanahan, D. & Joyce, J. A. (2007) Inhibition

Benes, P.; Vetwicka, V.& Fusek, M. (2008) Cathepsin D – Many Functions of One Aspartic Protease. *Critical Reviews in Oncology/Hemathology* Vol. 68 (No. 1): 12-28. Berdowska, I. (2004) Proteases as Disease Markers. *Clinica Chimica Acta* Vol. 342 (No. 1-2):

Bhongade, B. A. & Gadad, A. K. (2006) Insight into the Structural Requirements of

45 (No. 12): 5919-5925.

(No. 5): 1638-1650.

*Chemistry* Vol. 53 (No. 1): 509-513

Vol. 144 (No. 6): 875-886.

41-69.

*Reviews* Vol. 26 (No. 1): 88-130.

Cancer. *Cancer Research* Vol. 67 (No. 15): 7378-7385.

20S β5-Subunit, *Biochemical Journal* Vol. 430 (No. 3): 461-476.

*Medicinal Chemistry* Vol. 48 (No. 1): 330-334.

(2010) Structure-based Approach to Nanomolar, Water Soluble Matrix Metalloproteinases Inhibitors (MMPIs). *European Journal of Medicinal Chemistry* Vol.

Hydrazino-azapeptoids as Peptidomimetics of Proteasome Inhibitors. *Journal of* 

Tomatis, R. (2010) α,β-Unstaurated *N-*Acylpyrrole Peptidyl Derivatives: New Proteasome Inhibitors. *Journal of Medicinal Chemistry* Vol. 53 (No. 17): 6511-6515. Barbosa, J. A. R. G.; Silva, R. P.; Teles, R. C. L.; Esteves, G. F.; Azevedo, R. B.; Ventura, M. M.

& de Freitas, S. M. (2007) Crystal Structure of tyhe Bowman-Birk Inhibitor from *Vigna unguiculata* Seeds in Complex with β-Chymotripsin at 1,55 Å Resolution and Its Structural Properties in Association with Proteinases. *Biophysical Journal* Vol. 92

P.; Sobczak-Thépot, J.; Reboud-Ravaux, M. & Vidal, J. (2007) Linear TMC-95 Based Proteasome Inhibitors. *Journal of Medicinal Chemistry* Vol. 50 (No. 12): 2842-2850. Basse, N.; Montes, M.; Maréchal, X.; Qin, L.; Bouvier-Durand, M.; Genin, E.; Vidal, J. ;

Villoutreix, B. O. & Reboud-Ravaux, M. (2010) Novel Organic Proteasome Inhibitors Idnetified by Virtual and in Vitro Screening. *Journal of Medicinal* 

Baumgartner, R.; Oduncu, F. & Schmidmaier, R. (2009) The Peptide-semicarbazone S-2209, a Representative of New Class of Proteasome Inhibitors, Induces Apoptosis and Cell Growth Arrest in Multiple Myeloma Cells. *British Journal of Haemathology* 

Chemistry, Biological Evaluations and Therapeutic Prospects. *Medicinal Research* 

of Cysteine Cathepsin Protease Activity Enhances Chemotherapy Regimens by Decreasing Tumor Growth and Invasiveness in a Mouse Model of Multistage

Urokinase-Type; Plasminogen Activator Inhibitors Based on 3D QSAR CoMFA/CoMSIA Models, *Journal of Medicinal Chemistry* Vol. 49 (No. 2): 475-489. Blackburn, C.; Gigstad, K. M.; Hales, P.; Garcia, K.; Jones, M.; Bruzzese, M. J.; Barrett, C.;

Liu, J. X.; Soucy, T. A.; Sappal, D. S.; Bump, N.; Olhava, E. J.; Fleming, P.; Dick, L. R.; Tsu, C.; Sintchak, M. D. & Blank J. L. (2010) Characterization of a New Series of Non-Covalent Proteasome Inhibitors with Exquisite Potency and Selectivity for the

## **7. Conclusion**

Looking back at the progress made with anticancer therapies using inhibitors of various proteinases it is hard to consider it as particularly successful. Today the major successful areas in protease-targeted therapies are the cardiovascular, inflammatory and infectious diseases (mostly anti-HIV), however, the intensive studies on therapies of cancer and neurodegradative disorders are predicted. This is a good prognosis if taking into account that the annual spending for protease-directed drugs amounts close to US\$ 10 billion annually (Turk, et al., 2006).

It is worth to note that past drug failures are not worthless. They provide not only invaluable lessons but are also a useful resource of data, which could still be used.

In order to achieve more satisfactory results, better understanding of the proteolytic network in tumor envinroment and increased knowledge in protease biology based on comprehensive analysis of protease activity in physiologically relevant conditions are required. The fact that tumor cells are only one part of the tumor environment and that extracellular matrix components and stromal cells are important contributors to the proteolytic activity of tumors should also be taken into consideration. For example, the use of transgenic animals may help in elucidation of the role of individual components of this complex networks.

Also the techniques of inhibitor design are developing significantly with *in silico* structurebased ligand design and various types of high-throughput screening being the major ones. Today's strategy in inhibitor design is to provide compounds complementary to active sites of the inhibited proteins, while the other concepts are used scarcely. One of the solutions is to design allosteric inhibitors altering proteinase activity by binding outside the enzyme active site, most likely in the cavity lacking any physiological role. The development of computer-aided methods for drug design (especially docking procedures) might be very helpful in this respect.

## **8. Acknowledgment**

This work is dedicated to Professor Francisco Palacios from University of Basque Country at Vitoria on the occasion of his 60th birthday.

Authors thank Polish Ministry of Science and Higher Education for financial support.

## **9. References**


Looking back at the progress made with anticancer therapies using inhibitors of various proteinases it is hard to consider it as particularly successful. Today the major successful areas in protease-targeted therapies are the cardiovascular, inflammatory and infectious diseases (mostly anti-HIV), however, the intensive studies on therapies of cancer and neurodegradative disorders are predicted. This is a good prognosis if taking into account that the annual spending for protease-directed drugs amounts close to US\$ 10 billion

It is worth to note that past drug failures are not worthless. They provide not only

In order to achieve more satisfactory results, better understanding of the proteolytic network in tumor envinroment and increased knowledge in protease biology based on comprehensive analysis of protease activity in physiologically relevant conditions are required. The fact that tumor cells are only one part of the tumor environment and that extracellular matrix components and stromal cells are important contributors to the proteolytic activity of tumors should also be taken into consideration. For example, the use of transgenic animals may help in elucidation of the role of individual components of this

Also the techniques of inhibitor design are developing significantly with *in silico* structurebased ligand design and various types of high-throughput screening being the major ones. Today's strategy in inhibitor design is to provide compounds complementary to active sites of the inhibited proteins, while the other concepts are used scarcely. One of the solutions is to design allosteric inhibitors altering proteinase activity by binding outside the enzyme active site, most likely in the cavity lacking any physiological role. The development of computer-aided methods for drug design (especially docking procedures) might be very

This work is dedicated to Professor Francisco Palacios from University of Basque Country at

Abbenante, G.; Fairlie, D. P. (2005) Protease Inhibitors in the Clinic. *Medicinal Chemistry* Vol.

Adams, J. (2002) Development of the Proteasome Inhibitor PS-341. *Oncologist* Vol. 7 (No. 1):

Adams, J. & Stein, R. (1996) Novel Inhibitors of the Proteasome and Their Therepeutic Use in Inflammation. *Annual Reports in Medicinal Chemistry* Vol. 31: 279-288. Arastu-Kapur, S.; Anderl, J. L.; Karus, M.; Parlati, F.; Shenk, K. D.; Lee, S. J.; Muchamuel, T.;

Adverse Effects. *Clinical Cancer Research* Vol. 17 (No. 9): 2734-2743.

Bennett, M. K.; Driessen, C.; Ball, A. J. & Kirk, C. J. (2011) Non-proteasomal Targets of the Proteasome Inhibitors Bortezomib and Carfilzomib: a Link to Clinical

Authors thank Polish Ministry of Science and Higher Education for financial support.

invaluable lessons but are also a useful resource of data, which could still be used.

**7. Conclusion** 

annually (Turk, et al., 2006).

complex networks.

helpful in this respect.

**8. Acknowledgment** 

**9. References** 

9-16.

Vitoria on the occasion of his 60th birthday.

1 (No. 1): 71-104


Inhibitors of Proteinases as Potential Anti-Cancer Agents 67

Collinsová, M. & Jiraček, J. (2000) Phosphinic Acid Compounds in Biochemistry, Biology

De Bettignies, G. & Coux, O. (2010) Proteasome Inhibitors: Dozens of Molecules and Still

Decros, J. G.; Baudy Floc'h, M.; Prignet, C. & Arlot-Bonnemais, Y. (2003) Proteasome

Devel, L.; Czarny, B.; Beau, F.; Georgiadis, D.; Stura, E. & Dive, V. (2010) Third Generation

Dia, V. P. & de Mejia, E. G. (2010) Lunasin Promotes Apoptosis in Human Colon Cancer

Dive, V.; Georgiadis, D.; Matziari, M.; Makaritis, A.; Beau, F.; Cuniasse, P. & Yiotakis, A.

Elie, B. T.; Gocheva, V.; Shree, T.; Dalrymple, S. A. Holsinger, L. J. & Joyce, J. A. (2010)

Elofsson, G.; Splittgerber, U.; Myung, J.; Mohan, R. & Crews, C. M. (1999) Towards Subunit-

Fayard, B.; Bianchi, F.; Dey, J.; Moreno, E.; Djaffer, S.; Hynes, N. E. & Monard, D. (2009) The

Fleber, L. M.; Kündig, C.; Borgoño, C. A.; Chagas, J. R.; Tasinato, A.; Jichlinski, P.; Gygi, C.

Fiorucci, L. & Ascoli, F. (2004) Mast Cell Tryptase, a Still Enigmatic Enzyme. *Cellular and* 

Fisher, J. J. & Mobashery, S. (2006) Recent Advances in MMP Inhibitor Design. *Cancer* 

Gialeli, C.; Theocharis, A. D. & Karamanos, N. K. (2011) Roles of Matrix Metalloproteinases

Goldberg, A. (2007) Functions of the Proteasome: from Protein Degradation to Immune

Gräwert, M. A.; Gallastegui, N.; Stein, M.; Schmidt, B.; Kloetzel, P.-M.; Huber, R. & Groll, M.

Inhibitors as Therapeutic Agents: *Current Medicinal Chemistry* Vol. 10 (No.6): 479-

of Matrix Matalliproteinase Inhibitors: Gain in Selectivity by Targeting the Depth of

Cells by Mitochondrial Pathway Activation and Induction of Nuclear Clusterin

(2004) Phosphinic Peptides as Zinc Metalloproteinase Inhibitors. *Cellular and* 

Identification and Pre-Clinical Testing of a Reversible Cathepsin Protease Inhibitor Reveals Anti-Tumor Efficacy in a Pancreatic Cancer Model. *Biochimie* Vol. 92

Specific Proteasome Inhibitors: Synthesis and Evaluation of Peptide α',β'-

Serine Protease Inhibitor Protease Nexin-1 Controls Mammary Cancer Metastasis through LPR-1-Mediated MMP9 Expression. *Cancer Research* Vol. 69 (No. 14): 5690-

M.; Leisinger, H.-J.; Diamandis, E. P.; Deperthes, D. & Cloutier, S. M. (2006)Mutant Recombinant Serpins as Highly Specific Inhibitors of Human Kallikrein 14. *FASEB* 

in Cancer Progression and Their Pharmacological Targeting. *FEBS Journal* Vol. 276

Surveillance to Cancer Therapy. *Biochemical Society Transactions* Vol. 35 (No. 1): 12-

(2010) Elucidation of the α-Keto Aldehyde Binding Mechanism: A Lead Structure Motif for Proteasome Inhibition. *Angewandte Chemie International Edition* Vol. 50

and Medicine. *Current Medicinal Chemistry* Vol. 7 (No. 6): 629-647.

Counting. *Biochimie* Vol. 92 (No. 11): 1530-1545.

S' Cavity. *Biochemie* Vol. 92 (No. 11): 1501-1508.

Expression. *Cancer Letters* Vol. 295 (No. 1): 44-53. Di Cera, E. (2009) Serine Proteases. *IUBMB Life* Vol. 15 (No. 5): 510-515.

*Molecular Life Science* Vol. 16 (August 2004): 2010–2019.

Epoxyketones. *Chemistry & Biology* Vol. 6 (No. 11): 811-822.

503.

(No.11): 1618-1624.

*Journal* Vol. 273 (No. 11): 2505-2514.

*Molecular Life Sciences* Vol. 61 (No.11): 1278-1295.

*Metastasis Review* Vol. 25 (No. 1): 115-136.

5698.

(No. 1): 16-27.

(No. 2): 542-544

17.


Brouwer, A. J.; Bunschoten, A. & Liskamp, R. M. J. (2007) Synthesis and Evaluation of

Candia, B. J.; Hines, W. C.; Heaphy, C. N.; Griffith, J. K. & Orlando, R. A. (2006) Protease

Carter, S. A.; Lees, W. A.; Hill, J.; Brzin, J.; Kay, J. & Phylip, L. H. (2002) Aspartic Protease

Casini, A.; Gabbiani, C.; Sorrentino, S.; Rigobello, M. P.; Bindoli, A.; Geldbach, T. J.;

Castro-Guillén, J. L.; Garcia-Gasca, T & Blanco-Labra, A. (2010) Chapter V. Protease

Catanzaro, J. M.; Guierriero, J. L.;Liu, J.; Ullman, E.; Sheshadri, N.; Chen, J. J. & Zong, W.-X.

Chen, J. W.; Nelson, F. C.; Levin, J. I.; Mobilio, D.; Moy, F. J.; Nilakantan, R.; Zask, A. &

Choi, S.-Y.; Bertram, S.; Głowacka, I. ; Park, Y. W. & Pöhlmann, S. (2009) Type II

Clerc, J.; Groll, M.; Illich, D. J.; Bachmann, A. S.; Huber, R.; Schellenberg, B.; Dudler, R. &

Coleman, C. S.; Rocetes, J. D., Park, D. J.; Wallick, C. J.; Warn-Cramer, B. J.; Michael, K.;

*Journal of American Chemical Society* Vol. 122 (No. 40): 9648-9654.

Inhibitors. *Bioorganic & Medicinal Chemistry* Vol. 15 (No. 22): 6985-6993. Brouwer, A. J.; Ceylan, T.; Jonker, A. M.; van der Linden, T & Liskamp R. M. J. (2011)

*Medicinal Chemistry* Vol. 19 (No. 7): 2397-2406.

*Molecular Enzymology* Vol. 1596 (No. 1): 76-82.

*Medicinal Chemistry* Vol. 51 (No. 21): 6773-6781.

16 (31 May 2006): 16.

61728-304-9

*Molecules* Vol. 3 (No. 1): 5-14.

*Medicine* Vol. 15 (No. 7): 303-312.

*Proliferation* Vol. 39 (No. 6) 599-609.

(No. 16): 6507-6512.

Chloromethyl Sulfoxides as a New Class of Selective Irreversible Cysteine Protease

Synthesis and Biological Evaluation of Novel Irreversible Serine Protease Inhibitors Using Amino Acid Based Sulfonyl Fluorides as an Electrophilic Trap. *Bioorganic &* 

Nexin I Expression Is Altered in Human Breast Cancer. *Cancer Cell International* Vol.

Inhibitors from Tomato and Potato are More Potent Against yeast Proteinase A than Cathepsin D. *Biochimica and Biophysica Acta (BBA) – Protein Structure anhd* 

Marrone, A.; Re, N.; Hartinger, C. G.; Dyson, P. J. &Messori, L. (2008) Emerging Protein Targets for Anticancer Metallodrugs: Inhibition of Thioredoxin Reductase and Cathepsin B by Antitumor Ruthenium (II) – Arene Compounds. *Journal of* 

Inhibitors as Anticancer Agents. In: *New Approaches in the Treatment of Cancer.* V. C. Mejia Vazquez & S. Navarro (Eds.), 91-124, Nova Science Publishers, ISBN 978-1-

(2011) Elevated Expression of Squamus Cell Carcinoma Antigen (SCAA) Is Associated with Human Breast Carcinoma. *PloS ONE* Vol. 6 (No. 4): 1-8, e19096 Chang, W.-S. W.; Wu, H.-R.; Wu, C.-W. & Chang, J.-Y. (2007) Lysosomal Cysteine Protease

Cathepsin S as a Potential Target for Anti-cancer Therapy. *Journal of Cancer* 

Powers, A. (2000) Structure-based Design of a Novel, Potent and Selective Inhibitor for MMP-13Utilizing NMR Spectroscopy and Computer-Aided Molecular Design.

Transmembrane Serine Proteases in Cancer and Viral Infections. *Trends in Molecular* 

Kaiser, M. (2009) Synthetic and Structural Studies on Syringolins A and B Reveal Critical Determinants of Selectivity and Potency of Proteasome Inhibition. *Proceedings of the National Academy of Sciences of the United States of America* Vol.106

Dudler, R. & Bachmann, A. S. (2006) Syringolin A, a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis. *Cell* 


Inhibitors of Proteinases as Potential Anti-Cancer Agents 69

Kessler, B. M.; Tortorella, D.; Altun. M.; Kisselev, A. M.; Fiebiger, E.; Hekking, B. G.; Ploegh,

Klinghofer, V.; Steward, K.; McGonigal, T.; Smith, R.; Sarthy, A.; Nienaber, V.; Butler, C.;

Koblinski, J. E.; Abram, M. & Sloane, B. F. (2000) Unraveling the Role of Proteases In Cancer.

Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, A.; Okuda, T.; Ohnuki, T. & Komatsubara, S.

Kopitz, C.; Anton, M. Gansbacher, B. & Krüger, A. (2005) Reduction of Experimental

Overexpression in the Host. *Cancer Research* Vol. 65 (no. 19): 8608-8612. Krige, D.; Needham, L. A.; Bawden, L. J.; Flores, N.; Farmer, H.; Miles, L. E. C.; Stone, E.;

in Human Leukemic Cells. *Cancer Research* Vol. 68 (No. 16): 6669-6679. Kumar, G. D. K.; Chavarria, G. E.; Charlton-Sevcik, A.; Arispe, W. M.; MacDonough, M. T.;

Kwan, J. C.; Eksioglu, E. A.; Liu, C.; Paul, V. J. & Leusch, H. (2009) Grassystatins A-C from

Leban, J.; Blisse, M.; Krauss, B.; Rath, S.; Baumgartner, R. & Seifert, M. H. J. (2008)

Lee, M.; Fridman, R. & Mobashery, S. (2004) Extracellular Proteases as Targets for Treatment of Cancer Metastases. *Chemical Society Reviews* Vol*.* 33 (No. 7): 401-409. Lee, T. K.; Murthy, S. R. K.; Cawley, N. X.; Dhanvantari, S.; Hewitt, S. M.; Lou, H.; Lau, T.;

Cancers, *The Journal of Clinical Investigation* Vol. 121 (No. 3): 880-892. Li, X.; Yin, S.; Meng, Y.; Sakr, W. & Sheng, S (2006) Endogenous Inhibition of Histone

Presentation. *Journal of Medicinal Chemistry* Vol. 52 (No. 18): 5732-5747. Lavelin, I.; Beer, A.; Kam, Z.; Rotter, V.; Oren, M.; Navon, A. & Geiger, B. (2009) Discovery

Subunits. *Chemistry & Biology* Vol. 8 (No. 9): 913-929.

*Clinica Chimica Acta* Vol. 291 (No. 2): 113-135.

4): 1415-1419.

9329.

*PloS ONE* Vol. 4 (No. 12): e8503.

Vol. 16 (No. 18): 4579-4588.

Urokinase Inhibitors. *Biochemistry* Vol. 40 (No. 31): 9125-9131.

Activities. *Journal of Antibiotics (Tokyo)* Vol. 53 (No. 2): 105-109.

H. L. & Overkleeft, H. S. (2001) Extended Peptide-based Inhibitors Efficiently Target the Proteasome an Reveal Overlapping Specificities of the Catalytic β–

Dorwin, S.; Richardson, P.; Weitzberg, M.; Wendt, M.; Rockway, T.; Zhao, Z.; Hulkower, K. L. & Giranda, V. L. (2001) Species Specificity of Aminidine-Based

(2000) TMC-95A, B, C and D, Novel Proteasome Inhibitors Produced by *Apiospora montagnei* Sacc. TC 1093. Taxonomy, Production, Isolation, and Biological

Human Fibrosarcoma Lung Metastasis in Mice by Adenovirus-mediated Cystatin C

Callaghan, J.; Chandler, S.; Clark, V. L.; Kirwin-Jones, P.; Legris, V.; Owen, J.; Patel, T.; Wood, S.; Box, G.; Laber, D.; Odedra, R.; Wright, A.; Wood, L. M.; Eccles, S. A.; Bone, E. A.; Ayscough, A. & Drummond, A. H. (2008) CHR-2797: An Antiproliferative Aminopeptidase Inhibitor that Leads to Amino Acid Deprivation

Strecker, T. E.; Chen, S.-E.; Siim, B. G.; Chaplin, D. J.; Trawick, M. L. & Pinney, K. G. (2010) Design, Synthesis and Biological Evaluation of potent Thiosemicarbazone Based Cathepsin L Inhibitors. *Bioorganic & Medicinal Chemistry Letters* Vol.20 (No.

Marine Cyanobacteria, Potent Cathepsin E Inhibitors That Reduce Antigen

of Novel Proteasome Inhibitors Using a High Content Cell Base Screening System.

Proteasome Inhibition by Peptide Semicarbazones. *Bioorganic & Medicinal Chemistry* 

Ma, S.; Huynh, T.; Wesley, R. A.; Ng, I. O.; Pacak, K.; Poon, R. T. & Loh, Y. P. (2011)An N-Terminal Truncated Carboxypeptidase E Splice Isoform Induces Tumor Growth and Is a Biomarker for Predicting Future Metastasis in Human

Deacylase 1 by Tumor-Suppressive Maspin. *Cancer Research* Vol. 66 (No. 18): 9323-


Greenspan, P. D.; Clark, P. N.; Tommasi, R. A.; Cowen, S. D.; McQuire, L.W. Farley D. L.;

Grzywa, R.; Dyguda-Kazimierowicz, E.; Sieńczyk, M.; Feliks, M.; Sokalski, W. A. &

Hanada, M.; Sugawara, K.; Kaneta, K.; Toda, S.; Nishiyama, Y.; Tomita, K.; Yamamoto, H.;

Hanada, K.; Tamai, N.; Yamagishi, N.; Ohmura, S. Sawada, J. & Tanaka, I. (1978) Isolation

Hassan, H. T. (2004) Ajoene (Natural Garlic Compound): A New Anti-Leukaemia Agent for

Hsieh, C. C.; Hernández-Ledesma, B.; Jeong, H. J.; Park, J. H. & de Lumen, B. O. (2010)

Joanitti, G. A.; Azevedo, R. B. & Freitas, S. M. (2010) Apoptosis and Lysosome Membrane

Joossens, J.; Van der Veken, P.; Surpetanu, G.; LAmbeit, A.-M.; El-Sayed, I.; Ali, O. M.;

Joyce, J. A.; Baruch, A.; Chehade, K.; Meyer-Morse, N.; Giraudo, E.; Tsai, F. Y.; Greenbaum,

Katunuma, N. (2011) Structure-based Development of Specific Inhibitors for Individual

Kemik, O.; Kemik, A. S.; Sumer, A.; Beğenik, H.; Dügler, A. C.; Purisa, S. & Tuzun, S. (2011)

Keppler, D. (2006) Mini Review. Towards Novel Anti-Cancer Strategies Based on Cystatin

Preventive Lunasin Bioavailable. *Plos ONE* Vol. 5 (No.1): 1-9, e8890. Huai, Q.; Zhou, A.; Lin, L.; Mazar, A. P.; Parry, G. C.; Callahan, J.; Shaw, D. E.; Furie, B.;

*Journal of Medicinal Chemistry* Vol. 44 (No. 26): 4524-4534.

Affinity. *Journal of Molecular Modeling* Vol. 13 (No. 6-7): 677-683.

Origin. *Journal of Antibiotics (Tokyo)* Vol. 45 (No. 11): 1746-1752.

*Biological Chemistry Tokyo* Vol. 42 (No. 3): 523-528.

*Medicinal Chemistry* Vol. 49 (No. 19): 5785-5793.

Tumorigenesis. *Cancer Cell* Vol. 5 (No. 5): 443-453.

Function. *Cancer Letters* Vol. 235 (No. 2): 159-175.

*Series B, Physical and Biological Sciences* Vol. 87 (No.2): 29-38.

*Biology* Vol. 15 (No. 4): 422-423.

published on April 18, 2011.

73-81.

AML Therapy. *Leukemia Research* Vol. 28 (No. 7): 667-671.

van Dauzer, J. H.; Goldber, R. L.; Zhou, H.; Du, Z.; Fitt, J. J.; Coppa, D.E.; Fang, Z.; Macchia, W.; Zhu, L.; Capparelli, M. P.; Goldstein, R.; Wigg, A. M.; Doughty, J. R.; Bohacek, R. S. & Knap.A. K. (2001) Identification of Dipeptidyl Nitriles as Potent and Selective Inhibitors of Cathepsin B through Structure-based Drug Design.

Oleksyszyn, J. (2007) The Molecular Basis of Urokinase Inhibition: from the Nonempirical Analysis of Instramolecular Interactions to the Prediction of Binding

Konishi, M. & Oki, T. (1992) Epoxomycin, a New Antitumor Agent of Microbial

and Identification of E-64, A New Thiol Protease Inhibitor. *Agricultural and* 

Complementary Roles in Cancer Prevention: Protease Inhibitor Makes the Cancer

Furie, B. C. & Huang, M. (2008) Crystal Structures of Two Human Vironectin, Urokinase and UrokinaseReceptor Complexes. *Nature Structural and Molecular* 

Permeabilization Induction on Breast Cancer Cells by Anticancerogenic Bowman-Birk Protease Inhibitor from *Vigna unguiculata* Seeds. *Cancer Letters* Vol. 293 (No. 1):

Augustyns, K. & Haemers, A. (2006) Diphenyl Phosphonate Inhibibtors for the Urokinase-Type Plasminogen Activator: Optimization of the P4 Position. *Journal of* 

D. C.; Harger, J. H.; Bogyo, M. & Hanahan, D. (2004) Cathepsin Cysteine Proteases are Effectors of Invasive Growth and Angiogenesis During Multistage

Cathepsins and Their Medical Applications. *Proceedings of Japan Academy of Sciences,* 

The Relationship Among Acute-phase Response Proteins, Cytokines, and Hormones in Various Gastrointestinal Cancer Types Patients with Cachectic. *Human & Experimental Toxicology* Vol. 30: doi: 10.1177/0960327111405864, first


Inhibitors of Proteinases as Potential Anti-Cancer Agents 71

Naffara, N. I.; Andreu, P. & Coussens, L. M. (2009) Delineating Protease Functions During

Naimuddin, M.; Kitamura, K.; Kinoshita, Y.; Honda-Takahashi, Y.; Murakami, M.; Ito, M.;

Niestroj, A. J.; Feuβner, K.; Heiser, U.; Dando, P. M.; Barrett, A.; Gerhardtz, B. & Demuth, H.

Omura S.; Fujimoto, T.; Matsuzaki, K.; Moriguchi, R.; Tanaka, H. & Sasaki, Y. (1991)

Otlewski, J.; Jeleń, F.; Zakrzewska, M. & Oleksy. A (2005) The Many Faces of Protease-Protein Inhibitor Interaction. *The EMBO Journal* Vol. 24 (No. 7): 1303-1310. Ovat, A.; Li, Z. Z.; Hampton, C. Y.; Asress, S. A.; Fernández, F. M.; Glass, J. D. & Powers, J.

Ozaki, N.; Ohmuraya, N.; Hirota, M.; Ida, S.; Wang, J.; Takamori, H.; Higashiyama, S.; Baba,

Pahl, H. L.; Krauss, B.; Schulze-Osthoff, B.; Decker, T.; Traenckner, E. B.; Vogt, M.; Myers, C.;

Palermo, C. & Joyce, J. A. (2007) Cysteine Cathepsin Proteases as Pharmacological Targets in

Pallares, I.; Bonet, R.; Garcia-Castellanos, R.; Ventura, S.; Avilés, F. X.; Vendrell, J. & Gomis-

Pandey, R.; Patilo, N. & Rao, M. (2007) Proteases and Protease Inhibitors: Implications in

Piovan, L.; Alves, M. F. M.; Juliano, L.; Brömme, D.; Cunha R. L. O. R. & Andrade, L. H.

Neuroblastoma Cells. *Journal of Antibiotics* Vol. 44 (No.1): 113-116.

Receptor. *Molecular Cancer Research* Vol. 7 (No. 9): 1572-1581.

Cancer. *Trends in Pharmacological Sciences* Vol. 29 (No. 1): 22-28

*States of America* Vol. 102 (No. 11): 3978-3983.

Halomethylketones *Biological Chemistry* Vol. 383 (No. 7-8): 1205-1214. Nuti, E.; Casalini, F.; Avramova, S. I.; Santamaria, S.; Fabbi, M.; Ferrini, S.; Marinelli, L.; La

T. H. Bugge (Eds.) 1-33, Humana Press, New York.

*Molecular Recognition* Vol. 20 (No. 1): 58-69.

*Chemistry* Vol.53 (No.17): 6326-6336.

2635.

1840.

Vol.17, (No. 1): 67-82.

(No. 6): 2009-2014.

Cancer Development. In: *Proteases and Cancer. Methods and Protocols.* T. M. Antalis &

Yamamoto, K.; Hanada, K.; Husimi, Y. & Nishigaki, K. (2007) Selection-by-function: Efficient Enrichement of Cathepsin E Inhibitors from a DNA Library. *Journal of* 

U. (2002) Inhibition of Mammalian Legumain by Michaels Acceptors and AzaAsn-

Pietra, V.; Limongelli, V.; Novellino, E.; Cercignani, G.; Orlandini, E.; Nencetti, S. & Rosello, A. (2010) Potent Arylsulfonamide Inhibitora of Tumor Necrosis Factor-α Converting Enzyme Able to Reduce Activated Leucocyte Cell Adhesion Molecule Shedding in Cancer Cell Models. *Journal of Medicinal Chemistry* Vol. 53 (No. 6): 2622-

Lactacystin, a Novel Microbial Metabolite, Induces Neuritogenesis of

C. (2010) Peptidyl α–Ketoamides with Nucleobases, Methylpiperazine and Dimethylaminoalkyl Substituents as Calpain Inhibitors. *Journal of Medicinal* 

H. & Yamamura, K.-I. (2009) Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells through the Epidermal Growth Factor

Parks, T.; Warring, P.; Mühlbacher, A.; Czernilofsky, A. P. & Baeuerle, P. A (1996) The Immosuppresive Fungal Metabolite Gliotoxin Specifically Inhibits Transcription Factor NF-κB. *Journal of Experimental Medicine* Vol. 183 (No. 4): 1829-

Rüth, F. X. (2005) Structure of Human Carboxypeptidase A4 with Its Endogenous Protein Inhibitor, Latexin. *Proceedings of the National Academy of Sciences of the United* 

Antitumorigenesis and drug Development. *International Journal of Human Genetics* 

(2011) Structure-Activity Relationship of Hypervalent Organochalcogenanes as Inhibitors of Cysteine Cathepsins V and S. *Bioorganic & Medicinal Chemistry* Vol. 19


Li, Y.; Dou, D.; He, G.; Lushington, G. H. & Groutas, W. C. (2009) Mechanism-Based

Lindeman, J.H.; Hanemaaijer, R.; Mulder, A.; Dijkstra, P. D.; Szuhai, K.; Bromme, D.;

Ma, X.-Q.; Zhang, H.-J.; Hang, Y.-H.; Chen, Y.-H.; Wu, F.; Du, J.-Q.; Yu, H.-P.; Zhou, Z.-L.;

Magdolen, U.; Krol, J.; Sato, S.; Mueller, M. M.; Sperl, S.; Krüger, A.; Schmidtt, M. &

Manello, F. (2006) Natural Bio-Drugs as Natural Matrix Metalloprotreinase Inhibitors: New

Maskos, K. (2005) Crystal Structures of MMPs in Complex with Physiological and

Mason, S. D. & Joyce, J. A (2011) Proteolytic Networks in Cancer. *Trends in Cell Biology* Vol.

McConnell, R. M.; Godwil, W. E.; Stefan, A.; Newton, C.; Myers, N. & Hatfield, S. E. (2003).

Moore, H. E.; Davenport, E. L.; Smith E. M.; Mularikrishnan, S.; Dunlop, A. S.; Walker, B. A.;

Mroczkiewicz, M.; Winkler, K.; Nowis, D.; Placha, G.; Golob, J. & Ostaszewski, R. (2010)

Mucha, A.; Drąg, M.; Dalton, J. & Kafarski. P. Metallo-peptidase Inhibitors. *Biochimie* Vol. 92

Mussap, M & Plebani, M. (2004) Biochemistry and Clinical Role of Human Cystatin C. *Critical Reviews in Clinical Laboratory Sciences* Vol. 41 (No. 5-6): 467-550.

Cyclic Tertiary Amines. *Letters in Peptide Science* Vol. 10 (No. 2): 69-78. Miller, E. K.-I.; Trabi, M.; Masci, P.P.; Lavin, M. F.; de Jersey, J. & Guddast, L. W. Crystal

Pharmacological Inhibitors. *Biochimie* Vol. 87 (No. 3-4): 249-263.

*Molecular Cancer Therapeutics* Vol.8 (No. 4): 762-770.

*Chemical Society* Vol. 126 (No. 33): 10271-10277.

*and Oncology* Vol. 36 (No. 2): 131-143.

22-27.

(No. 1): 56-65.

(No. 1):91-103

21 (No. 4): 228-237.

(No. 11): 3162-3175.

(No. 11): 1509-1529.

1518.

Inhibitors of Serine Proteases with High Selectivity Through Optimization of S' Subsite Binding. *Bioorganic & Medicinal Chemistry* Vol. 17 (No. 10): 5336-5342. Lim, I. T.; Meroueh, S. O.; Lee, M.; Heeg, M. J. & Mobashery, S. (2004) Strategy in Inhibition

of Cathepsin B, A Target in Tumor Invasion and Metastasis. *Journal of American* 

Verheijen, J. H. & Hogendoorn P. C. (2004) Cathepsin K is Principal Protease in Giant Cell Tumor of Bone. *American Journal of Pathology* Vol. 165 (No.2): 593-600. Linington, R. G.; Edwards, D. J.; Shuman, C. F.; McPhail, K. L.; Matainaho, T. & Gerwick, W.

H. (2008) Symploxacine A, a Potent Cytotoxin and Chymotrypsin Inhibitor from the Marine Cyanobacterium *Symploca* sp. *Journal of Natural Products* Vol. 71 (No. 1):

Li, J.-Y.; Nan, F.-J. & Li. J. (2007) Novel Irreversible Caspase-1 Inhibitor Attenuates the Maturation of Intracellular Interleukin -1β. *Biochemistry & Cell Biology* Vol. 85

Magdolen, V. (2002) Natural Inhibitors of Tumor-Associated Proteases. *Radiology* 

Perspectives on The Horison? *Recent Patents on Anti-cancer Drug Discovery* Vol. 1

Synthesis an Cathepsin D Inhibition of Peptide-hydroxyethyl Amine Isosteres with

Structure of Textinilin-1, a Kunitz-Type Serine Protease Inhibitor from the Venom of the Australian Common Brown Snake (*Pseudonaja textilis*). *FEBS Journal* Vol. 276

Krige, D.; Drummond, A. H.; Hooftman, L.; Morgan, G. J. & Davies, F. E. (2009) Aminopeptidase Inhibition as a Targeted Treatment Strategy in Myeloma.

Studies on the Synthesis of All Stereoisomers of MG-132 Proteasome Inhibitors in the Tumor Targeting Approach. *Journal of Medicinal Chemistry* Vol. 53 (No. 4): 1509-


Inhibitors of Proteinases as Potential Anti-Cancer Agents 73

Singh, J. P.; Tamang, S.; Rajamohanan, P. R.; Jima, N. C.; Chakraborty, G.; Kundu, G. C.;

Skillman, A. G.; Lin, B.; Lee, C. E.; Kunz, I. D.; Ellman, J. A. & Lynch, G. (2000) Plasminogen

Skrzydlewska, E.; Sulkowska, M.; Koda, M. & Sulkowski S. (2005) Proteolytic-antiproteolytic

Sperl, S.; Jacob, U.; Arroya de Parad, N.; Stürzerbecher, J.; Wilhelm, O. G.; Bode W.;

Stroup, G. B.; Lark, M. W.; Veber, D. F.; Battacharyya, A.; Blake, S.; Dare, L. C.; Erhard, K. F.;

Primate. *Journal of Bone and Mineral Research* Vol.16 (No. 10): 1739-1746. Sugawara,K.; Hatori, M.; Nishiyama, Y.; Tomita, K.; Kamei, H.; Konishi, M. & Oki, T. (1990)

*Medicinal Chemistry* Vol. 53 (No. 14): 5121-5128.

*Neurochemistry* Vol. 74 (No. 4): 1459-1477.

11 (No. 9): 1251-1266.

(No. 10): 5113-5118.

518-522.

23 (No. 5): 259-262.

29 (No. 1): 97-99.

Gaikwad, S. M. & Khan, M. I. (2010) Isolation, Structure and Functional Elucidation of Modified Pentapeptide, Cysteine Protease Inhibitor (CPI-2081) from *Streptomyces Species* 2081 that Exhibit Inhibitory Effect on Cancer Cell Migration. *Journal of* 

Activator Inhibitors Based on 3D QSAR CoMFA/CoMSIA Models; Journal of Medicinal Chemistry Vol. 49 (no. 2) 457-489. Novel Cathepsin D Inhibitors Block the Formation of Hyperphosphorylated Tau Fragments in Hippocampus. *Journal of* 

Balance and Its Regulation in Carcinogenesis. *World Journal of Gastroenterology* Vol.

Magdolen, V.; Huber, R. & Moroder, L. (2000) 4-(Amidomethyl)phenylguanidine Derivatives as Non Peptidic Highly Selective Inhibitors of Human Urokinase. *Proceedings of the National Academy of Sciences of the United States of America* Vol. 97

Hoffman, S. J.; James, I. E.; Marquis, R. W.; Ru, Y.; Vasco-Moser, J. A.; Smith, B. R.; Tomaszek, T. & Gowen, M. (2001) Potent and Selective Inhibition of Human Cathepsin K Lead to Inhibition of Bone Resorption in Vivo in a Non-Human

Eponemycin, a New Antibiotic Active Against B16 Melanoma. I. Production, Isolation, Structure and Biological Activity*. Journal of Antibiotics (Tokyo)* Vol. 43 (No. 1): 8-18. Takai, S.; Jin, D.; Muramatsu, M. & Miyazaki, M. (2004) Cymase as a Novel Target for the

Prevention of Vascular Diseases. *Trends in Pharmacological Science.* Vol. 25 (No. 10):

New Pepsin Inhibitor Produced by Actinomycetes. *Journal of Antibiotics (Tokyo)* Vol.

of Aminopeptidase B, Produced by Actinomycetes. *Journal of Antibiotics (Tokyo)* Vol.

Napsin A Suppresses Tumor Growth Independent on its Catalytic Activity.

Peptide Inhibitors of and Active Site Probes of Papain Family Cysteine Proteases.

Madelmont, J. C. (2005) Preliminary studies of New Proteasome Inhibitors in the

Turk, B. (2006) Targeting Proteases: Successes, Failures and Future Prospects. *Nature* 

Turk, V.; Kos, J. & Turk, B. (2004) Cysteine Cathepsins (Proteases) - on The Main Stage of

Umezawa, H.; Aoyagi, T.; Morishima, H.; Matsuzaki, H. & Hamada, M. (1970) Pepstatin a

Umezawa, H.; Aoyagi, T.; Suda, H.; Hamada, M. & Takeuchi, T. (1976) Bestatin, an Inhibitor

Ueno, T.; Elmberger, G.; Weaver, T. E.; Toi, M & Linder, S. (2008) The Aspartic Protease

Velhelst, S. H. L.; Witte, M. D.; Arastu-Kapur, S.; Fonovic, M. & Bogyo, M. (2006) Novel Aza

Vivier, M.; Jarrousse, A.-S.; Bouchon, B.; Galmier, M.-J.; Auzeloux, P.; Sauzieres, J. &

*Reviews: Drug Discovery* Vol. 5 (September 2006): 785-799.

Cancer? *Cancer Cell* Vol. 5 (No. 5): 409-410.

*Laboratory Investigation* Vol. 88 (No. 3): 256-263.

*ChemBioChem* Vol. 7 (No. 5): 824-827


Powers, J. C.; Asqian, J. L.; Ekici, O. D. & James, K. E. (2002) Irreversible Inhibitors of Serine, Cysteine and Thereonine Proteases. *Chemical Reviews* Vol.102 (No. 12): 4639-4650. Puente, X. S.; Sanchez,L. M.; Overall, C. M. & Lopez-Otin, C. (2003). Human and Mouse

Puxbaum, V & Mach, L. (2009) Proteinases and Their Inhibitors in Liver Cancer. *World* 

Roth, P.; Kissel, M.; Herrmann, C.; Eisele, G.; Leban, J.; Weller, M. & Schmidt, F. (2009)

Sakuhari, N.; Suzuki, K.; Sano, Y.; Saito, T.; Yoshimura, H.; Nishimura, Y.; Yano, T.;

Saleh, Y.; Wnukiewicz, J.; Andrzejak, R. Trziszka, T. Siewiński, M. Ziolkowski, P. & Kopeć,

Sato, T.; Takahashi, S.; Mizumoto, T.; Harao, M.; Akizuki, M.; Takasugi, M.; Fukutomi, T. &

Schmideberg, N.; Schmitt, M.; Rölz, C.; Truffault, V.; Sukopp, M.; Bürgle, M.; Wilhelm, O.

Selvakumar, P.; Lakshmikuttayamma, A.; Dimmock, J. R. & Sharma, R. K. (2006) Methionine

Sieńczyk, M & Oleksyszyn, J. (2006) Inhibition of Trypsin and Urokinase by Cbz-Amino(4-

Sieńczyk, M. & Oleksyszyn, J. (2009) Irreversible Inhibition of Serine Proteases – Design and

Sieńczyk M.; Winiarski, Ł.; Kasperkiewicz, P.; Psurski, M.; Wietrzyk, J. & Oleksyszyn, J.

Sierko, E.; Wojtukiewicz, M. Z. & Kisiel, W. (2007) The Role of Tissue Factor Pathway

Cystatin. *Journal of Cancer Molecules* Vol. 2 (No. 2): 67-72.

*In vitro* and *In vivo. Clinical Cancer Research* Vol. 15 (No. 21): 6609-6618. Sahab, Z. J.; Hall, M. D.; Me Sung, Y.; Dakshanamurthy, S.; Ji, Y.; Kumar, D. & Byers, S. W.

7): 544-558.

1219-1228.

903-907.

(No. 4): 217-222.

1765 (No. 2): 148-154.

*Letters* Vol. 16 (No. 11): 2886-2890

*Chemistry* Vol. 16 (No. 13): 1673-1687.

*Medicinal Chemistry Letters* Vol. 21 (No. 5): 1310-1314.

4894-4994.

7): 653-659.

*Journal of Hepatology* Vol. 31 (No. 1): 28-34.

Proteases: A Comparative Genomic Approach. *Nature Reviews: Genetics* Vol. 4 (No.

SC68896, a Novel Small Molecule Proteasome Inhibitor, Exerts Antiglioma Activity

(2011) Tumor Suppressor RARRES 1 Interacts with Cytoplasmic Carboxypeptidase AGBL2 to Regulate α–Tubulin Tyrosination Cycle. *Cancer Research* Vol. 71 (No. 4):

Sadzuka, Y. & Asano, R. (2008) Effect of a Single Dose Administration of Bowman-Birk Inhibitor Concentrate on Anti-Proliferation and Inhabitation of Metastasis in M5076 Ovarian Sacroma-Bearing Mice. *Molecular Medicine Reports* Vol. 1 (No. 5):

W. (2006) Cathepsin B and Cysteine Protease Inhibitors In Human Tongue Cancer: Correlation with Tumor Staging and In Vitro Inhibition of Cathepsin B by Chicken

Yamashita, J.-I. (2006) Nautrophil Elastase and Cancer. *Surological Oncology* Vol. 15

G.; Schmalix, W.; Magdolen, V & Kessler, H. (2002) Synthesis, Solution Structure, and Biological Evaluation of Urokinase Type Plasminogen Activator (uPA)-Derived Receptor Binding Domain Mimetics. *Journal of Medicinal Chemistry* Vol. 45 (No. 23):

Aminopeptise 2 and Cancer. *Biochimica et Biophysica Acta – Reviews on Cancer* Vol.

guanidinophenyl)methanephosphonate Aromatic Ester Derivatives: The Influence of the Ester Group on Their Biological Activity. *Bioorganic & Medicinal Chemistry* 

in Vivo Activity of Diaryl α–Aminophosphonate Derivatives. *Current Medicinal* 

(2011) Simple Phosphonic Inhibitors of Human Neutrophil Elastase. *Bioorganic &* 

Inhibitor-2 in Cancer Biology. *Seminars in Thrombosis and Homeostasis* Vol. 33 (No.


**4** 

*University of Buenos Aires* 

*Argentina* 

**Histamine Receptors as Potential Therapeutic** 

Although research over the last decade has led to new and improved therapies for a variety of different diseases, anticancer drug therapy continues to have undesirable outcomes, including both poor response and severe toxicity. In addition to the critical need to discover new drugs, it is important to optimize existing therapies in order to minimize adverse

In the context of the complexity of cancer disease processes, future anticancer treatments will have to take into account the tumour microenvironment and aim to target the different cellular and molecular participants encompassed in a tumour, as well as their specific

In the present chapter we aimed to briefly summarize current knowledge on histamine and histamine receptors involvement in cancer, focusing on some recent evidence that points them out as a promising molecular targets and avenue for cancer drug development. On the basis of the role on immune system, it has been reported the efficiency of histamine as an adjuvant to tumour immunotherapy. In addition, we present here novel findings, suggesting the potential application of histamine and its ligands as adjuvants to tumour

It is generally acknowledged that histamine is an important regulator of a plethora of (patho) physiological conditions and exerts its actions through the interaction with four histamine receptor subtypes. All these receptors belong to the family of heptahelical Gprotein coupled receptors (GPCR) and they are the H1, H2, H3 and H4 histamine receptors (H1R, H2R, H3R, H4R). Based on the classical pharmacological analysis H1R was proposed in 1966 by Ash and Schild (Ash & Schild, 1966) and H2R was described in 1972 by Black et al. (Black et al., 1972). The third histamine receptor was discovered in 1983 by a traditional pharmacological approach, consisting of assessing the inhibitory effect of histamine on its own release from depolarized rat brain slices (Arrang et al., 1983). It was not until 2000-2001

**1. Introduction** 

interactions.

radiotherapy.

**2. Histamine receptors** 

reactions and maximize efficacy.

Vanina A. Medina1,2, Diego J. Martinel Lamas1, Pablo G. Brenzoni1,

**Targets for Cancer Drug Development** 

Noelia Massari1, Eliana Carabajal1 and Elena S. Rivera1 *1Laboratory of Radioisotopes, School of Pharmacy and Biochemistry,* 

*2National Scientific and Technical Research Council (CONICET)* 

Tumor Targeting Approach: Synthesis and in Vitro Toxicity. *Journal of Medicinal Chemistry* Vol. 48 (No. 21): 6731-6740.


## **Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development**

Vanina A. Medina1,2, Diego J. Martinel Lamas1, Pablo G. Brenzoni1, Noelia Massari1, Eliana Carabajal1 and Elena S. Rivera1 *1Laboratory of Radioisotopes, School of Pharmacy and Biochemistry, University of Buenos Aires 2National Scientific and Technical Research Council (CONICET) Argentina* 

## **1. Introduction**

74 Drug Development – A Case Study Based Insight into Modern Strategies

Ward, Y. D.; Thomson, D. S.; Frye, L. L.; Cywin, C. L.; Morwick, T.; Emmanuel, M. J.;

Wickström, M.; Larsson, R.; Nygren, P. & Gullbo, J. (2011) Aminopeptidase N (CD13) as a Target for Cancer Chemotherapy. *Cancer Science* Vol. 102 (No. 3): 501-508. Wieczerzak, E.; Drabik, P.; Łankiewicz, L.; Ołdziej, S.; Grzonka, Z.; Abrahamson, M.; Grubb,

Wieczerzak, E.; Rodziewicz-Motowidło, S.; Jankowska, E.; Giełdoń, A. & Ciarkowski, J.

Xuan, Q.; Yang, X.; Mo, L.; Huang, F.; Pang, Y.; Qin, M.; Chen, Z.; He, M.; Wang, Q. & Mo,

Yabe, K. & Koide, T. (2009) Inhibition of the 20S Proteosome by a Protein Proteinase

Yang, H.; Chen, D.; Cui, Q. C.; Yuan, X. & Dou, Q. P. (2006) Celastrol, a Triterpene Extracted

Yang, Z.-Q.; Kwok, B. H. B.; Lin, S.; Koldobskiy, M. A.; Crews, C. M. & Danishefsky, S. J.

Zhou, H.-J.; Aujay, M. A. ; Bennett, M.K. ; Dajee, M. ; Demo, S. D.; Fang, Y.; Ho, M. N.;

Zhu, M.; Gokhale, V. M.; Szabo, L.; Munoz, R. M.; Baek, H.; Bashyam, S.; Hurley, L. H.; Von

Amino Acids. *Journal of Medicinal Chemistry* Vol. 53 (No. 5): 1990-1999.

Proteasome Inhibitory Activity. *ChemBioChem* Vol. 4 (No. 6); 508-513 Yotaklis, A.; & Dive, V. (2008) Synthesis and Site Directed Inhibitors of Metzicind:

Inhibitors. *Journal of Medicinal Chemistry* Vol. 45 (No. 25): 5471-5482.

*Chemistry* Vol. 48 (No. 21): 6731-6740.

*Medicinal Chemistry* Vol. 45 (No. 19): 4202-4211.

*Journal of Peptide Science* Vol. 13 (No. 8): 536-543.

*Laboratory Medicine* Vol. 132 (No. 11): 1796-1801.

(No. 9): 4758-4765.

Vol. 52 (No. 9): 3028-3028.

Proteinase. *Journal of Biochemistry* Vol. 145 (No. 2): 217-227.

Tumor Targeting Approach: Synthesis and in Vitro Toxicity. *Journal of Medicinal* 

Zindell, L.; McNell, D.; Bekkall, Y.; Girardot, M.; Hrapchak, M.; De Turi, M.; Crane, K.; White, D.; Pav, S.; Wang, Y.; Hao, M. H.; Grygon, C. A.; Labadia, M. E.; Freeman, D. M.; Davidson, W.; Hopkins, J. L.; Brown, M. L. & Spero, D. M. (2002) Design and Synthesis of Dipeptide Nitriles as Reversible and Potent Cathepsin S

A. & Brömme, D. (2002) Azapeptides Structurally Based upon Inhibitory Sites of Cystatins as Potent and Selective Inhibitors of Cysteine Proteases. *Journal of* 

(2007) An Enormously Active and Selective Azapeptide Inhibitors of Cathepsin B.

Z.-N (2008) Expression of Serine Protease Kallikrein 7 and Its Inhibitor Antileukoprotease is Decreased in Prostate Cancer. *Archives of Pathology and* 

Inhibitor: Evidence that Natural Serine Proteinase Inhibitor Can Inhibit a Threonine

from the Cinese "Thunder of God Vine", Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice. *Cancer Research* Vol. 66

(2003) Simplified Synthetic TMC-95A/B Analogues Retain the Potency of

Achievement and Perspectives. *Molecular Ascpects of Medicine* Vol. 29 (No. 2): 329-338.

Jiang. J.; Kirk, C. J.; Laiding, G. J.; Lewis. E. R.; Lu, Y.; Muchamuel, T.; Parlati, F.; Ring, E.; Shenk, K. D.; Shields, J.; Showonek, P. J.; Stanton, T.; Sun, C. M.; Sylvain, C.; Woo, T. M. & Yang, J. (2009) Design and Synthesis of Orally Bioavailable and Selective Epoxyketone Proteasome Inhibitor (PR-047). *Journal of Medicinal Chemistry* 

Hoff, D. D. & Han, H. (2007) Identification of Novel Inhibitor of Urokinase-Type Plasminogen Activator. *Molecular Cancer Therapeutics* Vol. 6 (No. 4): 1348-1356. Zhu, Y.; Zhu, XC.; Wu, G.; Ma, Y.; Li, Y.; Zhao, X.; Yuan, Y.; Yang, J.; Yu, S.; Shao, F.; Li, R.;

Ke. Y.; Lu, A.; Liu, Z. & Zhang, L. (2010) Synthesis in Vitro and in Vivo Biologial Evaluation, Docking Studies and Structure – Activity Relationship (SAR) Discussion of Dipeptidyl Boronic Acid Proteasome Inhibitors Composed of β–

Although research over the last decade has led to new and improved therapies for a variety of different diseases, anticancer drug therapy continues to have undesirable outcomes, including both poor response and severe toxicity. In addition to the critical need to discover new drugs, it is important to optimize existing therapies in order to minimize adverse reactions and maximize efficacy.

In the context of the complexity of cancer disease processes, future anticancer treatments will have to take into account the tumour microenvironment and aim to target the different cellular and molecular participants encompassed in a tumour, as well as their specific interactions.

In the present chapter we aimed to briefly summarize current knowledge on histamine and histamine receptors involvement in cancer, focusing on some recent evidence that points them out as a promising molecular targets and avenue for cancer drug development. On the basis of the role on immune system, it has been reported the efficiency of histamine as an adjuvant to tumour immunotherapy. In addition, we present here novel findings, suggesting the potential application of histamine and its ligands as adjuvants to tumour radiotherapy.

#### **2. Histamine receptors**

It is generally acknowledged that histamine is an important regulator of a plethora of (patho) physiological conditions and exerts its actions through the interaction with four histamine receptor subtypes. All these receptors belong to the family of heptahelical Gprotein coupled receptors (GPCR) and they are the H1, H2, H3 and H4 histamine receptors (H1R, H2R, H3R, H4R). Based on the classical pharmacological analysis H1R was proposed in 1966 by Ash and Schild (Ash & Schild, 1966) and H2R was described in 1972 by Black et al. (Black et al., 1972). The third histamine receptor was discovered in 1983 by a traditional pharmacological approach, consisting of assessing the inhibitory effect of histamine on its own release from depolarized rat brain slices (Arrang et al., 1983). It was not until 2000-2001

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 77

contains 487 amino acids and is a Gαq/11-coupled protein with a very large third intracellular loop and a relatively short C-terminal tail. The most important signal induced by ligand binding is the activation of phospholipase C (PLC)-generating inositol 1,4,5 triphosphate (Ins (1,4,5) P3) and 1,2-diacylglycerol leading to increased cytosolic calcium. In addition to the inositol signalling system, H1R activation could lead to additional secondary signalling pathways. This rise in intracellular calcium levels seems to account for the various pharmacological activities promoted by the receptor, such as nitric oxide production, vasodilatation, liberation of arachidonic acid from phospholipids and increased cyclic guanosine-3',5'-monophosphate (cGMP). Additionally, it was reported that H1R can directly increase the cyclic adenosine-3',5'-monophosphate (cAMP) levels (Davio et al., 1995). H1R also activates NF-kB through Gαq11 and Gβγ upon agonist binding, while constitutive activation of NF-kB occurs only through the Gβγ (Bakker et al., 2001; Leurs et al., 1995; Smit et al., 1999). Recently, it was reported that the stimulation of H1R induced H1R gene expression through protein kinase C δ (PKCδ) activation, resulting in receptor upregulation

The H2R principal action from a clinical point of view is its role in stimulating gastric acid secretion, thus H2R antagonists are used in the relief of symptoms of gastro-oesophageal reflux disease treatment. The human H2R intronless gene, encodes a protein of 359 amino acids and is located on chromosome 5. The H2R has a ubiquitous expression as the H1R. It is expressed in gastric parietal cells, heart, endothelial cells, nerve cells, airway and vascular smooth muscle, hepatocytes, chondrocytes and immune cells, such as neutrophils, monocytes, eosinophils, DC, and T and B lymphocytes (Black et al., 1972; Dy & Schneider, 2004; Leurs et al., 1995). The H2R is coupled both to adenylate cyclase via a GTP-binding protein Gs, and phosphoinositide second messenger systems by separate GTP-dependent mechanisms. However, H2R-dependent effects of histamine are predominantly mediated by cAMP that activates protein kinase A (PKA) enzymes phosphorylating a wide variety of proteins involved in regulatory processes. Activation of H2R is also associated with other additional signal transduction pathways including activation of c-Fos, c-Jun, PKC and

p70S6kinase (Davio et al., 1995; Fitzsimons et al., 2002; Fukushima et al., 1997).

The H3R has initially been identified in both central and peripheral nervous system as a presynaptic receptor controlling the release of histamine and other neurotransmitters (dopamine, serotonine, noradrenalin, γ-aminobutyric acid and acetylcholine) (Arrang et al., 1983; Bongers et al., 2007; Leurs et al., 2005; Lovenberg et al., 1999). The H3R has gained pharmaceutical interest as a potential drug target for the treatment of various important disorders like obesity, myocardial ischemia, migraine, inflammatory diseases and several CNS disorders like Alzheimer's disease, attention-deficit hyperactivity disorder and schizophrenia. Pitolisant (BF2.649, 1-{3-[3-(4-chlorophenyl)propoxy]propyl} piperidine, hydrochloride) is the first H3R inverse agonist to be introduced in the clinics. Its wakepromotion activity was evidenced in excessive diurnal sleepiness of patients with narcolepsy, Parkinson's disease or obstructive sleep apnea/hypopnea (Bongers et al., 2007; Lebois et al., 2011; Leurs et al., 2005; Schwartz, 2011). The human H3R gene consists of either three exons and two introns, or four exons and three introns spanning 5.5 kb on

(Mizuguchi et al., 2011).

**2.2 Histamine H2R** 

**2.3 Histamine H3R** 

that by using the H3R DNA sequence, several independent research groups identified the novel H4R highly expressed in immune cells (Coge et al. 2001b; Lui et al., 2001; Morse et al. 2001; Nakamura et al., 2000; Nguyen et al. 2001; Oda et al., 2000).

Recent studies employing human genetic variance and mice lacking specific receptors or the ability to generate histamine, have shown functions for the histamine pathway that extend well beyond the established roles. As a result, antihistamines may have wider applications in the future than previously predicted (Smuda & Bryce, 2011).


Table 1. Compounds most widely used in histamine receptor investigation

Like most other GPCR, histamine receptors exist as equilibrium between their inactive and active conformations. Constitutive activity has now been shown for all four types of histamine receptors, leading to the reclassification of some antagonists as inverse agonists. These members of the GPCR family may exist as homo- and hetero-oligomers at the cell surface, which could have different pharmacological and physiological effects (Bongers et al., 2007; Fukushima et al., 1997; Hancock et al., 2003; Leurs et al., 2002, 2009). Moreover, the affinity of histamine binding to different histamine receptors varies significantly. Thus, the effects of histamine and receptor ligands upon receptor stimulation are rather complex. Pharmacologic agents are summarized in table 1.

## **2.1 Histamine H1R**

Since histamine is considered to be the most important mediator in allergies such as allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, asthma and anaphylaxis, the most commonly used drugs to treat these pathological disorders are antihistamines acting on the H1R. In the lung, it mediates bronchoconstriction and increased vascular permeability. The H1R is expressed in a wide variety of tissues, including airway and vascular smooth muscle, endothelia, gastrointestinal tract, liver, genitourinary and cardiovascular systems, central nervous system (CNS), adrenal medulla, chondrocytes and in various immune cells including neutrophils, monocytes, eosinophils, dendritic cells (DC), as well as T and B lymphocytes, in which it mediates the various biological manifestations of allergic responses. The coding sequence of the human H1R is intronless and is located in the chromosome 3 (Bakker et al., 2001; Dy & Schneider, 2004; Leurs et al., 1995). The human H1R contains 487 amino acids and is a Gαq/11-coupled protein with a very large third intracellular loop and a relatively short C-terminal tail. The most important signal induced by ligand binding is the activation of phospholipase C (PLC)-generating inositol 1,4,5 triphosphate (Ins (1,4,5) P3) and 1,2-diacylglycerol leading to increased cytosolic calcium. In addition to the inositol signalling system, H1R activation could lead to additional secondary signalling pathways. This rise in intracellular calcium levels seems to account for the various pharmacological activities promoted by the receptor, such as nitric oxide production, vasodilatation, liberation of arachidonic acid from phospholipids and increased cyclic guanosine-3',5'-monophosphate (cGMP). Additionally, it was reported that H1R can directly increase the cyclic adenosine-3',5'-monophosphate (cAMP) levels (Davio et al., 1995). H1R also activates NF-kB through Gαq11 and Gβγ upon agonist binding, while constitutive activation of NF-kB occurs only through the Gβγ (Bakker et al., 2001; Leurs et al., 1995; Smit et al., 1999). Recently, it was reported that the stimulation of H1R induced H1R gene expression through protein kinase C δ (PKCδ) activation, resulting in receptor upregulation (Mizuguchi et al., 2011).

## **2.2 Histamine H2R**

76 Drug Development – A Case Study Based Insight into Modern Strategies

that by using the H3R DNA sequence, several independent research groups identified the novel H4R highly expressed in immune cells (Coge et al. 2001b; Lui et al., 2001; Morse et al.

Recent studies employing human genetic variance and mice lacking specific receptors or the ability to generate histamine, have shown functions for the histamine pathway that extend well beyond the established roles. As a result, antihistamines may have wider applications

H2R Amthamine, impromidine, arpromidine Famotidine, ranitidine, cimetidine,

Like most other GPCR, histamine receptors exist as equilibrium between their inactive and active conformations. Constitutive activity has now been shown for all four types of histamine receptors, leading to the reclassification of some antagonists as inverse agonists. These members of the GPCR family may exist as homo- and hetero-oligomers at the cell surface, which could have different pharmacological and physiological effects (Bongers et al., 2007; Fukushima et al., 1997; Hancock et al., 2003; Leurs et al., 2002, 2009). Moreover, the affinity of histamine binding to different histamine receptors varies significantly. Thus, the effects of histamine and receptor ligands upon receptor stimulation are rather complex.

Since histamine is considered to be the most important mediator in allergies such as allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, asthma and anaphylaxis, the most commonly used drugs to treat these pathological disorders are antihistamines acting on the H1R. In the lung, it mediates bronchoconstriction and increased vascular permeability. The H1R is expressed in a wide variety of tissues, including airway and vascular smooth muscle, endothelia, gastrointestinal tract, liver, genitourinary and cardiovascular systems, central nervous system (CNS), adrenal medulla, chondrocytes and in various immune cells including neutrophils, monocytes, eosinophils, dendritic cells (DC), as well as T and B lymphocytes, in which it mediates the various biological manifestations of allergic responses. The coding sequence of the human H1R is intronless and is located in the chromosome 3 (Bakker et al., 2001; Dy & Schneider, 2004; Leurs et al., 1995). The human H1R

H3R R-()-methylhistamine, imetit, immepip Clobenpropit, thioperamide,

**Inverse agonists** 

iodoproxyfan

roxatidine, zolantidine

Mepyramine, cetirizine, terfenadine diphenhydramine, loratadine

Thioperamide, JNJ7777120, VUF 6002, A-987306, A-940894

2001; Nakamura et al., 2000; Nguyen et al. 2001; Oda et al., 2000).

in the future than previously predicted (Smuda & Bryce, 2011).

4-methylhistamine, R-()-methylhistamine,

Table 1. Compounds most widely used in histamine receptor investigation

H1R Histaprodifens, 2-(3-trifluoromethylphenyl)

H4R Clobenpropit, VUF 8430, imetit,

Pharmacologic agents are summarized in table 1.

**2.1 Histamine H1R** 

OUP-16, clozapine

histamine

**Agonists Antagonists/** 

The H2R principal action from a clinical point of view is its role in stimulating gastric acid secretion, thus H2R antagonists are used in the relief of symptoms of gastro-oesophageal reflux disease treatment. The human H2R intronless gene, encodes a protein of 359 amino acids and is located on chromosome 5. The H2R has a ubiquitous expression as the H1R. It is expressed in gastric parietal cells, heart, endothelial cells, nerve cells, airway and vascular smooth muscle, hepatocytes, chondrocytes and immune cells, such as neutrophils, monocytes, eosinophils, DC, and T and B lymphocytes (Black et al., 1972; Dy & Schneider, 2004; Leurs et al., 1995). The H2R is coupled both to adenylate cyclase via a GTP-binding protein Gs, and phosphoinositide second messenger systems by separate GTP-dependent mechanisms. However, H2R-dependent effects of histamine are predominantly mediated by cAMP that activates protein kinase A (PKA) enzymes phosphorylating a wide variety of proteins involved in regulatory processes. Activation of H2R is also associated with other additional signal transduction pathways including activation of c-Fos, c-Jun, PKC and p70S6kinase (Davio et al., 1995; Fitzsimons et al., 2002; Fukushima et al., 1997).

### **2.3 Histamine H3R**

The H3R has initially been identified in both central and peripheral nervous system as a presynaptic receptor controlling the release of histamine and other neurotransmitters (dopamine, serotonine, noradrenalin, γ-aminobutyric acid and acetylcholine) (Arrang et al., 1983; Bongers et al., 2007; Leurs et al., 2005; Lovenberg et al., 1999). The H3R has gained pharmaceutical interest as a potential drug target for the treatment of various important disorders like obesity, myocardial ischemia, migraine, inflammatory diseases and several CNS disorders like Alzheimer's disease, attention-deficit hyperactivity disorder and schizophrenia. Pitolisant (BF2.649, 1-{3-[3-(4-chlorophenyl)propoxy]propyl} piperidine, hydrochloride) is the first H3R inverse agonist to be introduced in the clinics. Its wakepromotion activity was evidenced in excessive diurnal sleepiness of patients with narcolepsy, Parkinson's disease or obstructive sleep apnea/hypopnea (Bongers et al., 2007; Lebois et al., 2011; Leurs et al., 2005; Schwartz, 2011). The human H3R gene consists of either three exons and two introns, or four exons and three introns spanning 5.5 kb on

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 79

2009; van Rijn et al., 2008). In addition, H4R dimeric structures that include homo- and hetero-oligomer formation and post-translational changes of the receptor might contribute to added pharmacological complexity for H4R ligands (Leurs et al., 2009; van Rijn et al.,

An estimated 1 million cases of breast cancer are diagnosed annually worldwide. Breast cancer is the most common neoplastic disease in women, and despite advances in early detection, about 30% of patients with early-stage breast cancer have recurrent disease, which is metastatic in most cases and whose cure is very limited showing a 5-year survival rate of

Histamine plays a critical role in the pathologic and physiologic aspects of the mammary gland, regulating cell growth, differentiation and functioning during development, pregnancy and lactation. Among monoamines, histamine demonstrates the greatest proliferative activity in breast cancer (Davio et al., 1994; Malinski et al., 1993; Wagner et al., 2003). Furthermore, histamine is increased in plasma and cancerous tissue derived from breast cancer patients compared to healthy group which is associated to an enhanced histidine decarboxylase (HDC) activity and a reduced diaminooxydase (DAO) activity that determine an imbalance between the synthesis and degradation of this monoamine. Histamine plasma level is dependent on the concentration of histamine in the tissues of ductal breast cancers, suggesting the participation of this monoamine in the development of this neoplasia (Reynolds et al., 1998; Sieja et al., 2005; von Mach-Szczypiński et al., 2009). A pilot study revealed that in samples of the same invasive ductal carcinoma patient, histamine peripheral blood levels tended to be reduced post-operatively (Kyriakidis et al., 2009). It was reported that in experimental mammary carcinomas, histamine becomes an autocrine growth factor capable of regulating cell proliferation via H1R and H2R, as one of the first steps responsible for the onset of malignant transformation. In this light, the *in vivo* treatment with H2R antagonists produced the complete remission of 70% of experimental tumours (Cricco et al., 1994; Davio et al., 1995; Rivera et al., 2000). Many reports indicate the presence of H1R and H2R in normal and malignant tissues as well as in different cell lines derived from human mammary gland. H2R produced an increase in cAMP levels while H1R was coupled to PLC activation in benign lesions. On the other hand, H1R was invariably linked to PLC pathway but H2R stimulated both transductional pathways in carcinomas (Davio et al., 1993, 1996). However, the clinical trials with H2R antagonists demonstrated

controversial results for breast cancer (Bolton et al., 2000; Parshad et al., 2005).

Recently, it was demonstrated that H3R and H4R are expressed in cell lines derived from human mammary gland (Medina et al., 2006). Histamine is capable of modulating cell proliferation exclusively in malignant cells while no effect on proliferation or expression of oncogenes related to cell growth is observed in non-tumorigenic HBL-100 cells (Davio et al., 2002; Medina et al., 2006). Furthermore, histamine modulated the proliferation of MDA-MB-231 breast cancer cells in a dose-dependent manner producing a significant decrease at 10 μmol.L-1 concentration whereas at lower concentrations increased proliferation moderately. The negative effect on proliferation was associated to the induction of cell cycle arrest in G2/M phase, differentiation and a significant increase in the number of apoptotic cells (Medina et al., 2006; Medina & Rivera, 2010b). Accordingly, by using pharmacological tools, results demonstrated that histamine increased MDA-MB-231 cell proliferation and also

2006, 2008).

**3. Histamine receptors in breast cancer** 

20% (Ferlay et al., 2010; Gonzalez-Angulo et al., 2007).

chromosome 20. Alternatively, the most 3' intron has been proposed to be a pseudo-intron as it is retained in the hH3R(445) isoform, but deleted in the hH3R(413) isoform. Overall similarity between the H3R and the H1R and H2R amounts to only 22% and 20%, respectively (Bongers et al., 2007; Coge et al., 2001a; Dy & Schneider, 2004; Leurs et al., 2005; Tardivel-Lacombe et al., 2001; Wellendorph et al., 2002).

The cloning of the human H3R has led to the discovery of many H3R isoforms generated through alternative splicing of the H3R mRNA. H3R can activate several signal transduction pathways, including Gi/o-dependent inhibition of adenylate cyclase that leads to inhibition of cAMP formation, activation of mitogen activated protein kinase pathway (MAPK), phospholipase A2, and Akt/protein kinase B, as well as the inhibition of the Na+/H+ exchanger and inhibition of K+-induced Ca2+ mobilization (Bongers et al., 2007; Coge et al., 2001a; Leurs et al., 2005; Wellendorph et al., 2002). A negative coupling to phosphoinositide turnover in the human gastric cell line HGT has also been described (Cherifi et al., 1992). Moreover, at least 20 isoforms of the human H3R have been described and they vary in the length of the third intracellular loop, their distinct CNS localization, differential signalling pathways and ligand binding affinity, which contribute to the heterogeneity of H3R pharmacology (Bongers et al., 2007; Coge et al., 2001a; Hancock et al., 2003; Leurs et al., 2005).

#### **2.4 Histamine H4R**

The identification by genomics-based approach of the human H4R by several groups has helped refine our understanding of histamine roles. It appeared to have a selective expression pattern restricted to medullary and peripheral hematopoietic cells including eosinophils, mast cells, DC, T cells and monocytes. Therefore, growing attention is directed towards the therapeutic development of H4R ligands for inflammation and immune disorders. Several lines of evidence suggest a role of the H4R in chronic inflammatory skin disease and the H4R might be a therapeutic target for diseases such as atopic dermatitis (Gutzmer et al., 2011). In addition, H4R was reported to be present on other cell types including intestinal epithelium, spleen, lung, stomach, CNS, nerves of nasal mucosa, enteric neurons and interestingly in cancer cells (Cianchi et al., 2005; Coge et al. 2001b; Connelly et al., 2009; Leurs et al. 2009; Lui et al., 2001; Medina et al., 2006; Morse et al. 2001; Nakamura et al., 2000; Nguyen et al. 2001; Oda et al., 2000). The significance of the H4R presence in various human tissues remains to be elucidated and therefore, new roles of H4R are still unrevealed (Leurs et al., 2009; Zampeli & Tiligada, 2009). The H4R cDNA was finally identified in the human genome database on the basis of its overall homology (37%, 58% in transmembrane regions) to the H3R sequence and it has a similar genomic structure. On the other hand, the homology with H1R and H2R is of approximately 19%. The human H4R gene that mapped to chromosome 18 is interrupted by two large introns and encodes a protein of 390 amino acids (Coge et al., 2001b; Leurs et al., 2009). H4R is coupled to Gαi/o proteins, inhibiting forskolin-induced cAMP formation (Nakamura et al., 2000; Oda et al., 2000). Additionally, stimulation of H4R leads to activation of MAPK and also increased calcium mobilization via pertussis toxin-sensitive pathway (Leurs et al., 2009; Morse et al., 2001). Isoforms have been described for the H4R which have different ligand binding and signalling characteristics. H4R splice variants [H4R (67) and H4R (302)] have a dominant negative effect on H4R (390) functionality, being able to retain it intracellularly and to

inactivate a population of H4R (390) presumably via hetero-oligomerization (Leurs et al.,

2009; van Rijn et al., 2008). In addition, H4R dimeric structures that include homo- and hetero-oligomer formation and post-translational changes of the receptor might contribute to added pharmacological complexity for H4R ligands (Leurs et al., 2009; van Rijn et al., 2006, 2008).

## **3. Histamine receptors in breast cancer**

78 Drug Development – A Case Study Based Insight into Modern Strategies

chromosome 20. Alternatively, the most 3' intron has been proposed to be a pseudo-intron as it is retained in the hH3R(445) isoform, but deleted in the hH3R(413) isoform. Overall similarity between the H3R and the H1R and H2R amounts to only 22% and 20%, respectively (Bongers et al., 2007; Coge et al., 2001a; Dy & Schneider, 2004; Leurs et al., 2005;

The cloning of the human H3R has led to the discovery of many H3R isoforms generated through alternative splicing of the H3R mRNA. H3R can activate several signal transduction pathways, including Gi/o-dependent inhibition of adenylate cyclase that leads to inhibition of cAMP formation, activation of mitogen activated protein kinase pathway (MAPK), phospholipase A2, and Akt/protein kinase B, as well as the inhibition of the Na+/H+ exchanger and inhibition of K+-induced Ca2+ mobilization (Bongers et al., 2007; Coge et al., 2001a; Leurs et al., 2005; Wellendorph et al., 2002). A negative coupling to phosphoinositide turnover in the human gastric cell line HGT has also been described (Cherifi et al., 1992). Moreover, at least 20 isoforms of the human H3R have been described and they vary in the length of the third intracellular loop, their distinct CNS localization, differential signalling pathways and ligand binding affinity, which contribute to the heterogeneity of H3R pharmacology (Bongers et al., 2007; Coge et al., 2001a; Hancock et al., 2003; Leurs et al.,

The identification by genomics-based approach of the human H4R by several groups has helped refine our understanding of histamine roles. It appeared to have a selective expression pattern restricted to medullary and peripheral hematopoietic cells including eosinophils, mast cells, DC, T cells and monocytes. Therefore, growing attention is directed towards the therapeutic development of H4R ligands for inflammation and immune disorders. Several lines of evidence suggest a role of the H4R in chronic inflammatory skin disease and the H4R might be a therapeutic target for diseases such as atopic dermatitis (Gutzmer et al., 2011). In addition, H4R was reported to be present on other cell types including intestinal epithelium, spleen, lung, stomach, CNS, nerves of nasal mucosa, enteric neurons and interestingly in cancer cells (Cianchi et al., 2005; Coge et al. 2001b; Connelly et al., 2009; Leurs et al. 2009; Lui et al., 2001; Medina et al., 2006; Morse et al. 2001; Nakamura et al., 2000; Nguyen et al. 2001; Oda et al., 2000). The significance of the H4R presence in various human tissues remains to be elucidated and therefore, new roles of H4R are still unrevealed (Leurs et al., 2009; Zampeli & Tiligada, 2009). The H4R cDNA was finally identified in the human genome database on the basis of its overall homology (37%, 58% in transmembrane regions) to the H3R sequence and it has a similar genomic structure. On the other hand, the homology with H1R and H2R is of approximately 19%. The human H4R gene that mapped to chromosome 18 is interrupted by two large introns and encodes a protein of 390 amino acids (Coge et al., 2001b; Leurs et al., 2009). H4R is coupled to Gαi/o proteins, inhibiting forskolin-induced cAMP formation (Nakamura et al., 2000; Oda et al., 2000). Additionally, stimulation of H4R leads to activation of MAPK and also increased calcium mobilization via pertussis toxin-sensitive pathway (Leurs et al., 2009; Morse et al., 2001). Isoforms have been described for the H4R which have different ligand binding and signalling characteristics. H4R splice variants [H4R (67) and H4R (302)] have a dominant negative effect on H4R (390) functionality, being able to retain it intracellularly and to inactivate a population of H4R (390) presumably via hetero-oligomerization (Leurs et al.,

Tardivel-Lacombe et al., 2001; Wellendorph et al., 2002).

2005).

**2.4 Histamine H4R** 

An estimated 1 million cases of breast cancer are diagnosed annually worldwide. Breast cancer is the most common neoplastic disease in women, and despite advances in early detection, about 30% of patients with early-stage breast cancer have recurrent disease, which is metastatic in most cases and whose cure is very limited showing a 5-year survival rate of 20% (Ferlay et al., 2010; Gonzalez-Angulo et al., 2007).

Histamine plays a critical role in the pathologic and physiologic aspects of the mammary gland, regulating cell growth, differentiation and functioning during development, pregnancy and lactation. Among monoamines, histamine demonstrates the greatest proliferative activity in breast cancer (Davio et al., 1994; Malinski et al., 1993; Wagner et al., 2003). Furthermore, histamine is increased in plasma and cancerous tissue derived from breast cancer patients compared to healthy group which is associated to an enhanced histidine decarboxylase (HDC) activity and a reduced diaminooxydase (DAO) activity that determine an imbalance between the synthesis and degradation of this monoamine. Histamine plasma level is dependent on the concentration of histamine in the tissues of ductal breast cancers, suggesting the participation of this monoamine in the development of this neoplasia (Reynolds et al., 1998; Sieja et al., 2005; von Mach-Szczypiński et al., 2009). A pilot study revealed that in samples of the same invasive ductal carcinoma patient, histamine peripheral blood levels tended to be reduced post-operatively (Kyriakidis et al., 2009). It was reported that in experimental mammary carcinomas, histamine becomes an autocrine growth factor capable of regulating cell proliferation via H1R and H2R, as one of the first steps responsible for the onset of malignant transformation. In this light, the *in vivo* treatment with H2R antagonists produced the complete remission of 70% of experimental tumours (Cricco et al., 1994; Davio et al., 1995; Rivera et al., 2000). Many reports indicate the presence of H1R and H2R in normal and malignant tissues as well as in different cell lines derived from human mammary gland. H2R produced an increase in cAMP levels while H1R was coupled to PLC activation in benign lesions. On the other hand, H1R was invariably linked to PLC pathway but H2R stimulated both transductional pathways in carcinomas (Davio et al., 1993, 1996). However, the clinical trials with H2R antagonists demonstrated controversial results for breast cancer (Bolton et al., 2000; Parshad et al., 2005).

Recently, it was demonstrated that H3R and H4R are expressed in cell lines derived from human mammary gland (Medina et al., 2006). Histamine is capable of modulating cell proliferation exclusively in malignant cells while no effect on proliferation or expression of oncogenes related to cell growth is observed in non-tumorigenic HBL-100 cells (Davio et al., 2002; Medina et al., 2006). Furthermore, histamine modulated the proliferation of MDA-MB-231 breast cancer cells in a dose-dependent manner producing a significant decrease at 10 μmol.L-1 concentration whereas at lower concentrations increased proliferation moderately. The negative effect on proliferation was associated to the induction of cell cycle arrest in G2/M phase, differentiation and a significant increase in the number of apoptotic cells (Medina et al., 2006; Medina & Rivera, 2010b). Accordingly, by using pharmacological tools, results demonstrated that histamine increased MDA-MB-231 cell proliferation and also

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 81

dependence on the grade of malignancy as they found to be significantly higher in those classified as high-grade malignant (Belcheva & Mishkova, 1995). Immunostaining and ELISA method also confirmed the presence of histamine in the cytoplasm of acute lymphocytic leukaemia (ALL) cells, and H1R antihistamines inhibited their clonogenic growth. There was no correlation between the clonogenic growth of ALL cells and their histamine content, suggesting that while histamine may be important for the clonogenic growth of ALL cells; other factors also affect their clonogenity (Malaviya et al., 1996). Furthermore, leukaemia cell lines such as U937, expressed histamine receptors and a switch of histamine receptor expression from H2R to H1R during differentiation of monocytes into

Most patients with acute myeloid leukaemia (AML) achieve complete remission after induction chemotherapy. Despite ensuing courses of consolidation chemotherapy, a large fraction of patients will experience relapses with poor prospects of long-term survival. Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) are effective activators of lymphocytes with anti-neoplastic properties, such as T-cells or natural killer (NK) cells, constituting the basis for their widespread used as immunotherapeutic agents in human neoplastic disease. The functions of intratumoural lymphocytes in many human malignant tumours are inhibited by reactive oxygen species (ROS), generated by adjacent monocytes/macrophages. *In vitro* data suggest that those immunotherapeutic cytokines only weakly activate T cells or NK cells in a reconstituted environment of oxidative stress and inhibitors of ROS formation or ROS scavengers synergize with IL-2 and IFN-alpha to activate T cells and NK cells. Recently, IL-2 therapy for solid neoplastic diseases and haematopoietic cancers has been supplemented with histamine dihydrochloride (Ceplene), a synthetic derivative of histamine, with the aim of counteracting immunosuppressive signals from monocytes/macrophages. Histamine dihydrochloride inhibits the formation of ROS that suppress the activation of T cells and NK cells by suppressing the activity of NADPH oxidase via H2R. When administered in addition to IL-2, histamine dihydrochloride enables the activation of these lymphocytes by the cytokine, resulting in tumour cell killing. This combination was recently approved within the EU as a remission maintenance immunotherapy in AML, as histamine dihydrochloride reduces myeloid cell-derived suppression of anti-leukemic lymphocytes, improving NK and T-cell activation. Further research in this area will shed light on the role of histamine with the aim to improve cancer immunotherapy efficacy (Hellstrand et al., 2000; Martner et al., 2010; Yang & Perry, 2011).

Gynaecologic cancers encompass a remarkably heterogeneous group of tumours: cervical, ovarian, uterine, vaginal, and vulvar cancer. It has been postulated that histamine plays a critical role in proliferation of normal and cancer tissues, including the mammary gland,

In the murine uterus, the rapidly dividing epithelial cells of the endometrium can be defined as the major sources of histamine. In these cells the level of HDC expression is controlled mainly by progesterone-mediated signals which, interestingly, induce maximal level of

*In vitro* studies showed that histamine may play an important role in follicular development and ovulation via H1R and H2R in women, acting as apoptosis inducer, taking part in the selection process of the dominant follicle and stimulating ovulation (Szukiewicz et al., 2007).

macrophages is observed (Wang et al., 2000).

**5. Histamine receptors in gynaecologic cancers** 

HDC expression on the day of implantation (Pós et al., 2004).

ovarian and endometrium.

migration via H3R. In contrast, clobenpropit and VUF8430 treatments significantly decreased proliferation. This outcome was associated to an induction of apoptosis determined by Annexin-V staining and TdT-mediated UTP-biotin Nick End labelling (TUNEL) assay, which was blocked by the specific H4R antagonist JNJ7777120. Also H4R agonists exerted a 2.5-fold increase in the cell senescence while reduced migration (Medina et al., 2008, 2010c, 2011b). Furthermore, histamine differentially regulates expression and activity of matrix metalloproteinases, cell migration and invasiveness through H2R and H4R in MDA-MB-231 cells modulating H2O2 intracellular levels (Cricco et al., 2011).

In addition, histamine at all doses tested, decreased the proliferation of a more differentiated breast cancer cell line, MCF-7, through the stimulation of the four histamine receptor subtypes exhibiting a higher effect through the H4R. Treatment of MCF-7 cells with the H4R agonists, inhibited cell proliferation and increased apoptosis and senescence (Medina et al., 2011b). These results represent the first report about the expression of H3R and H4R in human breast cells and interestingly show that the H4R is involved in the regulation of breast cancer cell proliferation, apoptosis, senescence, migration and invasion.

Recent results obtained with the orthotopic xenograft tumours of the highly invasive human breast cancer line MDA-MB-231 in immune deficient nude mice indicate that the H4R was the major histamine receptor expressed in the tumour. Remarkably, *in vivo* JNJ7777120 treatment (10 mg.kg-1, *p.o.*, daily administration) significantly decreased lung metastases, indicating that H4R may be involved in the metastatic process (Medina & Rivera, 2010b). In addition, *in vivo* clozapine treatment (1 mg.kg-1, *s.c.*, daily administration) significantly decreased tumour growth while enhanced survival of bearing tumour mice (Martinel Lamas et al., unpublished data).

Recent data indicate that H3R and H4R are expressed in human biopsies of benign lesions and breast carcinomas being the level of their expression significantly higher in carcinomas, confirming that H3R and H4R are present not only in cell lines but also in the human breast tissue. Furthermore, the expression of H3R is highly correlated with proliferation and histamine production in malignant lesions while the 50% of malignant lesions expressed H4R, all of them corresponding to metastases or high invasive tumours (Medina et al., 2008).

The identification of histamine receptor subtypes and the elucidation of their role in the development and growth of human mammary carcinomas may represent an essential clue for advances in breast cancer treatment. The presented evidences contribute to the identification of molecules involved in breast carcinogenesis and confirm the role of H4R in the regulation of breast cancer growth and progression representing a novel molecular target for new therapeutic approach.

### **4. Histamine receptors in lymphomas and leukaemia**

There is increasing evidence that histamine plays a role in cell differentiation and proliferation in several of normal tissues and in a wide range of tumours, including haematological neoplasias.

After an initial work in the late 1970s showing that histamine is able to induce haematopoietic stem cell proliferation via H2R (Byron, 1977), a real rush broke out in searching for further effects of histamine in haematopoiesis and haematological neoplasias. The histamine levels were determined in lymph nodes of patients with malignant lymphomas, Hodking´s disease (HD) or non-Hodking lymphomas (NHL), and in all cases the values were higher than in controls. In patients with NHL, these levels showed

migration via H3R. In contrast, clobenpropit and VUF8430 treatments significantly decreased proliferation. This outcome was associated to an induction of apoptosis determined by Annexin-V staining and TdT-mediated UTP-biotin Nick End labelling (TUNEL) assay, which was blocked by the specific H4R antagonist JNJ7777120. Also H4R agonists exerted a 2.5-fold increase in the cell senescence while reduced migration (Medina et al., 2008, 2010c, 2011b). Furthermore, histamine differentially regulates expression and activity of matrix metalloproteinases, cell migration and invasiveness through H2R and H4R

In addition, histamine at all doses tested, decreased the proliferation of a more differentiated breast cancer cell line, MCF-7, through the stimulation of the four histamine receptor subtypes exhibiting a higher effect through the H4R. Treatment of MCF-7 cells with the H4R agonists, inhibited cell proliferation and increased apoptosis and senescence (Medina et al., 2011b). These results represent the first report about the expression of H3R and H4R in human breast cells and interestingly show that the H4R is involved in the regulation of

Recent results obtained with the orthotopic xenograft tumours of the highly invasive human breast cancer line MDA-MB-231 in immune deficient nude mice indicate that the H4R was the major histamine receptor expressed in the tumour. Remarkably, *in vivo* JNJ7777120 treatment (10 mg.kg-1, *p.o.*, daily administration) significantly decreased lung metastases, indicating that H4R may be involved in the metastatic process (Medina & Rivera, 2010b). In addition, *in vivo* clozapine treatment (1 mg.kg-1, *s.c.*, daily administration) significantly decreased tumour growth while enhanced survival of bearing tumour mice (Martinel Lamas

Recent data indicate that H3R and H4R are expressed in human biopsies of benign lesions and breast carcinomas being the level of their expression significantly higher in carcinomas, confirming that H3R and H4R are present not only in cell lines but also in the human breast tissue. Furthermore, the expression of H3R is highly correlated with proliferation and histamine production in malignant lesions while the 50% of malignant lesions expressed H4R, all of them corresponding to metastases or high invasive tumours (Medina et al., 2008). The identification of histamine receptor subtypes and the elucidation of their role in the development and growth of human mammary carcinomas may represent an essential clue for advances in breast cancer treatment. The presented evidences contribute to the identification of molecules involved in breast carcinogenesis and confirm the role of H4R in the regulation of breast cancer growth and progression representing a novel molecular

There is increasing evidence that histamine plays a role in cell differentiation and proliferation in several of normal tissues and in a wide range of tumours, including

After an initial work in the late 1970s showing that histamine is able to induce haematopoietic stem cell proliferation via H2R (Byron, 1977), a real rush broke out in searching for further effects of histamine in haematopoiesis and haematological neoplasias. The histamine levels were determined in lymph nodes of patients with malignant lymphomas, Hodking´s disease (HD) or non-Hodking lymphomas (NHL), and in all cases the values were higher than in controls. In patients with NHL, these levels showed

in MDA-MB-231 cells modulating H2O2 intracellular levels (Cricco et al., 2011).

breast cancer cell proliferation, apoptosis, senescence, migration and invasion.

et al., unpublished data).

target for new therapeutic approach.

haematological neoplasias.

**4. Histamine receptors in lymphomas and leukaemia** 

dependence on the grade of malignancy as they found to be significantly higher in those classified as high-grade malignant (Belcheva & Mishkova, 1995). Immunostaining and ELISA method also confirmed the presence of histamine in the cytoplasm of acute lymphocytic leukaemia (ALL) cells, and H1R antihistamines inhibited their clonogenic growth. There was no correlation between the clonogenic growth of ALL cells and their histamine content, suggesting that while histamine may be important for the clonogenic growth of ALL cells; other factors also affect their clonogenity (Malaviya et al., 1996). Furthermore, leukaemia cell lines such as U937, expressed histamine receptors and a switch of histamine receptor expression from H2R to H1R during differentiation of monocytes into macrophages is observed (Wang et al., 2000).

Most patients with acute myeloid leukaemia (AML) achieve complete remission after induction chemotherapy. Despite ensuing courses of consolidation chemotherapy, a large fraction of patients will experience relapses with poor prospects of long-term survival. Interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) are effective activators of lymphocytes with anti-neoplastic properties, such as T-cells or natural killer (NK) cells, constituting the basis for their widespread used as immunotherapeutic agents in human neoplastic disease. The functions of intratumoural lymphocytes in many human malignant tumours are inhibited by reactive oxygen species (ROS), generated by adjacent monocytes/macrophages. *In vitro* data suggest that those immunotherapeutic cytokines only weakly activate T cells or NK cells in a reconstituted environment of oxidative stress and inhibitors of ROS formation or ROS scavengers synergize with IL-2 and IFN-alpha to activate T cells and NK cells. Recently, IL-2 therapy for solid neoplastic diseases and haematopoietic cancers has been supplemented with histamine dihydrochloride (Ceplene), a synthetic derivative of histamine, with the aim of counteracting immunosuppressive signals from monocytes/macrophages. Histamine dihydrochloride inhibits the formation of ROS that suppress the activation of T cells and NK cells by suppressing the activity of NADPH oxidase via H2R. When administered in addition to IL-2, histamine dihydrochloride enables the activation of these lymphocytes by the cytokine, resulting in tumour cell killing. This combination was recently approved within the EU as a remission maintenance immunotherapy in AML, as histamine dihydrochloride reduces myeloid cell-derived suppression of anti-leukemic lymphocytes, improving NK and T-cell activation. Further research in this area will shed light on the role of histamine with the aim to improve cancer immunotherapy efficacy (Hellstrand et al., 2000; Martner et al., 2010; Yang & Perry, 2011).

## **5. Histamine receptors in gynaecologic cancers**

Gynaecologic cancers encompass a remarkably heterogeneous group of tumours: cervical, ovarian, uterine, vaginal, and vulvar cancer. It has been postulated that histamine plays a critical role in proliferation of normal and cancer tissues, including the mammary gland, ovarian and endometrium.

In the murine uterus, the rapidly dividing epithelial cells of the endometrium can be defined as the major sources of histamine. In these cells the level of HDC expression is controlled mainly by progesterone-mediated signals which, interestingly, induce maximal level of HDC expression on the day of implantation (Pós et al., 2004).

*In vitro* studies showed that histamine may play an important role in follicular development and ovulation via H1R and H2R in women, acting as apoptosis inducer, taking part in the selection process of the dominant follicle and stimulating ovulation (Szukiewicz et al., 2007).

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 83

a decreased of H1R and H4R protein levels in colorectal cancer while the levels of the H2R

It was described that the H1R antagonist, loratadine, inhibited proliferation and enhanced radiosensitivity in human colon cancer cells (Soule et al., 2010). Also the H2R seems to be implicated in the proliferation of colon cancer. In 1994 Adams, showed that *in vivo* and in two human colonic adenocarcinoma cell lines, C170 and LIM2412, cell proliferation induced by histamine in a dose dependent manner was blocked by H2R antagonist, cimetidine (Adams et al., 1994). Ranitidine, another H2R antagonist, also showed to extend the survival of patients who were under surgery of colorectal cancer (Nielsen et al., 2002). It is well known the effects of histamine in the immune system, according to this it was demonstrated that patients receiving cimetidine or famotidine before curative resection augmented the probabilities of having tumour infiltrating lymphocytes in their tumours than control patients (Adams & Morris, 1996; Kapoor et al., 2005). Furthermore, earlier studies demonstrated that histamine induced *in vitro* and *in vivo* cell proliferation and this outcome was blocked by H2R antagonists (Adams et al., 1994; Cianchi et al., 2005). This effect was associated with the attenuation of anti-tumour cytokine expression in the tumour microenvironment exerted by histamine, thus resulting in stimulated colorectal cancer growth (Takahashi et al., 2001; Tomita & Okabe, 2005). In addition, H2R antagonist significantly suppressed the growth of tumour implants in mice by inhibiting angiogenesis

As it was described above, the expression of the H4R seems to be suppressed in human colorectal cancer. It was also demonstrated that the levels of the H4R are reduced in advanced colorectal cancer compared with those in an initiating state, which suggest that the H4R expression is regulated during the progression of the disease (Fang et al., 2011). The stimulation *in vitro* of the H4R by a specific agonist induced an augmented expression of the p21Cip1 and p27 Kip1 proteins, producing an increase of arrested cells in the G1 phase. It has been proposed that prostaglandin E2 (PGE-2), the main product of the cyclooxygenase-2 activity, is implicated in colorectal cancer development. In this line, it has been demonstrated that histamine is fully implicated in the production of PGE-2 by its two receptors H2R and H4R in two human colon carcinoma cell lines (Cianchi et al., 2005). Histamine effect can be blocked by zolantidine, an H2R antagonist, and also by JNJ7777120, an H4R antagonist, whereas mepyramine, an H1R antagonist, has no effect on the production of PGE-2. Furthermore, JNJ7777120 inhibited the cell growth induced by histamine in three different human colon cancer cell lines and also inhibited the histamine-mediated increase in VEGF in two cell lines. Combined treatment with zolantidine (an H2R antagonist) and JNJ7777120 determined an additive effect on reducing the histamine-induced VEGF production and histamine-stimulated proliferation (Cianchi et al., 2005), suggesting the

Malignant melanoma arises from epidermal melanocytes and despite being the cause of less than 5% of skin cancers, it is responsible for the large majority of skin cancer deaths (Ferlay et al., 2010). Early detection is vital for long-term survival, given that there is a direct

Melanoma cells but not normal melanocytes contain large amounts of histamine that has been found to accelerate malignant growth (Pós et al., 2004). The absence of expression of

correlation between tumour thickness and mortality (Cummins et al., 2006).

were not modified compared to normal colon mucosa (Boer et al., 2008).

via reducing VEGF expression (Tomita et al., 2003).

involvement of H4R in colon carcinogenesis (Boer et al., 2008).

**7. Histamine receptors in melanoma** 

Interestingly, histamine content increased unequivocally in ovarian, cervical and endometrial carcinoma in comparison with their adjoining normal tissues, suggesting the participation of histamine in carcinogenesis. Besides, exogenous histamine, at micromolar concentration, stimulated proliferation of human ovarian cancer cell line SKOV-3 (Batra & Fadeel, 1994; Chanda & Ganguly, 1995). Preliminary results show that H4R is expressed in primary and metastatic ovarian carcinoma and also in gallbladder cancer (Medina & Rivera, 2010b).

Histamine levels within ovarian tissue during the oestrus may correspond to cyclic changes of mast cells content and distribution in the ovary, suggesting an involvement of these cells in local regulation of ovarian function (Adyin et al., 1998; Nakamura et al., 1987). Interestingly, mast cells can typically be found in the peritumoural stroma of cervix carcinomas, as well as in many other cancers. Furthermore, high numbers of active, degranulated mast cells have been described in HPV infections and cervical intraepithelial neoplasias (Cabanillas-Saez et al., 2002; Demitsu et al., 2002). Hence, a functional relationship between mast cells and tumour cells has been proposed, where mast cells are involved in stimulating tumour growth and progression by enhancing angiogenesis, immunosuppression, mitogenesis, and metastasis (Chang et al., 2006). Mast cell activation leads to the release of inflammatory mediators, including histamine. Increased histamine levels have been described in the cervix lesions, where they have been associated with tumour growth and progression. Moreover, histamine receptors have been reported in different cell lines and tissues derived from experimental and human cervical neoplasias. The functional significance of immune cell infiltration of a tumour, specifically of mast cells located at the periphery of several neoplasias, is still a matter of controversy. Histamine acting via H1R in cervical cancer cells could be pro-migratory, but when acting via H4R could inhibit migration. On the other hand, other results also showed that cervical carcinoma cell mediators can activate mast cells to degranulate, demonstrating an active and dynamic cross-talk between tumour cells and infiltrating mast cells as shown in morphologic studies of neoplastic tissues (Rudolph et al., 2008).

In the light of these results, further investigations have to be done in order to elucidate the physiological role of histamine receptors on cell proliferation, as well as its implication in gynaecologic cancer progression with a potential interest for cancer treatment.

## **6. Histamine receptors in colorectal cancer**

Colorectal cancer is one of the leading causes of cancer death among both men and women worldwide (Ferlay et al., 2010). It has been previously described that the histamine catabolising enzymes, DAO or histamine N-methyltransferase (HNMT), activities were significantly lower in adenoma tissue than in healthy mucosa in the same patients (Kuefner et al., 2008). Furthermore, HDC expression and its activity are increased in many human tumours including colorectal cancer (Cianchi et al., 2005; Masini et al., 2005; Reynolds et al., 1997). The levels of histamine were elevated in colon carcinoma and this is directly related to an increase in HDC expression and a decrease in DAO activity (Chanda & Ganguly, 1987). Also, the distribution of histamine receptors in the normal intestinal tract was reported (Sander et al., 2006). It was showed the expression pattern of H1R, H2R and H4R in intestinal tract, receptors that were over expressed in the colon of patients with irritable bowel syndrome and food allergies. Furthermore, the H3R was not detected in intestinal tissue (Sander et al., 2006). This data was further confirmed by Boer K et al, that also demonstrated

Interestingly, histamine content increased unequivocally in ovarian, cervical and endometrial carcinoma in comparison with their adjoining normal tissues, suggesting the participation of histamine in carcinogenesis. Besides, exogenous histamine, at micromolar concentration, stimulated proliferation of human ovarian cancer cell line SKOV-3 (Batra & Fadeel, 1994; Chanda & Ganguly, 1995). Preliminary results show that H4R is expressed in primary and metastatic ovarian carcinoma and also in gallbladder cancer (Medina & Rivera,

Histamine levels within ovarian tissue during the oestrus may correspond to cyclic changes of mast cells content and distribution in the ovary, suggesting an involvement of these cells in local regulation of ovarian function (Adyin et al., 1998; Nakamura et al., 1987). Interestingly, mast cells can typically be found in the peritumoural stroma of cervix carcinomas, as well as in many other cancers. Furthermore, high numbers of active, degranulated mast cells have been described in HPV infections and cervical intraepithelial neoplasias (Cabanillas-Saez et al., 2002; Demitsu et al., 2002). Hence, a functional relationship between mast cells and tumour cells has been proposed, where mast cells are involved in stimulating tumour growth and progression by enhancing angiogenesis, immunosuppression, mitogenesis, and metastasis (Chang et al., 2006). Mast cell activation leads to the release of inflammatory mediators, including histamine. Increased histamine levels have been described in the cervix lesions, where they have been associated with tumour growth and progression. Moreover, histamine receptors have been reported in different cell lines and tissues derived from experimental and human cervical neoplasias. The functional significance of immune cell infiltration of a tumour, specifically of mast cells located at the periphery of several neoplasias, is still a matter of controversy. Histamine acting via H1R in cervical cancer cells could be pro-migratory, but when acting via H4R could inhibit migration. On the other hand, other results also showed that cervical carcinoma cell mediators can activate mast cells to degranulate, demonstrating an active and dynamic cross-talk between tumour cells and infiltrating mast cells as shown in

In the light of these results, further investigations have to be done in order to elucidate the physiological role of histamine receptors on cell proliferation, as well as its implication in

Colorectal cancer is one of the leading causes of cancer death among both men and women worldwide (Ferlay et al., 2010). It has been previously described that the histamine catabolising enzymes, DAO or histamine N-methyltransferase (HNMT), activities were significantly lower in adenoma tissue than in healthy mucosa in the same patients (Kuefner et al., 2008). Furthermore, HDC expression and its activity are increased in many human tumours including colorectal cancer (Cianchi et al., 2005; Masini et al., 2005; Reynolds et al., 1997). The levels of histamine were elevated in colon carcinoma and this is directly related to an increase in HDC expression and a decrease in DAO activity (Chanda & Ganguly, 1987). Also, the distribution of histamine receptors in the normal intestinal tract was reported (Sander et al., 2006). It was showed the expression pattern of H1R, H2R and H4R in intestinal tract, receptors that were over expressed in the colon of patients with irritable bowel syndrome and food allergies. Furthermore, the H3R was not detected in intestinal tissue (Sander et al., 2006). This data was further confirmed by Boer K et al, that also demonstrated

gynaecologic cancer progression with a potential interest for cancer treatment.

morphologic studies of neoplastic tissues (Rudolph et al., 2008).

**6. Histamine receptors in colorectal cancer** 

2010b).

a decreased of H1R and H4R protein levels in colorectal cancer while the levels of the H2R were not modified compared to normal colon mucosa (Boer et al., 2008).

It was described that the H1R antagonist, loratadine, inhibited proliferation and enhanced radiosensitivity in human colon cancer cells (Soule et al., 2010). Also the H2R seems to be implicated in the proliferation of colon cancer. In 1994 Adams, showed that *in vivo* and in two human colonic adenocarcinoma cell lines, C170 and LIM2412, cell proliferation induced by histamine in a dose dependent manner was blocked by H2R antagonist, cimetidine (Adams et al., 1994). Ranitidine, another H2R antagonist, also showed to extend the survival of patients who were under surgery of colorectal cancer (Nielsen et al., 2002). It is well known the effects of histamine in the immune system, according to this it was demonstrated that patients receiving cimetidine or famotidine before curative resection augmented the probabilities of having tumour infiltrating lymphocytes in their tumours than control patients (Adams & Morris, 1996; Kapoor et al., 2005). Furthermore, earlier studies demonstrated that histamine induced *in vitro* and *in vivo* cell proliferation and this outcome was blocked by H2R antagonists (Adams et al., 1994; Cianchi et al., 2005). This effect was associated with the attenuation of anti-tumour cytokine expression in the tumour microenvironment exerted by histamine, thus resulting in stimulated colorectal cancer growth (Takahashi et al., 2001; Tomita & Okabe, 2005). In addition, H2R antagonist significantly suppressed the growth of tumour implants in mice by inhibiting angiogenesis via reducing VEGF expression (Tomita et al., 2003).

As it was described above, the expression of the H4R seems to be suppressed in human colorectal cancer. It was also demonstrated that the levels of the H4R are reduced in advanced colorectal cancer compared with those in an initiating state, which suggest that the H4R expression is regulated during the progression of the disease (Fang et al., 2011). The stimulation *in vitro* of the H4R by a specific agonist induced an augmented expression of the p21Cip1 and p27 Kip1 proteins, producing an increase of arrested cells in the G1 phase. It has been proposed that prostaglandin E2 (PGE-2), the main product of the cyclooxygenase-2 activity, is implicated in colorectal cancer development. In this line, it has been demonstrated that histamine is fully implicated in the production of PGE-2 by its two receptors H2R and H4R in two human colon carcinoma cell lines (Cianchi et al., 2005). Histamine effect can be blocked by zolantidine, an H2R antagonist, and also by JNJ7777120, an H4R antagonist, whereas mepyramine, an H1R antagonist, has no effect on the production of PGE-2. Furthermore, JNJ7777120 inhibited the cell growth induced by histamine in three different human colon cancer cell lines and also inhibited the histamine-mediated increase in VEGF in two cell lines. Combined treatment with zolantidine (an H2R antagonist) and JNJ7777120 determined an additive effect on reducing the histamine-induced VEGF production and histamine-stimulated proliferation (Cianchi et al., 2005), suggesting the involvement of H4R in colon carcinogenesis (Boer et al., 2008).

#### **7. Histamine receptors in melanoma**

Malignant melanoma arises from epidermal melanocytes and despite being the cause of less than 5% of skin cancers, it is responsible for the large majority of skin cancer deaths (Ferlay et al., 2010). Early detection is vital for long-term survival, given that there is a direct correlation between tumour thickness and mortality (Cummins et al., 2006).

Melanoma cells but not normal melanocytes contain large amounts of histamine that has been found to accelerate malignant growth (Pós et al., 2004). The absence of expression of

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 85

mast cell activation initiates upon ultraviolet-B irradiation, which triggers histamine

Moreover, the role of histamine in local immune reactions was further supported by the results of Hellstrand et al., who found that histamine can inhibit the ROS formation of monocytes/macrophages in the tumour (Hellstrand et al., 2000). This may explain the clinical benefit demonstrated by histamine (Ceplene) as an adjuvant to immunotherapy with IL-2 in several phase II and III clinical trials in metastatic melanoma (Agarwala, 2002). The addition of histamine dihydrochloride to an outpatient regimen of IL-2 is safe and well tolerated and demonstrates a survival advantage over IL-2 alone (9.4 vs. 5.1 months) in melanoma patients with liver metastases (Agarwala, 2002). However, a second confirmatory

phase III study failed to show any survival benefit for those patients (Naredi, 2002).

of antioxidant enzymes leads to the cell proliferation inhibition (Medina et al., 2009).

these cell lines but also in human melanoma tissue (Massari et al., 2011).

potential target for new drug development for the treatment of this disease.

**8. Histamine as a potential adjuvant to radiotherapy** 

**8.1 Radioprotectors** 

Besides, Medina et al. showed that exogenous histamine modulated the activity of the antioxidant enzymes, increasing superoxide dismutase while decreasing catalase activity in WM35 melanoma cells. Accordingly, histamine treatment markedly augmented the levels of hydrogen peroxide and diminished those of superoxide anion, indicating that the imbalance

Furthermore, it was demonstrated that WM35 and M1/15 melanoma cells express H4R at the mRNA and protein level. By using histamine agonists and antagonists it was shown that the inhibitory effect of histamine on proliferation was in part mediated through the stimulation of the H4R. Treatment with a specific H4R antagonist, JNJ7777120 and the use of siRNA specific for H4R mRNA blocked the decrease in proliferation triggered by the H4R agonists. Furthermore, the decrease in proliferation exerted by H4R agonists was associated with a 2-fold induction of cell senescence and an increase in melanogenesis that is a differentiation marker on these cells (Massari et al., 2011). Current studies indicate that the H4R is expressed in the 42% of human melanoma biopsies of different histopathological types, showing cytoplasmic localization and confirming that the H4R is present not only in

The *in vivo* subcutaneous daily 1 mg.kg-1 histamine or 1 mg.kg-1 clozapine (H4R agonist) injections of M1/15 melanoma cell tumour bearing nude mice showed a survival increase vs. control group (treated with saline solution). Besides, results showed an antitumour effect of histamine and clozapine, including suppression of tumour growth (Massari et al., unpublished data). Further studies are needed to corroborate the H4R importance as

Radiotherapy is the most common modality for treating human cancers and relies on ionising radiation induced DNA damage to kill malignant cells. Eighty percent of cancer patients need radiotherapy at some time or other, either for curative or palliative purpose. To optimise results, a cautious balance between the total dose of radiotherapy delivered and the threshold limit of the surrounding normal critical tissues is required. In order to obtain better tumour control with a higher dose, the normal tissues should be protected against radiation damage. Therefore, the role of radioprotective compounds is of utmost importance in clinical radiotherapy *(*Hall & Giaccia, 2006; Mah et at., 2011). Ionising radiation causes damage to living tissues through a series of molecular events. DNA double-strand breaks (DSBs), which are exceptionally lethal lesions, can be formed either by direct energy

secretion acts as a cellular immunity suppressor (Chang et al., 2006).

HDC in Mel-5 positive melanocytes isolated from skin samples of healthy persons, suggest that the level of HDC is strongly associated with malignancy in the skin (Haak-Frendscho et al., 2000). As a functional consequence of the inhibition of HDC protein synthesis, specific antisense oligonucleotide strongly (> 50%) decreased the proliferation rate of both WM938/B and HT168/91 human melanoma cells. Similar effects were found with other two melanoma cell lines WM35 and M1/15, suggesting that endogenous histamine may act as an autocrine growth factor (Hegyesi et al., 2000). On the other hand, overexpression of HDC markedly accelerated tumour growth and increased metastatic colony-forming potential along with rising levels of local histamine production that was correlated with tumour H2R and rho-C expression in mouse melanoma (Pós Z et al., 2005).

It has been previously reported the expression of H1R, H2R and H3R in melanoma cell lines (Hegyesi et al., 2005). In addition, it was described that in human melanoma cells, histamine acting through the H1R decreases cell proliferation, whereas it enhances growth when acting through the H2R (Lázar-Molnar et al., 2002). Furthermore, there is no evidence of mitogenic signalling through the H3R in human melanoma (Hegyesi et al., 2005).

H1R function is involved in chemotaxis via PLC activation, and its subsequent intracellular calcium mobilization. Proliferation assays showed that histamine exerted a concentration dependent dual effect on proliferation of the WM35 primary melanoma cell line. High concentrations of histamine (10-5 M) had an inhibitory effect while lower concentrations (10- 7 M) increased colony formation. Similar results were achieved when using H1R agonist 2-(3 fluoromethylphenyl)histamine and H2R agonist arpromidine, respectively. The use of ranitidine, famotidine and cimetidine, all H2R specific antagonists, abolished the stimulatory effect of histamine on cell proliferation, indicating the participation of H2R in this mitogenic role of histamine. Second messenger measurement indicated that H2R are linked to cAMP production, thus suggesting an involvement of PKA in the mitogenic pathway triggered in this system, which is corroborated by the fact that forskolin and permeable cAMP analogues also produce a dose-dependent increase on cell proliferation (Lázar-Molnar et al., 2002).

Numerous *in vivo* studies employing animal models bearing syngenic or xenogenic melanoma grafts demonstrated that both endogenous and exogenous histamine have the ability to stimulate tumour growth while H2R antagonists (e.g. cimetidine, famotidine, roxatidine) inhibited this effect (Pós et al., 2005; Szincsák et al., 2002; Tomita et al., 2005; Uçar, 1991). Additionally, H2R antagonists stimulated melanogenesis and inhibited proliferation in B16-C3 mouse melanoma cells (Uçar, 1991). It was also found that melanoma tumour growth was not modulated by *in vivo* histamine treatment while treatment with terfenadine, an H1R antagonist, *in vitro* induced melanoma cell death by apoptosis and *in vivo* significantly inhibited tumour growth in murine models (Blaya et al., 2010).

Differences between melanoma cells in their capacity to produce and degrade histamine could explain the different sensitivities of melanoma cell types to exogenous histamine treatment. Moreover, there is evidence that cytokines can influence HDC expression and activity. It has been shown that there is a regulation loop between interleukin 6 (IL-6) and histamine: histamine increased IL-6 expression and secretion in metastatic lines via the H1R, and IL-6 treatment increased the HDC and histamine content in primary melanoma lines (Lázar-Molnar et al., 2002). Interferon-gamma (IFN-gamma) produced by surrounding immune cells decreases HDC expression, affecting melanoma growth and also impairs antitumour activity of the immune system, then contributing to the escape of melanoma cells from immunosurveillance (Horváth et al., 1999; Heninger et al., 2000). Furthermore, mast cell activation initiates upon ultraviolet-B irradiation, which triggers histamine secretion acts as a cellular immunity suppressor (Chang et al., 2006).

Moreover, the role of histamine in local immune reactions was further supported by the results of Hellstrand et al., who found that histamine can inhibit the ROS formation of monocytes/macrophages in the tumour (Hellstrand et al., 2000). This may explain the clinical benefit demonstrated by histamine (Ceplene) as an adjuvant to immunotherapy with IL-2 in several phase II and III clinical trials in metastatic melanoma (Agarwala, 2002). The addition of histamine dihydrochloride to an outpatient regimen of IL-2 is safe and well tolerated and demonstrates a survival advantage over IL-2 alone (9.4 vs. 5.1 months) in melanoma patients with liver metastases (Agarwala, 2002). However, a second confirmatory phase III study failed to show any survival benefit for those patients (Naredi, 2002).

Besides, Medina et al. showed that exogenous histamine modulated the activity of the antioxidant enzymes, increasing superoxide dismutase while decreasing catalase activity in WM35 melanoma cells. Accordingly, histamine treatment markedly augmented the levels of hydrogen peroxide and diminished those of superoxide anion, indicating that the imbalance of antioxidant enzymes leads to the cell proliferation inhibition (Medina et al., 2009).

Furthermore, it was demonstrated that WM35 and M1/15 melanoma cells express H4R at the mRNA and protein level. By using histamine agonists and antagonists it was shown that the inhibitory effect of histamine on proliferation was in part mediated through the stimulation of the H4R. Treatment with a specific H4R antagonist, JNJ7777120 and the use of siRNA specific for H4R mRNA blocked the decrease in proliferation triggered by the H4R agonists. Furthermore, the decrease in proliferation exerted by H4R agonists was associated with a 2-fold induction of cell senescence and an increase in melanogenesis that is a differentiation marker on these cells (Massari et al., 2011). Current studies indicate that the H4R is expressed in the 42% of human melanoma biopsies of different histopathological types, showing cytoplasmic localization and confirming that the H4R is present not only in these cell lines but also in human melanoma tissue (Massari et al., 2011).

The *in vivo* subcutaneous daily 1 mg.kg-1 histamine or 1 mg.kg-1 clozapine (H4R agonist) injections of M1/15 melanoma cell tumour bearing nude mice showed a survival increase vs. control group (treated with saline solution). Besides, results showed an antitumour effect of histamine and clozapine, including suppression of tumour growth (Massari et al., unpublished data). Further studies are needed to corroborate the H4R importance as potential target for new drug development for the treatment of this disease.

#### **8. Histamine as a potential adjuvant to radiotherapy**

#### **8.1 Radioprotectors**

84 Drug Development – A Case Study Based Insight into Modern Strategies

HDC in Mel-5 positive melanocytes isolated from skin samples of healthy persons, suggest that the level of HDC is strongly associated with malignancy in the skin (Haak-Frendscho et al., 2000). As a functional consequence of the inhibition of HDC protein synthesis, specific antisense oligonucleotide strongly (> 50%) decreased the proliferation rate of both WM938/B and HT168/91 human melanoma cells. Similar effects were found with other two melanoma cell lines WM35 and M1/15, suggesting that endogenous histamine may act as an autocrine growth factor (Hegyesi et al., 2000). On the other hand, overexpression of HDC markedly accelerated tumour growth and increased metastatic colony-forming potential along with rising levels of local histamine production that was correlated with tumour H2R

It has been previously reported the expression of H1R, H2R and H3R in melanoma cell lines (Hegyesi et al., 2005). In addition, it was described that in human melanoma cells, histamine acting through the H1R decreases cell proliferation, whereas it enhances growth when acting through the H2R (Lázar-Molnar et al., 2002). Furthermore, there is no evidence of mitogenic

H1R function is involved in chemotaxis via PLC activation, and its subsequent intracellular calcium mobilization. Proliferation assays showed that histamine exerted a concentration dependent dual effect on proliferation of the WM35 primary melanoma cell line. High concentrations of histamine (10-5 M) had an inhibitory effect while lower concentrations (10- 7 M) increased colony formation. Similar results were achieved when using H1R agonist 2-(3 fluoromethylphenyl)histamine and H2R agonist arpromidine, respectively. The use of ranitidine, famotidine and cimetidine, all H2R specific antagonists, abolished the stimulatory effect of histamine on cell proliferation, indicating the participation of H2R in this mitogenic role of histamine. Second messenger measurement indicated that H2R are linked to cAMP production, thus suggesting an involvement of PKA in the mitogenic pathway triggered in this system, which is corroborated by the fact that forskolin and permeable cAMP analogues also produce a dose-dependent increase on cell proliferation (Lázar-Molnar et al., 2002). Numerous *in vivo* studies employing animal models bearing syngenic or xenogenic melanoma grafts demonstrated that both endogenous and exogenous histamine have the ability to stimulate tumour growth while H2R antagonists (e.g. cimetidine, famotidine, roxatidine) inhibited this effect (Pós et al., 2005; Szincsák et al., 2002; Tomita et al., 2005; Uçar, 1991). Additionally, H2R antagonists stimulated melanogenesis and inhibited proliferation in B16-C3 mouse melanoma cells (Uçar, 1991). It was also found that melanoma tumour growth was not modulated by *in vivo* histamine treatment while treatment with terfenadine, an H1R antagonist, *in vitro* induced melanoma cell death by apoptosis and *in* 

and rho-C expression in mouse melanoma (Pós Z et al., 2005).

signalling through the H3R in human melanoma (Hegyesi et al., 2005).

*vivo* significantly inhibited tumour growth in murine models (Blaya et al., 2010).

Differences between melanoma cells in their capacity to produce and degrade histamine could explain the different sensitivities of melanoma cell types to exogenous histamine treatment. Moreover, there is evidence that cytokines can influence HDC expression and activity. It has been shown that there is a regulation loop between interleukin 6 (IL-6) and histamine: histamine increased IL-6 expression and secretion in metastatic lines via the H1R, and IL-6 treatment increased the HDC and histamine content in primary melanoma lines (Lázar-Molnar et al., 2002). Interferon-gamma (IFN-gamma) produced by surrounding immune cells decreases HDC expression, affecting melanoma growth and also impairs antitumour activity of the immune system, then contributing to the escape of melanoma cells from immunosurveillance (Horváth et al., 1999; Heninger et al., 2000). Furthermore, Radiotherapy is the most common modality for treating human cancers and relies on ionising radiation induced DNA damage to kill malignant cells. Eighty percent of cancer patients need radiotherapy at some time or other, either for curative or palliative purpose. To optimise results, a cautious balance between the total dose of radiotherapy delivered and the threshold limit of the surrounding normal critical tissues is required. In order to obtain better tumour control with a higher dose, the normal tissues should be protected against radiation damage. Therefore, the role of radioprotective compounds is of utmost importance in clinical radiotherapy *(*Hall & Giaccia, 2006; Mah et at., 2011). Ionising radiation causes damage to living tissues through a series of molecular events. DNA double-strand breaks (DSBs), which are exceptionally lethal lesions, can be formed either by direct energy

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 87

COMPOUND SIDE EFFECTS CHEMICAL STRUCTURE

mouth (tingling, tongue thickness) C721-H-1142-N-202-O204-S9

of warmth; hiccups, nausea, sneezing, vomiting

bleeding, liver problems, skin condition, decreased calcification of bone, seizures, broken

(rash; hives; itching; difficulty breathing; tightness in the chest; swelling of the mouth, face, lips, or tongue), loss of appetite, muscle weakness, nausea, slow reflexes, vomiting

Table 2. Radioprotectors. Extracted and modified from http://www.wolframalpha.com/

Mitigants are administered after radiotherapy but before the phenotypic expression of injury and are intended to ameliorate injury. The keratinocyte growth factor (KGF), palifermin, has been approved as a new, targeted therapy for the prevention of severe oral mucositis in patients with head and neck cancer undergoing post-operative radiochemotherapy and can be considered as the prototype mitigant (Weigelt et at., 2011) (Table 2). Palifermin, like the natural KGF, helps maintain the normal structure of the skin and gastrointestinal surface (lining) by stimulating cells to divide, grow and develop (Le et

Treatment is a strategy that is predominantly palliative and supportive in nature. Pharmacologic radioprotective strategies should be integrated with physical strategies such as intensity-modulated radiotherapy to realize their maximum clinical potential (Hall &

In addition, low-to-moderate doses of some agents such as nitroxides, adrenoceptor agonist, were found to have radioprotective activity in experiments but their application in clinic remains doubtful. Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) belongs to a class of water-soluble nitroxides which are membrane-permeable stable free radical compounds that confer protection against radiation-induced damage (Bennett et at., 1987; Mah et at., 2011; Muscoli et at., 2003) (Table 2). It is thought to elicit its effects through the oxidation of reduced transition metals, scavenging free radicals and mimicking superoxide dismutase

Despite many years of research there are surprisingly few radiation protectors in use today, whose clinical value is limited due to their toxicity; thus, the development of effective and nontoxic agents is yet a challenge for oncologists and radiobiologists (Hall &

Amifostine (WR-2721) Drowsiness, feeling of coldness, flushing/feeling

Cysteamine Depression, stomach or intestinal ulcer and

Palifermin Skin rash, flushing, unusual sensations in the

Tempol Constipation; diarrhoea, severe allergic reactions

entities/chemicals/palifermin/hs/j8/6k/; http://www.drugs.com

Cysteine Toxic, nausea, vomiting

at., 2011; Weigelt et at., 2011).

Giaccia, 2006; Le et al., 2011).

activity (Jiang et al., 2007).

Giaccia, 2006).

**8.2 Histamine as a radioprotector** 

bone, decreased white blood cells

deposition or indirectly through the radiolysis of water molecules, which generate clusters of ROS that react with DNA molecules. Because human tissues contain 80% water, the major radiation damage produced by low linear transfer energy (LET) radiation is due to the aqueous free radicals. DSBs are essentially two single stranded nicks in opposing DNA strands that occur in close proximity, severely compromising genomic stability *(*Grdina, 2002; Hall & Giaccia, 2006; Mah et at., 2011). A series of complex pathways collectively known as the DNA damage response (DDR) is responsible for the recognition, signalling and repair of DSBs in cells, ultimately resulting in either cell survival or cell death (Mah et at., 2011). These free radicals react not only with DNA but also with other cellular macromolecules, such as RNA, proteins, membrane, *etc*, and cause cell dysfunction and mortality. Unfortunately, these reactions take place in tumour as well as normal cells when exposed to radiation. Therefore, to improve the efficacy of radiotherapy there is an intense interest in combining this modality with ionising radiation modifiers, such as radioprotectors. These compounds mitigate damage to surrounding non-malignant tissue (Brizel, 2007; Grdina, 2002; Hall & Giaccia, 2006; Hosseinimehr, 2007).

The most remarkable group of true radioprotectors is the sulfhydryl compounds. The simplest is cysteine, a sulfhydryl compound containing a natural amino acid (Table 2). In 1948, Patt discovered that cysteine could protect mice from the effects of total-body exposure to X-rays if the drug was injected or ingested in large amounts before the radiation exposure. At about the same time, in Europe independently discovered that cysteamine could also protect animals from total-body irradiation (Table 2). However, cysteine is toxic and induces nausea and vomiting at the dose levels required for radioprotection. A developmental program was initiated in 1959 and conducted at the Walter Reed Institute of Research to identify and synthesize drugs capable of conferring protection to individuals in a radiation environment by the U.S. Army. Over 4.000 compounds were synthesized and tested and it was discovered that the covering of the sulfhydryl group by a phosphate group reduced toxicity (Grdina, 2002; Hall & Giaccia, 2006; Nucifora et al., 1972).

The concept of the therapeutic ratio is central to understanding the rationale for using radioprotectors. It relates tumour control probabilities and normal tissue complication probabilities to one another. An ideal radioprotector will reduce the latter without compromising the former and should also be minimally toxic itself. Radioprotective strategies can be classified under the categories of protection, mitigation, and treatment. Protectors are administered before radiotherapy and are designed to prevent radiationinduced injury. Amifostine is the prototype drug (Table 2). Amifostine is the only radioprotective agent that is approved by FDA for preventing of xerostomia induced by gamma irradiation in patients under radiotherapy (Grdina et al., 2009; Hall & Giaccia, 2006; Hosseinimehr, 2007; Kouvaris et al., 2007, Wasserman & Brizel, 2001). Its selectivity for normal tissue is due to its preferential accumulation in normal tissue compared to the hypoxic environment of tumour tissues with low pH and low alkaline phosphatase, which is required to dephosphorylate and activate amifostine (Calabro-Jones et al., 1985; Grdina, 2002; Mah et at., 2011). The active metabolite, WR-1065 scavenges free radicals and is oxidised, causing anoxia or the rapid consumption of oxygen in tissues. This sulfhydryl compound is one of the most effective radioprotectors known nowadays, but there are two main problems of its using. The first one is their toxicity and the second is the short-ranged activity. Amifostine is also the unique radioprotector widely used in clinic on chemotherapy applications (Grdina et al., 2009; Hall & Giaccia, 2006; Hosseinimehr, 2007).

deposition or indirectly through the radiolysis of water molecules, which generate clusters of ROS that react with DNA molecules. Because human tissues contain 80% water, the major radiation damage produced by low linear transfer energy (LET) radiation is due to the aqueous free radicals. DSBs are essentially two single stranded nicks in opposing DNA strands that occur in close proximity, severely compromising genomic stability *(*Grdina, 2002; Hall & Giaccia, 2006; Mah et at., 2011). A series of complex pathways collectively known as the DNA damage response (DDR) is responsible for the recognition, signalling and repair of DSBs in cells, ultimately resulting in either cell survival or cell death (Mah et at., 2011). These free radicals react not only with DNA but also with other cellular macromolecules, such as RNA, proteins, membrane, *etc*, and cause cell dysfunction and mortality. Unfortunately, these reactions take place in tumour as well as normal cells when exposed to radiation. Therefore, to improve the efficacy of radiotherapy there is an intense interest in combining this modality with ionising radiation modifiers, such as radioprotectors. These compounds mitigate damage to surrounding non-malignant tissue

The most remarkable group of true radioprotectors is the sulfhydryl compounds. The simplest is cysteine, a sulfhydryl compound containing a natural amino acid (Table 2). In 1948, Patt discovered that cysteine could protect mice from the effects of total-body exposure to X-rays if the drug was injected or ingested in large amounts before the radiation exposure. At about the same time, in Europe independently discovered that cysteamine could also protect animals from total-body irradiation (Table 2). However, cysteine is toxic and induces nausea and vomiting at the dose levels required for radioprotection. A developmental program was initiated in 1959 and conducted at the Walter Reed Institute of Research to identify and synthesize drugs capable of conferring protection to individuals in a radiation environment by the U.S. Army. Over 4.000 compounds were synthesized and tested and it was discovered that the covering of the sulfhydryl group by a phosphate group

The concept of the therapeutic ratio is central to understanding the rationale for using radioprotectors. It relates tumour control probabilities and normal tissue complication probabilities to one another. An ideal radioprotector will reduce the latter without compromising the former and should also be minimally toxic itself. Radioprotective strategies can be classified under the categories of protection, mitigation, and treatment. Protectors are administered before radiotherapy and are designed to prevent radiationinduced injury. Amifostine is the prototype drug (Table 2). Amifostine is the only radioprotective agent that is approved by FDA for preventing of xerostomia induced by gamma irradiation in patients under radiotherapy (Grdina et al., 2009; Hall & Giaccia, 2006; Hosseinimehr, 2007; Kouvaris et al., 2007, Wasserman & Brizel, 2001). Its selectivity for normal tissue is due to its preferential accumulation in normal tissue compared to the hypoxic environment of tumour tissues with low pH and low alkaline phosphatase, which is required to dephosphorylate and activate amifostine (Calabro-Jones et al., 1985; Grdina, 2002; Mah et at., 2011). The active metabolite, WR-1065 scavenges free radicals and is oxidised, causing anoxia or the rapid consumption of oxygen in tissues. This sulfhydryl compound is one of the most effective radioprotectors known nowadays, but there are two main problems of its using. The first one is their toxicity and the second is the short-ranged activity. Amifostine is also the unique radioprotector widely used in clinic on chemotherapy

(Brizel, 2007; Grdina, 2002; Hall & Giaccia, 2006; Hosseinimehr, 2007).

reduced toxicity (Grdina, 2002; Hall & Giaccia, 2006; Nucifora et al., 1972).

applications (Grdina et al., 2009; Hall & Giaccia, 2006; Hosseinimehr, 2007).


Table 2. Radioprotectors. Extracted and modified from http://www.wolframalpha.com/ entities/chemicals/palifermin/hs/j8/6k/; http://www.drugs.com

Mitigants are administered after radiotherapy but before the phenotypic expression of injury and are intended to ameliorate injury. The keratinocyte growth factor (KGF), palifermin, has been approved as a new, targeted therapy for the prevention of severe oral mucositis in patients with head and neck cancer undergoing post-operative radiochemotherapy and can be considered as the prototype mitigant (Weigelt et at., 2011) (Table 2). Palifermin, like the natural KGF, helps maintain the normal structure of the skin and gastrointestinal surface (lining) by stimulating cells to divide, grow and develop (Le et at., 2011; Weigelt et at., 2011).

Treatment is a strategy that is predominantly palliative and supportive in nature. Pharmacologic radioprotective strategies should be integrated with physical strategies such as intensity-modulated radiotherapy to realize their maximum clinical potential (Hall & Giaccia, 2006; Le et al., 2011).

In addition, low-to-moderate doses of some agents such as nitroxides, adrenoceptor agonist, were found to have radioprotective activity in experiments but their application in clinic remains doubtful. Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) belongs to a class of water-soluble nitroxides which are membrane-permeable stable free radical compounds that confer protection against radiation-induced damage (Bennett et at., 1987; Mah et at., 2011; Muscoli et at., 2003) (Table 2). It is thought to elicit its effects through the oxidation of reduced transition metals, scavenging free radicals and mimicking superoxide dismutase activity (Jiang et al., 2007).

#### **8.2 Histamine as a radioprotector**

Despite many years of research there are surprisingly few radiation protectors in use today, whose clinical value is limited due to their toxicity; thus, the development of effective and nontoxic agents is yet a challenge for oncologists and radiobiologists (Hall & Giaccia, 2006).

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 89

was associated with a reduction of submandibular gland wet weight and an alteration of epithelial architecture, vacuolization of acinar cells and partial loss of eosinophilic secretor granular material. It is worth noting that histamine treatment (0.1 mg.kg-1) completely reversed the reduced salivation induced by radiation, preserving glandular function and mass with normal structure organization of acini and ducts. Histamine prevented radiationinduced toxicity in submandibular gland essentially by suppressing apoptosis of ductal and

To summarize, histamine treatment can selectively modulate cellular damage produced by ionising radiation, thus preventing radiation induced damage on small intestine, bone marrow and salivary glands. Furthermore, histamine *in vitro* enhances the radiosensitivity of breast cancer cells (Medina et al., 2006) while does not modify that of melanoma (Medina et al., 2007). Despite histamine may be proliferative in some cancer cell types, it may still be beneficial as radioprotector in view of the fact that it is only administered for a short period of time to reduce the radiation induced damage. It is important to highlight that histamine radioprotective effect was demonstrated in two different rodent species, which suggests that histamine could exert a radioprotective action in other mammals. Also, no local or systemic

The presented evidences indicate that histamine is a potential candidate as a safe radioprotective agent that might increase the therapeutic index of radiotherapy for intraabdominal, pelvic, and head and neck cancers, and enhance patient quality of life by protecting normal tissue from radiation injury. However, the efficacy of histamine needs to

In this chapter, we have presented major findings of the most recent research in histamine cancer pharmacology. These data clearly indicate that histamine plays a key role as a mediator in most human tumours. Interestingly, histamine is not only involved in cancer cell proliferation, migration and invasion, but also the tumour microenvironment and immune system responses are tightly affected. In human neoplasias H3R and H4R seemed to be the main receptors involved in the control of the metabolic pathways responsible for tumour growth and progression, suggesting that H3R and H4R represent potential molecular targets for cancer drug development. Finally, a novel role for histamine as a selective radioprotector is highlighted, indicative of the potential application of histamine

This work has been supported by grants from the University of Buenos Aires 20020090300039 and 20020100100270, from the National Agency of Scientific and Technological Promotion PICT-2007-01022, and from the EU-FP7 COST Action BM0806.

Adams, W.J., Lawson, J.A., Morris, D.L. (1994). Cimetidine inhibits in vivo growth of human

*Medical Association,* Vol. 35, (11/94), pp. (1632-1636), ISSN 0017-5749

colon cancer and reverses histamine stimulated in vitro and in vivo growth. *British* 

acinar cells, reducing the number of apoptotic cells per field (Medina et al., 2011a).

side effects were observed upon histamine administration in both species.

be carefully investigated in prospective clinical trials.

and its ligands as adjuvants to radiotherapy.

**9. Conclusions** 

**10. Acknowledgment** 

**11. References** 

The acute effects of irradiation result from the death of a large number of cells in tissues with a rapid rate of turnover. These include effects in the epidermal layer or skin, gastrointestinal epithelium, and haematopoietic system, in which the response is determined by a hierarchical cell lineage, composed of stem cells and their differentiating offspring. In clinical radiotherapy, the tolerance of normal tissues for radiation depends on the ability of clonogenic cells to maintain a sufficient number of mature cells suitably structured to preserve organ function (Hall & Giaccia, 2006). During radiotherapy for intraabdominal and pelvic cancers, radiation seriously affects radiosensitive tissues such as small intestine and bone marrow (Erbyl et al., 2005; Hall & Giaccia, 2006). It was previously demonstrated that histamine treatment (daily subcutaneous injection, 0.1 mg.kg-1) significantly protects mouse small intestine against radiation-induced toxicity ameliorating histological injury and improving trophism of enterocytes (Medina et al., 2007). Histamine completely prevented the decrease in the number of crypts evoked by whole body irradiation, which is vital for small intestine restoration since the intestinal crypt contains a hierarchy of stem cells that preserve the potential to regenerate the stem cell population and the tissue after cytotoxic exposure (Potten et al., 2002). Histamine radioprotective effect on small intestine was related to an increased rate of proliferation as evidenced by the enhanced proliferation markers immunoreactivity [5-bromo-2'-deoxyuridine (BrdU), and proliferating cell nuclear antigen (PCNA)]. Additionally, this outcome was accompanied by a reduction in the number of apoptotic cells per crypt and a modification of antioxidant enzyme levels that could lead to enhance the antioxidant capacity of intestinal cells (Medina et al., 2007). Histamine also protects rat small intestine against ionising radiation damage and this effect was principally associated to a decrease in intestinal cell crypt apoptosis (Medina & Rivera, 2010a).

The bone marrow pluripotent stem cells, such as erythroblast, are particularly radiosensitive and, after whole body irradiation, an important grade of aplasia is observed increasing the possibility of haemorrhage and/or infection occurrence that could be lethal. The survival of stem cells determines the subsequent repopulation of bone marrow after irradiation (Hall & Giaccia, 2006). Results demonstrated that histamine (0.1 mg.kg-1) significantly reduced the grade of aplasia, ameliorating the oedema and vascular damage produced by ionising radiation while eliciting a significant conservation of the medullar progenies on bone marrow in mouse and rat species, increasing the number of megakaryocytes, myeloid, lymphoid and erythroid cells per mm2. The histamine effect is mediated at least in part by an increase in the rate of proliferation, as evidenced by the enhanced PCNA protein expression and BrdU incorporation, and is associated with an enhanced HDC expression in irradiated bone marrow cells (Medina et al., 2010; Medina & Rivera, 2010a). In this line, it was reported that a faster bone marrow repopulation was observed in wild type in comparison with HDC-deficient mice and that intracellular HDC and histamine content in regenerating bone marrow populations is increased after total-body irradiation (Horvath et al., 2006).

Despite improvements in the technology for delivering therapeutic radiation, salivary glands are inevitably injured during head and neck cancer radiotherapy, causing devastating side-effects which results in salivary hypofunction and consequent xerostomia (Burlage et al., 2008; Hall & Giaccia, 2006; Nagler, 2002). Salivary glands of rat are quite similar to human salivary glands in which salivary flow is rapidly reduced after radiation exposure (Nagler, 2002). Recent results demonstrated that histamine markedly prevented radiation injury on submandibular gland, ameliorating the histological and morphological alterations. Radiation significantly decreased salivation by approximately 35-40%, which was associated with a reduction of submandibular gland wet weight and an alteration of epithelial architecture, vacuolization of acinar cells and partial loss of eosinophilic secretor granular material. It is worth noting that histamine treatment (0.1 mg.kg-1) completely reversed the reduced salivation induced by radiation, preserving glandular function and mass with normal structure organization of acini and ducts. Histamine prevented radiationinduced toxicity in submandibular gland essentially by suppressing apoptosis of ductal and acinar cells, reducing the number of apoptotic cells per field (Medina et al., 2011a).

To summarize, histamine treatment can selectively modulate cellular damage produced by ionising radiation, thus preventing radiation induced damage on small intestine, bone marrow and salivary glands. Furthermore, histamine *in vitro* enhances the radiosensitivity of breast cancer cells (Medina et al., 2006) while does not modify that of melanoma (Medina et al., 2007). Despite histamine may be proliferative in some cancer cell types, it may still be beneficial as radioprotector in view of the fact that it is only administered for a short period of time to reduce the radiation induced damage. It is important to highlight that histamine radioprotective effect was demonstrated in two different rodent species, which suggests that histamine could exert a radioprotective action in other mammals. Also, no local or systemic side effects were observed upon histamine administration in both species.

The presented evidences indicate that histamine is a potential candidate as a safe radioprotective agent that might increase the therapeutic index of radiotherapy for intraabdominal, pelvic, and head and neck cancers, and enhance patient quality of life by protecting normal tissue from radiation injury. However, the efficacy of histamine needs to be carefully investigated in prospective clinical trials.

## **9. Conclusions**

88 Drug Development – A Case Study Based Insight into Modern Strategies

The acute effects of irradiation result from the death of a large number of cells in tissues with a rapid rate of turnover. These include effects in the epidermal layer or skin, gastrointestinal epithelium, and haematopoietic system, in which the response is determined by a hierarchical cell lineage, composed of stem cells and their differentiating offspring. In clinical radiotherapy, the tolerance of normal tissues for radiation depends on the ability of clonogenic cells to maintain a sufficient number of mature cells suitably structured to preserve organ function (Hall & Giaccia, 2006). During radiotherapy for intraabdominal and pelvic cancers, radiation seriously affects radiosensitive tissues such as small intestine and bone marrow (Erbyl et al., 2005; Hall & Giaccia, 2006). It was previously demonstrated that histamine treatment (daily subcutaneous injection, 0.1 mg.kg-1) significantly protects mouse small intestine against radiation-induced toxicity ameliorating histological injury and improving trophism of enterocytes (Medina et al., 2007). Histamine completely prevented the decrease in the number of crypts evoked by whole body irradiation, which is vital for small intestine restoration since the intestinal crypt contains a hierarchy of stem cells that preserve the potential to regenerate the stem cell population and the tissue after cytotoxic exposure (Potten et al., 2002). Histamine radioprotective effect on small intestine was related to an increased rate of proliferation as evidenced by the enhanced proliferation markers immunoreactivity [5-bromo-2'-deoxyuridine (BrdU), and proliferating cell nuclear antigen (PCNA)]. Additionally, this outcome was accompanied by a reduction in the number of apoptotic cells per crypt and a modification of antioxidant enzyme levels that could lead to enhance the antioxidant capacity of intestinal cells (Medina et al., 2007). Histamine also protects rat small intestine against ionising radiation damage and this effect was principally associated to a decrease in intestinal cell crypt apoptosis

The bone marrow pluripotent stem cells, such as erythroblast, are particularly radiosensitive and, after whole body irradiation, an important grade of aplasia is observed increasing the possibility of haemorrhage and/or infection occurrence that could be lethal. The survival of stem cells determines the subsequent repopulation of bone marrow after irradiation (Hall & Giaccia, 2006). Results demonstrated that histamine (0.1 mg.kg-1) significantly reduced the grade of aplasia, ameliorating the oedema and vascular damage produced by ionising radiation while eliciting a significant conservation of the medullar progenies on bone marrow in mouse and rat species, increasing the number of megakaryocytes, myeloid, lymphoid and erythroid cells per mm2. The histamine effect is mediated at least in part by an increase in the rate of proliferation, as evidenced by the enhanced PCNA protein expression and BrdU incorporation, and is associated with an enhanced HDC expression in irradiated bone marrow cells (Medina et al., 2010; Medina & Rivera, 2010a). In this line, it was reported that a faster bone marrow repopulation was observed in wild type in comparison with HDC-deficient mice and that intracellular HDC and histamine content in regenerating bone marrow populations is

Despite improvements in the technology for delivering therapeutic radiation, salivary glands are inevitably injured during head and neck cancer radiotherapy, causing devastating side-effects which results in salivary hypofunction and consequent xerostomia (Burlage et al., 2008; Hall & Giaccia, 2006; Nagler, 2002). Salivary glands of rat are quite similar to human salivary glands in which salivary flow is rapidly reduced after radiation exposure (Nagler, 2002). Recent results demonstrated that histamine markedly prevented radiation injury on submandibular gland, ameliorating the histological and morphological alterations. Radiation significantly decreased salivation by approximately 35-40%, which

(Medina & Rivera, 2010a).

increased after total-body irradiation (Horvath et al., 2006).

In this chapter, we have presented major findings of the most recent research in histamine cancer pharmacology. These data clearly indicate that histamine plays a key role as a mediator in most human tumours. Interestingly, histamine is not only involved in cancer cell proliferation, migration and invasion, but also the tumour microenvironment and immune system responses are tightly affected. In human neoplasias H3R and H4R seemed to be the main receptors involved in the control of the metabolic pathways responsible for tumour growth and progression, suggesting that H3R and H4R represent potential molecular targets for cancer drug development. Finally, a novel role for histamine as a selective radioprotector is highlighted, indicative of the potential application of histamine and its ligands as adjuvants to radiotherapy.

## **10. Acknowledgment**

This work has been supported by grants from the University of Buenos Aires 20020090300039 and 20020100100270, from the National Agency of Scientific and Technological Promotion PICT-2007-01022, and from the EU-FP7 COST Action BM0806.

## **11. References**

Adams, W.J., Lawson, J.A., Morris, D.L. (1994). Cimetidine inhibits in vivo growth of human colon cancer and reverses histamine stimulated in vitro and in vivo growth. *British Medical Association,* Vol. 35, (11/94), pp. (1632-1636), ISSN 0017-5749

Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 91

Bolton, E., King, J., & Morris, D.L. (2000). H2-antagonists in the treatment of colon and

Bongers, G., Bakker, R.A., & Leurs, R. (2007). Molecular aspects of histamine H3 receptor. *Biochemical pharmacology*, Vol. 73, No. 8, (04/07), pp. (1195-204), ISSN 0006-2952 Brizel, DM. (2007). Pharmacologic approaches to radiation protection. *World journal of clinical oncology*, Vol., 25, No. 26, (09/07), pp. (4084-9), ISSN 2218-4333 Burlage, F.R., Roesink, J.M., Kampinga, H.H., Coppes, R.P., Terhaard, C., Langendijk, J.A.,

Byron, J.W. (1977). Mechanism for histamine H2-receptor induced cell-cycle changes in the

Cabanillas-Saez, A., Schalper, J.A., Nicovani, S.M. & Rudolph, M.I. (2002). Characterization

Calabro-Jones, P., Fahey, R., Smoluk, G., & Ward, J. (1985). Alkaline phosphatase promotes

Chanda, R., Ganguly, A.K. (1987). Diamineoxidase activity and tissue histamine content of

Chanda, R. & Ganguly, A.K. (1995). Diamine-oxidase activity and tissue di- and poly-amine

Chang, S., Wallis, R.A., Yuan, L., Davis, P.F. & Tan, S.T. (2006). Mast cells and cutaneous malignancies. *Modern pathology*, Vol. 19, (01/06), pp. 149–159, ISSN 0893-3952 Cherifi, I., Pigeon, C., Le romancer, M., Bado, A., Reyl-Desmars, F., & Lewin, M.J.M. (1992).

Cianchi, F., Cortesini, C., Schiavone, N., Perna, F., Magnelli, L., Fanti, E., Bani, D., Messerini,

Coge, F., Guenin, S.P., Audinot, V., Renouard-Try, A., Beauverger, P., Macia, C., Ouvry, C.,

*Biochemical journal*, Vol. 355, No. Pt 2, (04/01), pp. (279-88), ISSN 0264-6021.

*research*, Vol. 11, No. 19 Pt 1, (10/05), pp. (6807-15), ISSN 1078-0432

Vol. 70, No. 1, (01/08), pp. (14-22), ISSN 0360-3016

(01/02), pp. 92–98, ISSN 1048-891X

(06/85), pp. (23-7), ISSN 0020-7616

89, No. 1, (02/95), pp. 23-8, ISSN 0304-3835

267, No. 35, (12/92), pp. (25315-20), ISSN 0021-9258

(207-12), ISSN 0304-3835

1044-579X

0065-4299

breast cancer. *Seminars in cancer biology*, Vol. 10, No. 1, (02/00), pp. (3-10), ISSN

van Luijk, P., Stokman, M.A., & Vissink, A. (2008). Protection of salivary function by concomitant pilocarpine during radiotherapy: a double-blind, randomized, placebo-controlled study. *International journal of radiation oncology, biology, physics*,

bone marrow stem cell. *Agents and actions,* Vol. 7, No. 2, (07/77), pp. 209-13, ISSN

of mast cells according to their content of tryptase and chymase in normal and neoplastic human uterine cervix. *International journal of gynecological cancer*, Vol. 12,

radioprotection and accumulation of WR-1065 in V79-171 cells incubated in medium containing WR-2721. *International journal of radiation biology*, Vol., 47, No. 1,

human skin, breast and rectal carcinoma. *Cancer letters,* Vol. 34, No. 2, (02/87), pp.

contents of human ovarian, cervical and endometrial carcinoma. *Cancer letters, Vol.* 

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1. *The Journal of biological chemistry*, Vol.

L., Fabbroni, V., Perigli, G., Capaccioli, S., & Masini, E. (2005). The role of cyclooxygenase-2 in mediating the effects of histamine on cell proliferation and vascular endothelial growth factor production in colorectal cancer. *Clinical cancer* 

Nagel, N., Rique, H., Boutin, J.A., & Galizzi, J.P. (2001a). Genomic organization and characterization of splice variants of the human histamine H3 receptor. *The* 


Adams, W., Morris, D. (1996). Cimetidine and colorectal cancer. *Diseases of the colon and* 

Agarwala, S.S., Glaspy, J., O'Day, S.J., Mitchell, M., Gutheil, J., Whitman, E., Gonzalez, R.,

Arrang, J.M., Garbarg, M., & Schwartz, J.C. (1983). Auto-inhibition of brain histamine

Ash, A.S., & Schild, H.O. (1966). Receptors mediating some actions of histamine. *British* 

Aydin, Y., Tunçel, N., Gürer, F., Tuncel, M., Koşar, M. & Oflaz, G. (1998). Ovarian, uterine

Bakker, R.A., Schoonus, S.B.J., Smit, M.J., Timmerman, H., & Leurs, R. (2001). Histamine H1-

Batra, S. & Fadeel, I. (1994). Release of intracellular calcium and stimulation of cell growth

Belcheva, A. & Mishkova, R. (1995). Histamine content in lymph nodes from patients with

Bennett, H., Swartz, H., Brown, Rr., & Koenig, S. (1987). Modification of relaxation of lipid

Black, J.W., Duncan, W.A., Durant, C.J., Ganellin, C.R., & Parsons, E.M. (1972).Definition

Blaya, B., Nicolau, G.F., Jangi, S.M., Ortega, M.I., Alonso, T.E., Burgos-Bretones, J, Pérez,

Boer, K., Helinger, E., Helinger, A., Pocza, P., Pos, Z., Demeter, P., Baranyai, Z., Dede, K.,

Hersh, E., Feun, L., Belt, R., Meyskens, F., Hellstrand, K., Wood, D., Kirkwood, J.M., Gehlsen, K.R., & Naredi, P. (2002). Results from a randomized phase III study comparing combined treatment with histamine dihydrochloride plus interleukin-2 versus interleukin-2 alone in patients with metastatic melanoma. *Journal of clinical* 

release mediated by a novel class (H3) of histamine receptor. *Nature*, Vol. 302, No.

*journal of pharmacology and chemotherapy*, Vol. 27, No. 2, (08/96), pp. (427-39), ISSN

and brain mast cells in female rats: cyclic changes and contribution to tissue histamine. *Comparative biochemistry and physiology. Part A, Molecular & integrative* 

receptor activation of nuclear factor-ΚB; roles for G gamma- and G alpha/11 subunits in constitutive and agonist-mediated signaling. *Molecular pharmacology*,

by ATP and histamine in human ovarian cancer cells (SKOV-3). *Cancer letters,* Vol.

malignant lymphomas. *Inflammation Research,* Vol. 44 Suppl 1, (04/95), pp. S86-7,

protons by molecular oxygen and nitroxides. *Investigative radiology*, No. 6, (06/87),

and antagonism of histamine H2-receptors. *Nature*, Vol. 236, No. 5347, (04/72), pp.

Y.G., Asumendi, A., & Boyano, M.D. (2010). Histamine and histamine receptor antagonists in cancer biology. *Inflammation & allergy drug targets,* Vol. 9, No. 3

Darvas, Z., Falus, A. (2008). Decreased expression of histamine H1 and H4 receptors suggests disturbance of local regulation in human colorectal tumours by histamine. *European journal of cell biology,* Vol. 87, (04/08), pp. (227–236), ISSN 0171-

*rectum,* Vol. 39, No. 1, (01/96), pp. (111-2), ISSN 0012-3706

*oncology* Vol. 20, No 1, (06/02), pp. (125–33), ISSN 0732-183X

*physiology*, Vol. 120, No. 2, (06/98), pp. 255-62, ISSN 1095-6433

Vol. 60, No. 5, (11/01), pp. (1133-42), ISSN 0026-895X

77, No. 1, (02/94), pp. 57-63, ISSN 0304-3835

5911, (04/83), pp. (832-7), ISSN 0028-0836

0366-0826

ISSN 1023-3830

9335

pp. (502-507), ISSN 0020-9996

,(07/10), pp. (146-57) ISNN 1871-5281

(385-90), ISSN 0028-0836


Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 93

Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., & Parkin, D.M. (2010). Estimates of

Fukushima, Y., Asano, T., Saitoh, T., Anai, M., Funaki, M., Ogihara, T., Katagiri, H.,

Fitzsimons, C., Engel, N., Policastro, L., Durán, H., Molinari, B., & Rivera, E. (2002).

Gonzalez-Angulo, A.M., Morales-Vasquez, F., & Hortobagyi, G.N. (2007). Overview of

Grdina, D.J., Murley, J.S., Kataoka, Y., Baker, K.L., Kunnavakkam, R., Coleman, M.C., &

Gutzmer, R., Gschwandtner, M., Rossbach, K., Mommert, S., Werfel, T., Kietzmann, M., &

Haak-Frendscho, M., Darvas, Z., Hegyesi, H., Kárpáti, S., Hoffman, R.L., László, V.,

Hall E.J., & Giaccia, A.J. (Eds.). (2006). *Radiobiology for the radiologist*, Lippincott Williams &

Hancock, A.A., Esbenshade, T.A., Krueger. K,M., & Yao, B.B. (2003). Genetic and

Hegyesi, H., Somlai, B., Varga, V.L., Toth, G., Kovacs, P., Molnar, E.L., Laszlo, V., Karpati, S.,

Hegyesi, H., Horváth, B., Pállinger, E., Pós, Z., Molnár, V., & Falus, A. (2005). Histamine

*dermatology*, Vol 115, No 3, (09/00), pp. (345-52), ISSN:0022-202X

*experimental medicine and biology,* Vol. 608, pp. (1-22), ISSN 0065-2598 Grdina, D.J., Murley, J.S., & Kataoka, Y. (2002). Radioprotectans: current status and new

directions. *Oncology*, Vol., 63, No. Suppl. 2-2 10, ISSN 0030-2414

*edition)*, Vol. 3, (06/11), pp. (985-94), ISSN 1945-0516

Wilkins, ISBN 978-0-7817-4151-4, Philadelphia

73, No. 24, (10/03), pp. (3043-72), ISSN 0024-3205

*cancer*, Vol. 127, No. 12, (12/10), pp. (2893-917), ISSN 0020-7136

2002; Vol. 63, No. 10, (05/02), pp. (1785-96), ISSN 0006-2952

(283-6), ISSN 0014-5793

ISSN 0360-3016

202X

5793

worldwide burden of cancer in 2008: GLOBOCAN 2008. *International journal of* 

Matsuhashi, N., Yazaki, Y., Sugano, K. (1997). Oligomer formation of histamine H2 receptors expressed in Sf9 and COS7 cells. *FEBS letters*, Vol. 409, No. 2, (06/97), pp.

Regulation of phospholipase C activation by the number of H(2) receptors during Ca(2+)-induced differentiation of mouse keratinocytes. *Biochemical pharmacology*

resistance to systemic therapy in patients with breast cancer. *Advances in* 

Spitz, D.R. (2009). Amifostine induces antioxidant enzymatic activities in normal tissues and a transplantable tumor that can affect radiation response. *International journal of radiation oncology, biology, physics*, Vol., 1-73, No. 3 (03/09), pp. (886-96),

Baeumer, W. (2011). Pathogenetic and therapeutic implications of the histamine H4 receptor in inflammatory skin diseases and pruritus. *Frontiers in bioscience (Scholar* 

Bencsáth, M., Szalai, C., Fürész, J., Timár, J., Bata-Csörgõ, Z., Szabad, G., Pivarcsi, A., Pállinger, E., Kemény, L., Horváth, A., Dobozy, A., & Falus, A. (2000). Histidine decarboxylase expression in human melanoma. *The Journal of investigative* 

pharmacological aspects of histamine H3 receptor heterogeneity. *Life sciences*, Vol.

Rivera, E., Falus, A., & Darvas, Z. (2000). Suppression of melanoma cell proliferation by histidine decarboxylase specific antisense oligonucleotides. *The Journal of investigative dermatology*, Vol 117, No 1, (07/00), pp. (151-3), ISSN:0022-

elevates the expression of Ets-1, a protooncogen in human melanoma cell lines through H2 receptor. *FEBS letters*, Vol 579, No 11, (04/05), pp. (2475-9), ISSN:0014-


Coge, F., Guenin, S.P., Rique, H., Boutin, J.A., & Galizzi, J.P. (2001b). Structure and

*research communications*, Vol. 284, No. 2, (06/01), pp. (301-9), ISSN 0006-291X Cricco, G.P., Davio, C.A., Fitzsimons, C.P., Engel, N., Bergoc, R.M., & Rivera, E.S. (1994).

Cricco, G., Mohamad, N., Sáez, M.S., Valli, E., Rivera, E.S., & Martín, G. Histamine and

Connelly, W.M., Shenton, F.C., Lethbridge, N., Leurs, R., Waldvogel, H.J., Faull, R.L., Lees,

Cummins, D.L., Cummins, J.M., Pantle, H., Silverman, M.A., Leonard, A.L., & Chanmugam,

Davio, C.A., Cricco, G.P., Andrade, N., Bergoc, R.M., & Rivera, E.S. (1993). H1 and H2

Davio, C.A., Cricco, G.P., Martin, G., Fitzsimons, C.P., Bergoc, R.M., & Rivera, E.S. (1994).

Davio, C.A., Cricco, G.P., Bergoc, R.M., & Rivera, E.S. (1995). H1 and H2 histamine receptors

*Biochemical pharmacology*, Vol. 50, No. 1, (06/95), pp. (91-6), ISSN 0006-2952 Davio, C., Madlovan, A., Shayo, C., Lemos, B., Baldi, A., & Rivera, E. (1996). Histamine

Demitsu, T., Inoue, T., Kakurai, M., Kiyosawa, T., Yoneda, K. & Manabe, M. (2002).

Erbil, Y., Oztezcan, S., Giris, M., Barbaros, U., Olgac, V., Bilge, H., Kücücük, H., & Toker, G.

Fang, Z., Yao, W., Xiong, Y., Li, J., Liu, L., Shi, L., Zhang, W., Zhang, C., Nie, L., Wan, J.

Vol. 78, No. 4, (12/05), pp. (376-820), ISSN 0024-3205

*research*, Vol. 45, No. Suppl. 1, (03/96), pp. (S62-S63), ISSN 1023-3830 Davio, C., Mladovan, A., Lemos, B., Monczor, F., Shayo, C., Rivera, E., & Baldi, A. (2002). H1

*and actions*, Vol. 41, (06/94), pp. (C115-C117), ISSN 0065-4299

*actions*, Vol. 43, No. 1-2, (11/94), pp. (17-20), ISSN 0065-4299

(05/09), pp. (55-63), ISSN 0007-1188

(04/06), pp. (500-7), ISSN:0025-6196

pp. (1-7), ISSN 1023-3830

410), ISSN 1359-6101

pp. (195), ISSN 1471-2407

(06/93), pp. (C172-C174), ISSN 0065-4299

expression of the human histamine H4-receptor gene. *Biochemical and biophysical* 

Histamine as an autocrine growth factor in experimental carcinomas. *Agents and* 

Breast Cancer: a New Role for a Well Known Amine, In: *Breast Cancer Cells / Book 1,* Mehmet Gunduz, pp. in press, Okayama University, ISBN 979-953-307-137-3, Japan

G., & Chazot, P.L. (2009). The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS. *British journal of pharmacology*, Vol. 157, No. 1,

A. (2006). Cutaneous malignant melanoma. *Mayo Clinic proceedings*, Vol 81, No 4,

histamine receptors in human mammary carcinomas. *Agents and actions*, Vol. 38,

Effect of histamine on growth and differentiation of the rat mammary gland. *Agents* 

in experimental carcinomas with an atypical coupling to signal transducers.

receptors in neoplastic transformation. Studies in human cell lines. *Inflammation* 

and H2 histamine receptors mediate the production of inositol phosphates but not cAMP in human breast epithelial cells. *Inflammation research*, Vol. 51, No. 1, (01/02),

Activation of mast cells within a tumor of angiosarcoma: ultrastructural study of five cases*. The Journal of dermatology,* Vol. 29, (05/02), pp. 280–289, ISSN 0385-2407 Dy, M., & Schneider, E. (2004). Histamine-cytokine connection in immunity and

hematopoiesis. *Cytokine and growth factor reviews*, Vol. 15, No. 5, (10/04), pp. (393-

(2005). The effect of glutamine on radiation-induced organ damage. *Life sciences*,

(2011). Attenuated expression of HRH4 in colorectal carcinomas: a potential influence on tumor growth and progression. *BMC Cancer*, Vol. 11, No. 1, (05/11),


Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 95

Lebois, E.P., Jones, C.K., & Lindsley, C.W. (2011). The evolution of histamine H3

Leurs, R., Smits, M.J., & Timmerman, H. (1995). Molecular pharmacological aspects of

Leurs, R., Church, M.K., & Taglialatela, M. (2002). H1-antihistamines: inverse agonism, anti-

Leurs, R., Bakker, R.A., Timmerman, H., & de Esch, I.J.P. (2005). The histamine H3 receptor:

Leurs, R., Chazot, P.L., Shenton, F.C., Lim, H.D., & de Esch, I.J. (2009). Molecular and

Liu, C., Ma, X.J., Jiang, X., Wilson, S.J., Hofstra, C.L., Blevitt, J., Pyati, J., Li, X., Chai, W.,

*Molecular pharmacology*, Vol. 59, No. 3, (03/01), pp. (420-6), ISSN 0026-895X Lovenberg, T.W., Roland, B.I., Wilson, S.J., Jiang, X., Pyati, J., Huvar, A., Jackson, M.R., &

Mah, L.J., Orlowski, C., Ververis, K., Vasireddy, R.S., El-Osta, A., & Karagiannis, T.C. (2011).

Malaviya, R., Morrison, A.R. & Pentland, A.P. (1996). Histamine in human epidermal cells is

Malinski, C., Kierska, D., Fogel, W., Kinnunum, A., & Panula, P. (1993). Histamine: its

Martner, A., Thorén, F.B., Aurelius, J., Söderholm, J., Brune, M. & Hellstrand, K. (2010).

Massari, N.A., Medina, V.A., Martinel Lamas, D.J., Cricco, G.P., Croci, M., Sambuco, L.,

Masini, E., Fabbroni, V., Giannini, L., Vannacci, A., Messerini, L., Perna, F., Cortesini, C.,

human melanoma. *Melanoma research*, in press, ISSN 0960-8931

*pharmacology*, Vol. 157, No. 1, (05/09), pp. (14-23), ISSN 0007-1188

*World journal of clinical oncology*, (06/11), ISSN 2218-4333

No. 4, (04/02), pp. (489-98), ISSN 0954-7894

2, (02/05), pp (107-22), ISSN 1474-1776

(06/11), pp. (3), ISSN 2041-9414

No. 4, (04/96), pp. 785-9, ISSN 0022-202X

(648-60), ISSN 1568-0266

63), ISSN 0163-7258

895X

0306-4492

91, ISSN 1747-4086

Advanced Head and Neck Cancer: A Randomized, Placebo-Controlled Study.

antagonists/inverse agonists. *Current topics in medicinal chemistry*, Vol. 11, No. 6, pp.

histamine receptors. *Pharmacology & therapeutics*, Vol. 66, No. 3, (06/95), pp. (413-

inflammatory actions and cardiac effects. *Clinical and experimental allergy*, Vol. 32,

from gene cloning to H3 receptor drugs. *Nature reviews. Drug discovery*, Vol. 4, No.

biochemical pharmacology of the histamine H4 receptor. *British journal of* 

Carruthers, N., & Lovenberg, T.W. (2001). Cloning and pharmacological characterization of a fourth histamine receptor (H4) expressed in bone marrow.

Erlander, M.G. (1999). Cloning and functional expression of the human histamine H3 receptor. *Molecular pharmacology*, Vol. 55, No. 6, (06/99), pp. (1101-7), ISSN 0026-

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks. *Genome integrity*, Vol., 2, No.1,

induced by ultraviolet light injury*. The Journal of investigative dermatology,* Vol. 106,

metabolism and localization in mammary gland. *Comparative biochemistry and physiology. C: Comparative pharmacology*, Vol. 105, No. 2, (06/93), pp. (269-73), ISSN

Immunotherapy with histamine dihydrochloride for the prevention of relapse in acute myeloid leukemia. *Expert review of hematology*, Vol 3, No. 4, (08/10) pp. 381-

Bergoc, R.M., & Rivera, E.S. Role of H4 receptor in histamine-mediated responses in

Cianchi, F. (2005). Histamine and histidine decarboxylase up-regulation in


Hellstrand, K., Brune, M., Naredi, P., Mellqvist, U.H., Hansson, M., Gehlsen, K.R. &

Heninger, E., Falus, A., Darvas, Z., Szalai, C., Zsinko, M., Pos, Z., & Hegyesi, H. (2000). Both

Horváth, B.V., Szalai, C., Mándi, Y., László, V., Radvány, Z., Darvas, Z., & Falus, A. (1999).

Horvath, Z., Pallinger, E., Horvath, G., Jelinek, I., Falus, A., & Buzas, E.I. (2006). Histamine

Hosseinimehr, S.J. (2007). Trends in the development of radioprotective agents. *Drug discovery today*, Vol., 12, No.19-20, (10/07), pp. (794-805), ISSN 1741-8364 Jiang, J., Kurnikov, I., Belikova, N.A., Xiao, J., Zhao, Q., Amoscato, A.A., Braslau, R., Studer,

*Cancer investigation*, Vol. 18, No. 4, pp. 347-55, ISSN 0735-7907

(08/00), pp. (393- 7), ISSN 1023-3830

9), ISSN:0165-2478

47), ISSN 1083-7159

(743-9), ISSN:0014-2972

8749

Hermodsson, S. (2000). Histamine: a novel approach to cancer immunotherapy.

interferon (IFN) alpha and IFN gamma inhibit histidine decarboxylase expression in the HT168 human melanoma cell line. *Inflammation research,* Vol. 49, No. 8,

Histamine and histamine-receptor antagonists modify gene expression and biosynthesis of interferon gamma in peripheral human blood mononuclear cells and in CD19-depleted cell subsets. *Immunology letters*, Vol 70, No 2, (11/99), pp. (95-

H1 and H2 receptors but not H4 receptors are upregulated during bone marrow regeneration. *Cellular immunology*, Vol. 244, No. 2, (12/06), pp. (110-5), ISSN 0008-

A., Fink, M.P., Greenberger, J.S., Wipf, P., & Kagan, V.E. (2007). Structural requirements for optimized delivery, inhibition of oxidative stress, and antiapoptotic activity of targeted nitroxides. *The Journal of pharmacology and experimental therapeutics*, Vol., 320, No. 3, (03/07), pp. (1050-60), ISSN 0022-3565 Kapoor, S., Pal, S., Sahni, P., Dattagupta, S., Kanti Chattopadhyay, T. (2005). Effect of pre-

operative short course famotidine on tumor infiltrating lymphocytes in colorectal cancer: a double blind, placebo controlled, prospective randomized study. *The Journal of surgical research*, Vol. 129, No. 2, (12/05), pp. (172-5), ISSN 0022-4804 Kouvaris, J.R., Kouloulias, V.E., & Vlahos, L.J. (2007). Amifostine: the first selective-target

and broad-spectrum radioprotector. *The oncologist*, Vol., 12, No. 6, (06/07), pp. (738-

catabolism in the colonic mucosa of patients with colonic adenoma. *Digestive* 

from women with ductal breast cancer: a pilot study *Inflammation research*, Vol. 58,

Martin, G., Bergoc, R., Darvas, Z., Rivera, E.S., & Falus, A. (2002). Inhibition of human primary melanoma cell proliferation by histamine is enhanced by interleukin-6. *European journal of clinical investigation*, Vol 32, No 10, (10/02), pp.

Hickey, M., Mo, M., Chen, M.G., Berger, D., Lizambri, R., & Henke, M. (2011). Palifermin Reduces Severe Mucositis in Definitive Chemoradiotherapy of Locally

Kuefner, M.A., Schwelberger, H.G., Hahn, E.G., Raithel, M. (2008). Decreased histamine

Lázár-Molnár, E., Hegyesi, H., Pállinger, E., Kovács, P., Tóth, S., Fitzsimons, C., Cricco, G.,

Le, Q.T., Kim, H.E., Schneider, C.J., Muraközy, G., Skladowski, K., Reinisch, S., Chen, Y.,

*diseases and sciences,* Vol. 53, No. 2, (02/08), pp. (436-42), ISSN 0163-2116 Kyriakidis, K., Zampeli, E., & Tiligada, E. (2009). Histamine levels in whole peripheral blood

No. Suppl. 1, (04/09), pp. (S73-4), ISSN 1023-3830

Advanced Head and Neck Cancer: A Randomized, Placebo-Controlled Study. *World journal of clinical oncology*, (06/11), ISSN 2218-4333


Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 97

Muscoli, C., Cuzzocrea, S., Riley, D., Zweier, J., Thiemermann, C., Wang, Z., & Salvemini, D.

Nagler, R.M. (2002). The enigmatic mechanism of irradiation-induced damage to the major salivary glands. *Oral diseases*, Vol. 8, No. 3, (05/02), pp. (141-6), ISSN 1354-523X Nakamura, T., Itadani, H., Hidaka, Y., Ohta, M., & Tanaka, K. (2000). Molecular cloning and

Nakamura, Y., Smith, M., Krishna, A. & Terranova, P.F. (1987). Increased number of mast

Naredi, P. (2002). Histamine as an adjunct to immunotherapy. *Seminars in oncology,* No. 3,

Nguyen, T., Shapiro, D.A., George, S.R., Setola, V., Lee, D.K., Cheng, R., Rauser, L., Lee, S.P.,

Nielsen, H.J., Christensen, I.J., Moesgaard, F., Kehlet, H. (2002). Ranitidine as adjuvant

Nucifora, G., Smaller, B., Remko, R., & Avery, E.C. (1972) Transient Radicals of DNA Bases

Oda, T., Morikawa, N., Saito, Y., Masuho, Y., & Matsumoto, S. (2000). Molecular cloning and

Parshad, R., Hazrah, P., Kumar, S., Gupta, S.D., Ray, R., & Bal, S. (2005). Effect of

Pós, Z., Sáfrány, G., Müller, K., Tóth, S., Falus, A., & Hegyesi, H. (2005). Phenotypic profiling

*journal of cancer*, Vol. 42, no. 4, (10-12/05), pp. (185-190), ISSN 0019-509X Pós, Z., Hegyesi, H., & Rivera, E. (2004). Histamine and cell proliferation, In: *Histamine* 

Suppl. 7, (06/02), pp. (31-4), ISNN 0093-7754

Radiation Research, Vol. 49, No. 1, (06/72), pp. 96-111

3565

291X

3363

33), ISSN 0026-895X

ISSN 0021-9258

pp. (1416-22), ISSN 0007-1323

ISBN 963-9456-39X, Hungary

Pt 11, (06/02), pp. (2381-8), ISSN 0021-9533

(445-60), ISSN 0007-1188

*and experimental therapeutics*, Vol. 296, No. 3, (03/01), pp. (1058-1066), ISSN 0022-

(2003). On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. *British journal of pharmacology*, Vol., 140, No. 3, (10/03), pp.

characterization of a new human histamine receptor, HH4R. *Biochemical and biophysical research communications*, Vol. 289, No. 2, (12/00), pp. (615-20), ISSN 0006-

cells in the dominant follicle of the cow: relationships among luteal, stromal, and hilar regions. *Biology of reproduction*, Vol. 37, No. 3, (10/87), pp. 546-9, ISSN 0006-

Lynch, K.R., Roth, B.L., & O'Dowd, B.F. (2001). Discovery of a novel member of the histamine receptor family. *Molecular pharmacology*, Vol. 59, No. 3, (03/01), pp. (427-

treatment in colorectal cancer. *The British journal of surgery*, Vol. 89, No. 1, (11/02),

by Pulse Radiolysis. Effects of Cysteine and Cysteamine as Radioprotectors.

characterization of a novel type of histamine receptor preferentially expressed in leukocytes. *The Journal of biological chemistry*, Vol. 275, No. 47, (11/00), pp. (36781-6),

preoperative short course famotidine on TILs and survival in breast cancer. *Indian* 

*Biology and Medical Aspects*, Falus A (editor), pp. (199-217), SpringMed Publishing,

of engineered mouse melanomas with manipulated histamine production identifies histamine H2 receptor and rho-C as histamine-regulated melanoma progression markers. *Cancer research*, Vol. 65, No 10, (05/05), pp. (4458-66), ISSN:0008-5472 Potten, C.S., Owen, G., & Booth, D. (2002). Intestinal stem cells protect their genome by

selective segregation of template DNA strands. *Journal of cell science*, Vol. 115, No.

colorectal cancer: correlation with tumor stage. 2. *Inflammation research : official journal of the European Histamine Research Society*, Vol. 54, No. Suppl 1, (40/05), pp. 80-1, ISSN 1023-3830


Medina, V., Cricco, G., Nuñez, M., Martín, G., Mohamad, N., Correa-Fiz, F., Sanchez-

Medina, V., Croci, M., Mohamad, N., Massari, N., Garbarino, G., Cricco, P., Núñez, M.,

Medina, V.A., Massari, N.A., Cricco, G.P., Martín, G.A., Bergoc, R.M., & Rivera, E.S. (2009).

Medina, V.A., Croci, C., Carabajal, E., Bergoc, R.M., & Rivera, E.S. (2010). Histamine protects

*immunopathology,* Vol. 6, No. 4, (11/10), pp. (357-70), ISSN 1573-3955 Medina, V.A., & Rivera, E.S. (2010b). Histamine receptors and cancer Pharmacology. *British journal of pharmacology*, Vol. 161, No. 4, (10/10), pp. (755-67), ISSN 0007-1188 Medina, V., Prestifilippo, J.P., Croci, M., Carabajal, E., Bergoc, R.M., Elverdin, J.C., & Rivera,

*biology*, Vol., 87, No. 3, (03/11), pp. (284-92), ISSN 0955-3002

*radiation biology*, Vol., 83, No. 10, (10/07), pp. (653-63), ISSN 0955-3002 Medina, V., Croci, M., Crescenti, E., Mohamad, N., Sanchez-Jiménez, F., Massari, N., Nuñez,

80-1, ISSN 1023-3830

(11/06), pp. (1462-71), ISSN 1538-4047

46, No. 11, (06/09), pp. (1510-5), ISSN 0891-5849

pp. (27-35), ISSN 1538-4047

ISSN 1945-0494

colorectal cancer: correlation with tumor stage. 2. *Inflammation research : official journal of the European Histamine Research Society*, Vol. 54, No. Suppl 1, (40/05), pp.

Jimenez, F., Bergoc, R., & Rivera, E. (2006). Histamine-mediated signaling processes in human malignant mammary cells*. Cancer biology & therapy*, Vol. 5, No. 11,

Martín, G., Crescenti, E., Bergoc, R., & Rivera, E. (2007). Mechanisms underlying the radioprotective effect of histamine on small intestine. *International journal of* 

M., Cricco, P., Martin, G., Bergoc, R., & Rivera, E. (2008). The Role of Histamine in Human Mammary Carcinogenesis. H3 and H4 Receptors as Potential Therapeutic Targets for Breast Cancer Treatment. *Cancer biology & therapy*, Vol. 7, No. 1, (06/08),

Involvement of hydrogen peroxide in histamine-induced modulation of WM35 human malignant melanoma cell proliferation. *Free radical biology & medicine*, Vol.

bone marrow against cellular damage induced by ionizing radiation. *International journal of radiation biology*, Vol., 86, No. 4, (04/10), pp. (283-90), ISSN 0955-3002 Medina, V.A., & Rivera, E.S. (2010a). Histamine as a potential adjuvant to immuno and

radiotherapy for cancer treatment. Discovering new functions for the oldest biogenic amine. *Current immunology reviews, special Issue; Advances in* 

E.S. (2011a). Histamine prevents functional and morphological alterations of submandibular glands exerted by ionising radiation. *International journal of radiation* 

Pignataro, O., & Rivera, E.S. (2011b). Role of histamine H4 receptor in breast cancer cell proliferation. *Frontiers in Bioscience (Elite Edition),* Vol. 3, (06/11), pp. (1042-60),

M., Takeda, N., & Fukui, H. (2011). Histamine H1 receptor gene as an allergic diseases-sensitive gene and its impact on therapeutics for allergic diseases.

Y., Hipkin, R.W., Gonsiorek, W., Shin, N., Gustafson, E.L., Qiao, X., Wang, S., Hedrick, J.A., Greene, J., Bayne, M., & Monsma, F.J. (2001). Cloning and characterization of a novel human histamine receptor *The Journal of pharmacology* 

Medina, V.A., Brenzoni, P.G., Martinel Lamas, D.J., Massari, N., Mondillo, C., Nuñez, M.A.,

Mizuguchi, H., Kitamura, Y., Kondo, Y., Kuroda, W., Yoshida, H., Miyamoto, Y., Hattori,

*Yakugaku Zasshi*, Vol. 131, No. 2, (02/11), pp. (171-8), ISSN 0031-6903 Morse, K.L., Behan, J., Laz, T.M., West, R.E., Greenfeder, S.A., Anthes, J.C., Umland, S., Wan, *and experimental therapeutics*, Vol. 296, No. 3, (03/01), pp. (1058-1066), ISSN 0022- 3565


Histamine Receptors as Potential Therapeutic Targets for Cancer Drug Development 99

Tardivel-Lacombe, J., Morisset, S., Gbahou, F., Schwartz, J.C., & Arrang, J.M. (2001).

*pharmacological sciences*, Vol. 93, No. 3, (11/03), pp. (321-30), ISSN 1347-8613 Tomita, K., & Okabe, S. (2005). Exogenous histamine stimulates colorectal cancer implant

Tomita, K., Nakamura, E., & Okabe, S. (2005). Histamine regulates growth of malignant

Uçar, K. (1991). The effects of histamine H2 receptor antagonists on melanogenesis and

van Rijn, R.M., van Marle, A., Chazot, P.L., Langemeijer, E., Qin, Y., Shenton, F.C., Lim,

von Mach-Szczypiński, J., Stanosz, S., Sieja, K., & Stanosz, M. (2009). Metabolism of

Wagner, W., Ichikawa, A., Tanaka, S., Panula, P., & Fogel, W.A. (2003). Mouse mammary

Wang, K.Y., Arima, N., Higuchi, S., Shimajiri, S., Tanimoto, A., Murata, Y., Hamada, T., &

Wasserman, T.H., & Brizel, D.M. (2001). The role of amifostine as a radioprotector. *Oncology (Williston Park,* N.Y.), Vol., 15, No. 10, (10/01), pp. (1349-54), ISSN 0890-9091 Weigelt, C., Haas, R., & Kobbe, G. (2011). Parmacokinetic evaluation of palifermin for

Wellendorph, P., Goodman, M.W., Burstein, E.S., Nash, N.R., Brann, M.R., & Weiner, D.M.

Yang, L.P. & Perry, C.M. (2011). Histamine dihydrochloride: in the management of acute myeloid leukaemia. *Drugs*, Vol. 71, No. 1, (01/11), pp. 109-22, ISSN 0012-6667

*experimental*, Vol. 58. No. 6, (06/09), pp. (867-70), ISSN 0026-0495

gene. *Neuroreport*, Vol. 12, No. 2, (02/01), pp. (321-4), ISSN 0959-4965 Tomita, K., Izumi, K., & Okabe, S. (2003). Roxatidine- and cimetidine-induced angiogenesis

No. 1, (01/05), pp. (116-23), ISSN 1347-8613

3, (05), pp. (281-9), ISSN:0925-4692

(08/08), pp. (121-31), ISSN 0264-6021

(06/03), pp. (211-23), ISSN 0867-5910

(05/00), pp. (345-8), ISSN 0014-5793

(929-40), ISSN 0028-3908

0026-895X

Chromosomal mapping and organization of the human histamine H3 receptor

inhibition suppresses growth of colon cancer implants in syngeneic mice. *Journal of* 

growth via immunosuppression in mice. *Journal of pharmacological sciences*, Vol. 97,

melanoma implants via H2 receptors in mice. *Inflammopharmacology*, Vol. 13, No 1-

cellular proliferation in melanoma cells in culture. *Biochemical and biophysical research communications*, Vol. 177, No 1, (05/91), pp. (545-50), ISSN:0006-291X van Rijn, R.M., Chazot, P.L., Shenton, F.C., Sansuk, K., Bakker, R.A., & Leurs, R. (2006).

Oligomerization of recombinant and endogenously expressed human histamine H(4) receptors. *Molecular pharmacology*, Vol. 70, No. 2, (08/06), pp. (604-15), ISSN

H.D., Zuiderveld, O.P., Sansuk, K., Dy, M., Smit, M.J., Tensen, C.P., Bakker, R.A., & Leurs, R. (2008). Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor. *The Biochemical journal*, Vol. 414, No. 1,

histamine in tissues of primary ductal breast cancer. *Metabolism: clinical and* 

epithelial histamine system. *Journal of physiology and pharmacology*, Vol. 54, No. 2,

Sasaguri, Y. (2000). Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages. *FEBS letters*, Vol. 473, No. 3,

mucosal protection from chemotherapy and radiation. Expert opinion on drug metabolism & toxicology, Vol., 7, No. 4, (04/11), pp. (505-15505-15) ISSN 1742-5255

(2002). Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H3 receptor. Neuropharmacology, Vol. 42, No. 7, (06/02), pp.


Reynolds, J.L., Akhter, J., Adams, W.J., Morris, D.L. (1997). Histamine content in colorectal

Reynolds, J.L., Akhter, J.A., Magarey, C.J., Schwartz, P., Adams, W.J., & Morris, D.L. (1998).

Rivera, E.S., Cricco, G.P., Engel, N.I., Fitzimons, C.P., Martin, G.A., & Bergoc, R.M. (2000).

Rudolph, M.I., Boza, Y., Yefi, R., Luza, S., Andrews, E., Penissi, A., Garrido, P. & Rojas, I.G.

Sander, L.E., Lorentz, A., Sellge, G., Coëffier, M., Neipp, M., Veres, T., Frieling, T., Meier,

Schwartz, J.C. (2011). The histamine H3 receptor: from discovery to clinical trials with

Sieja, K., Stanosz, S., Von Mach-Szczypinski, J., Olewniezak, S., & Stanosz, M. (2005).

cancer in women. *Breast*, Vol. 14, No. 3, (06/05), pp (236-41), ISSN 0960-9776 Smit, M.J., Hoffmann, M., Timmerman, H., & Leurs, R. (1999). Molecular properties and

Smuda, C., & Bryce, P.J. (2011). New developments in the use of histamine and histamine

Soule, B.P., Simone, N.L., DeGraff, W.G., Choudhuri, R., Cook, J.A., Mitchell, J.B. (2010).

Szincsák, N., Hegyesi, H., Hunyadi, J., Falus, A., & Juhász, I. (2002). Different h2 receptor

Szukiewicz, D., Klimkiewicz, J., Pyzlak, M., Szewczyk, G. & Maslinska, D. (2007). Locally

*Inflammation Research,* Vol. 56, Suppl 1, (04/07), pp. S33-4, ISSN 1023-3830 Takahashi, K., Tanaka, S., Ichikawa, A. (2001). Effect of cimetidine on intratumoral cytokine

*communications*, Vol. 281, No. 5, (03/01), pp. (1113-9), ISSN 0006-291X

*Association*, Vol. 55, No. 4, (04/06), pp. (498-504), ISSN 0017-5749

Vol. 29, No. Suppl. 3, (07/99), pp. (19-28), ISSN 0954-7894

7983

579X

18, ISSN 1347-8613.

0007-1188

ISSN 1529-7322

6), ISSN:1065-6995

(04/98), pp. (538-41), ISSN 0007-1323

cancer. Are there sufficient levels of histamine to affect lymphocyte function? *European journal of surgical oncology*, Vol. 23, No. 3, (06/97), pp. (224-7), ISSN 0748-

Histamine in human breast cancer. *The British journal of surgery*, Vol. 85, No. 4,

Histamine as an autocrine growth factor: an unusual role for a widespread mediator. *Seminars in cancer biology*, Vol. 10, No. 1, (02/00), pp. (15-23), ISSN 1044-

(2008). The influence of mast cell mediators on migration of SW756 cervical carcinoma cells. *Journal of pharmacological sciences,* Vol. 106, No. 2, (02/08), pp. 208-

P.N., Manns, M.P., Bischoff, S.C. (2006). Selective expression of histamine receptors H1R, H2R, and H4R, but not H3R, in the human intestinal tract. *British Medical* 

pitolisant. *British journal of pharmacology*, Vol. 163, No. 4, (06/11), pp. (713-21), ISSN

Concentration of histamine in serum and tissues of the primary ductal breast

signaling pathways of the histamine H1 receptor. *Clinical and experimental allergy*,

receptors. *Current allergy and asthma reports*, Vol. 11, No. 2, (04/11), pp. (94-100),

Loratadine dysregulates cell cycle progression and enhances the effect of radiation in human tumor cell lines. *Radiation oncology*, Vol. 5, No. 8, (02/10), ISSN 1748-717X

antihistamines dissimilarly retard the growth of xenografted human melanoma cells in immunodeficient mice. *Cell biology international*, Vol 26, No 9, (02), pp. (833-

secreted histamine may regulate the development of ovarian follicles by apoptosis.

expression in an experimental tumor*. Biochemical and biophysical research* 


**5** 

*S\*BIO Pte Ltd Singapore* 

**Histone Deacetylase Inhibitors as** 

The processes of absorption (A), distribution (D), metabolism (M) and excretion (E) (collectively referred as ADME) determine the pharmacokinetics (PK) of a compound. Lack of optimum PK is one of the major reasons for compounds to fail in the clinic resulting in high attrition rates. In the beginning of 1990, 39% of the drugs failed in the clinic due to poor PK emphasizing its importance in drug development (Waterbeemd and Gifford, 2003). In 1988, a study of the pharmaceutical companies in UK showed that non-optimal PK was one of the major reasons (~40%) for termination of drugs in development (Prentis et al., 1988). In the last two decades this number dropped to ~ 10% (Yengi et al., 2007). The main reasons for this significant drop in the number of compounds failing for PK reasons can be attributed to the following: a) application of concepts of drug metabolism and PK to design compounds in medicinal chemistry programs (Smith et al., 1996); b) development of *in vitro*  ADME assays that are predictive of *in vivo* behavior (PK) of drugs (Obach et al., 1997; Venkatakrishnan et al.,2003; Pelkonen and Raunio, 2005; Thompson,2000; Fagerholm, 2007); c) use of the Lipinski rule of 5 to design oral drugs (Lipinski, 2000); d) development of computer programs to predict the human PK parameters and profiles based on *in vitro* ADME properties of drugs (Jamei et al., 2009); e) PK/PD correlation studies in preclinical setting and f) high throughput screening of ADME properties in *in vitro* and *in vivo* assays for hundreds of compounds in the lead identification to lead optimization stages of drug discovery. The consequence of all the above mentioned developments in ADME have resulted in the frontloading of non-drug like compounds early in drug discovery and

Histone acetylases (HATs) and Histone deacetylases (HDACs) are enzymes that carry out acetylation and deacetylation, respectively, of histone proteins (Minucci and Pelicci, 2006). Histone proteins form a complex with DNA called as nucleosomes, which are the structural units of chromatin. The interplay of HATs and HDACs activities regulate the structure of chromatin and control gene expression. The aberrant expression of HDACs has been linked to the pathogenesis of cancer (Minucci and Pelicci, 2006). Histone deacetylase inhibitors

ultimately reducing the attrition rates of compounds in the clinic.

**1. Introduction** 

**Therapeutic Agents for Cancer** 

**Therapy: Drug Metabolism and** 

**Pharmacokinetic Properties** 

Ethirajulu Kantharaj and Ramesh Jayaraman

Zampeli, E., & Tiligada, E. (2009). The role of histamine H4 receptor in immune and inflammatory disorders. *British journal of pharmacology*, Vol. 157, No. 1, (05/09), pp. (24-33), ISSN 0007-1188

## **Histone Deacetylase Inhibitors as Therapeutic Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties**

Ethirajulu Kantharaj and Ramesh Jayaraman *S\*BIO Pte Ltd Singapore* 

## **1. Introduction**

100 Drug Development – A Case Study Based Insight into Modern Strategies

Zampeli, E., & Tiligada, E. (2009). The role of histamine H4 receptor in immune and

(24-33), ISSN 0007-1188

inflammatory disorders. *British journal of pharmacology*, Vol. 157, No. 1, (05/09), pp.

The processes of absorption (A), distribution (D), metabolism (M) and excretion (E) (collectively referred as ADME) determine the pharmacokinetics (PK) of a compound. Lack of optimum PK is one of the major reasons for compounds to fail in the clinic resulting in high attrition rates. In the beginning of 1990, 39% of the drugs failed in the clinic due to poor PK emphasizing its importance in drug development (Waterbeemd and Gifford, 2003). In 1988, a study of the pharmaceutical companies in UK showed that non-optimal PK was one of the major reasons (~40%) for termination of drugs in development (Prentis et al., 1988). In the last two decades this number dropped to ~ 10% (Yengi et al., 2007). The main reasons for this significant drop in the number of compounds failing for PK reasons can be attributed to the following: a) application of concepts of drug metabolism and PK to design compounds in medicinal chemistry programs (Smith et al., 1996); b) development of *in vitro*  ADME assays that are predictive of *in vivo* behavior (PK) of drugs (Obach et al., 1997; Venkatakrishnan et al.,2003; Pelkonen and Raunio, 2005; Thompson,2000; Fagerholm, 2007); c) use of the Lipinski rule of 5 to design oral drugs (Lipinski, 2000); d) development of computer programs to predict the human PK parameters and profiles based on *in vitro* ADME properties of drugs (Jamei et al., 2009); e) PK/PD correlation studies in preclinical setting and f) high throughput screening of ADME properties in *in vitro* and *in vivo* assays for hundreds of compounds in the lead identification to lead optimization stages of drug discovery. The consequence of all the above mentioned developments in ADME have resulted in the frontloading of non-drug like compounds early in drug discovery and ultimately reducing the attrition rates of compounds in the clinic.

Histone acetylases (HATs) and Histone deacetylases (HDACs) are enzymes that carry out acetylation and deacetylation, respectively, of histone proteins (Minucci and Pelicci, 2006). Histone proteins form a complex with DNA called as nucleosomes, which are the structural units of chromatin. The interplay of HATs and HDACs activities regulate the structure of chromatin and control gene expression. The aberrant expression of HDACs has been linked to the pathogenesis of cancer (Minucci and Pelicci, 2006). Histone deacetylase inhibitors

Histone Deacetylase Inhibitors asTherapeutic

H N

O N

O

<sup>H</sup> <sup>O</sup>

N

N H

H N

N N

S H N O

O

N

(Givinostat) HN

N

N

HN

O

O

N H

HN O

O

H N

O

N H

<sup>N</sup> Hydroxamic

O

O

O O

N H OH

OH

HN O

N H OH

S

O

Vorinostat (ZOLINZATM)

Romidepsin

MGCD0103 (Mocetinostat)

LBH589 (Panabinostat)

SB939 (Pracinostat)

ITF2357

PXD101 (Belinostat)

(Istodax) NH

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 103

**Compound name Structure Class Stage of clinical** 

O

N H

S

OH Hydroxamic

**development\***

Acid Approved (2006)

Cyclic peptide Approved (2009)

Acid Phase 2

Acid Phase 2

Acid Phase 2

Acid Phase 2

NH2 Benzamide Phase 2

Hydroxamic

Hydroxamic

OH Hydroxamic

(HDACi) are an emerging class of therapeutic agents that induce tumor cell cytostasis, differentiation and apoptosis in various hematologic and solid malignancies (Mercurio et al., 2010; Stimson et al., 2009). They are known to exert their anti-tumor activity by inhibiting the HDACs, which play an important role in controlling gene expression by chromatin remodeling, that affect cell cycle and survival pathways (Stimson et al., 2009). Inhibitors of histone deacetylases (HDACi) also show promising anti-inflammatory properties as demonstrated in a number of animal and cellular models of inflammatory diseases and for diabetes (Christensen et al., 2011). The HDACi Zolinza (Vorinostat/ Suberolyanilide hydroxamic acid [SAHA]) and Romidepsin (FK228) have been approved by the FDA (United States Food and Drug Administration) for the treatment of cutaneous T cell Lymphoma (CTCL) (Mann et al., 2007, Grant et al.,2010) and for peripheral T cell lymphoma (PTCL)(http://www.accessdata.fda.gov/drugsatfda\_docs/appletter/2011/022393s004ltr.p df) as such demonstrating clinical "proof-of-principle" for this class of compounds.

Four groups of HDAC inhibitors have been characterized: (i) short chain fatty acids (e.g., Sodium butyrate and phenylbutyrate), (ii) cyclic tetrapeptides (e.g., Depsipeptide and Trapoxin), (iii) benzamides (e.g. MGCD0103 (Mocetinostat), Cl-994 and MS-275 (Entinostat)), and (iv) hydroxamic acids (e.g., SAHA [Vorinostat/Zolinza]), LBH589 (Panabinostat), SB939 (Pracinostat), ITF2357 (Givinostat), PXD101 etc). Table 1 shows compounds that are currently in different stages of clinical development.

The clinical progress that has been made by hydroxamic acid derivatives as HDAC inhibitors is of particular interest because they are usually considered as non-druggable and are down-prioritized in lead identification campaigns attributing to their poor physicochemical and ADME properties. SB939 (Pracinostat) is a potent HDACi that was discovered and developed at S\*BIO (Wang et al., 2011; Novotny-Diermayr et al, 2011) to overcome some of the ADME and PK/PD (Pharmacokinetic/Pharmacodynamic) limitations of the current HDACi. The pharmacokinetics and drug metabolism aspects of the four classes of HDACi have not been reviewed extensively. In this article, we review the pharmacokinetic and drug metabolism properties of SB939 and the preclinical and clinical ADME aspects of other HDAC inhibitors in the clinic.

## **2. Short chain fatty acids**

### **2.1 Sodium butyrate (SB)**

Sodium butyrate is a short chain fatty acid inhibitor of HDAC enzymes that is in phase 2 clinical trials. The PK of SB in preclinical species was characterized by poor bioavailability, short t1/2 (< 5 min in mice and rabbits), leading to challenges in oral administration (Coradini et al, 1999; Daniel P et al, 1989). Butyrate was found to be transported by via a carrier mediated transport system MCT1 in Caco-2 cells suggesting that the absorption of SB might be saturable (Stein et al., 2000). SB has been reported to significantly increase the cytochrome P450 3A4 (CYP3A4) activity in Caco-2 cells transfected with CYP3A4 (Cummins et al; 2001) and induce P glycoprotein (PgP) *in vivo* (Machavaram et al., 2000). Due to its low potency very high doses were required to achieve pharmacological concentrations in animals and humans (Kim and Bae, 2011). In PK studies in mice and rats, SB showed rapid clearance (CL) with non-linear PK resulting from the high doses (up to 5 g/kg in mice), based on which the authors indicated that high doses would be problematic in humans (Egorin et al., 1999). In a clinical pharmacology study in leukemia patients, where SB was administered as continuous intravenous (IV) infusions (at a dose of 500 mg/kg/day) over a

(HDACi) are an emerging class of therapeutic agents that induce tumor cell cytostasis, differentiation and apoptosis in various hematologic and solid malignancies (Mercurio et al., 2010; Stimson et al., 2009). They are known to exert their anti-tumor activity by inhibiting the HDACs, which play an important role in controlling gene expression by chromatin remodeling, that affect cell cycle and survival pathways (Stimson et al., 2009). Inhibitors of histone deacetylases (HDACi) also show promising anti-inflammatory properties as demonstrated in a number of animal and cellular models of inflammatory diseases and for diabetes (Christensen et al., 2011). The HDACi Zolinza (Vorinostat/ Suberolyanilide hydroxamic acid [SAHA]) and Romidepsin (FK228) have been approved by the FDA (United States Food and Drug Administration) for the treatment of cutaneous T cell Lymphoma (CTCL) (Mann et al., 2007, Grant et al.,2010) and for peripheral T cell lymphoma (PTCL)(http://www.accessdata.fda.gov/drugsatfda\_docs/appletter/2011/022393s004ltr.p

df) as such demonstrating clinical "proof-of-principle" for this class of compounds.

compounds that are currently in different stages of clinical development.

ADME aspects of other HDAC inhibitors in the clinic.

**2. Short chain fatty acids 2.1 Sodium butyrate (SB)** 

Four groups of HDAC inhibitors have been characterized: (i) short chain fatty acids (e.g., Sodium butyrate and phenylbutyrate), (ii) cyclic tetrapeptides (e.g., Depsipeptide and Trapoxin), (iii) benzamides (e.g. MGCD0103 (Mocetinostat), Cl-994 and MS-275 (Entinostat)), and (iv) hydroxamic acids (e.g., SAHA [Vorinostat/Zolinza]), LBH589 (Panabinostat), SB939 (Pracinostat), ITF2357 (Givinostat), PXD101 etc). Table 1 shows

The clinical progress that has been made by hydroxamic acid derivatives as HDAC inhibitors is of particular interest because they are usually considered as non-druggable and are down-prioritized in lead identification campaigns attributing to their poor physicochemical and ADME properties. SB939 (Pracinostat) is a potent HDACi that was discovered and developed at S\*BIO (Wang et al., 2011; Novotny-Diermayr et al, 2011) to overcome some of the ADME and PK/PD (Pharmacokinetic/Pharmacodynamic) limitations of the current HDACi. The pharmacokinetics and drug metabolism aspects of the four classes of HDACi have not been reviewed extensively. In this article, we review the pharmacokinetic and drug metabolism properties of SB939 and the preclinical and clinical

Sodium butyrate is a short chain fatty acid inhibitor of HDAC enzymes that is in phase 2 clinical trials. The PK of SB in preclinical species was characterized by poor bioavailability, short t1/2 (< 5 min in mice and rabbits), leading to challenges in oral administration (Coradini et al, 1999; Daniel P et al, 1989). Butyrate was found to be transported by via a carrier mediated transport system MCT1 in Caco-2 cells suggesting that the absorption of SB might be saturable (Stein et al., 2000). SB has been reported to significantly increase the cytochrome P450 3A4 (CYP3A4) activity in Caco-2 cells transfected with CYP3A4 (Cummins et al; 2001) and induce P glycoprotein (PgP) *in vivo* (Machavaram et al., 2000). Due to its low potency very high doses were required to achieve pharmacological concentrations in animals and humans (Kim and Bae, 2011). In PK studies in mice and rats, SB showed rapid clearance (CL) with non-linear PK resulting from the high doses (up to 5 g/kg in mice), based on which the authors indicated that high doses would be problematic in humans (Egorin et al., 1999). In a clinical pharmacology study in leukemia patients, where SB was administered as continuous intravenous (IV) infusions (at a dose of 500 mg/kg/day) over a

Histone Deacetylase Inhibitors asTherapeutic

**2.2 Sodium phenyl butyrate (PB)** 

**2.3 Sodium valproate** 

patients with hyperammonemia (Gilbert et al., 2001).

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 105

10 day period, SB declined rapidly post infusion with a very short t1/2 (~ 6 min), with high systemic clearance (CL~5 L/h/kg) and low volume of distribution (Vd =0.74 L/kg) (Miller et al., 1987). The amount of unchanged SB in urine was minimal suggesting that SB's clearance was primarily by metabolism. The authors concluded that the lack of efficacy of SB in the leukemic patients was due to its low plasma levels and very short t1/2 (Miller et al., 1987).

Sodium phenyl butyrate (PB) is an aromatic fatty acid HDACi, with low potency of 0.5 mM that is in phase 2 trials for cancer. PB (Buphenyl) has already been approved by the FDA for

In a phase 1 study in patients with solid tumors, the PK of PB was characterized by rapid absorption (time of peak concentration [tmax] ~1.8 h), dose proportional increase in oral exposures between doses of 9 and 36 g/day, a short t1/2 of 1 h, with mean absolute oral bioavailability (F) of 78% (Gilbert et al., 2001). In the same study, the major circulating metabolites of PB were phenylacetate (PA) and phenyacetylglutamine (PG), the exposures of which were 46-66% and 70-100% respectively of PB, suggesting extensive metabolic clearance of PB in humans. The highest percentage of patients that showed stable disease was from the 36 g/day cohort, in which the time above 0.5 mM was ~ 4.0 h (Gilbert et al., 2001). In another phase 1 study in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML), where PB was dosed as IV infusions, PB showed non-linear PK between 125 and 500 mg/kg/day, with PA and PG being formed as major metabolites (Gore et al., 2001). The low potency of PB requires very high doses in humans, leading to non-linear kinetics, thus making it a less attractive chemotherapeutic agent. In another phase 1 study, where PB was evaluated as continuous IV infusions (120 h) in solid tumors, the PK of PB was best described by saturable elimination, and PG was the major metabolite found in urine which was indicative of extensive metabolic clearance of PB in humans (Carducci et al., 2001). In the same study the plasma clearance (CL) of PB increased during the infusion period in some patients at higher dose levels. In a dose escalation oral study of PB in patients with glioma, who also received anticonvulsants concomitantly, the mean CL of PB was significantly higher than in solid tumor patients, and the possible reason was attributed to the induction of cytochrome P450 (CYP450) enzymes by anticonvulsants (Phuphanich et al., 2005). Thus it appears that the

CYP450 metabolism might play a significant role in clearance of PB in humans.

http://www.accessdata.fda.gov/drugsatfda\_docs/label).

Sodium valproate is a short chain fatty acid that is currently in phase 1 and 2 clinical trials in patients with solid tumors and hematological malignancies (Federico and Bagella, 2011). Sodium valproate (Depakote) has been previously approved for use in epilepsy patients and is in medical use for the last 3 decades (Federico and Bagella, 2011). It is a moderately potent inhibitor of class 1 HDAC enzymes with promising antitumor effects *in vitro* and *in vivo*. The human ADME of sodium valproate is characterized by a) high plasma protein binding (PPB) of 90 % with concentration dependent PPB; b) weak inhibitor of some CYP450, epoxide hydrolase and glucoronosyl transferases; c) entirely metabolized by the liver via glucoronidation and β-oxidation pathways with less than 3% of unchanged parent drug found in the urine; d) minimum drug-drug interaction (DDI) potential with CYP450 inhibitors as CYP450 mediated oxidation is a minor pathway ; e) high absolute oral bioavailability (90%); f) mean terminal half-life of 9-16 h (Depakote prescribing information,


\* Reference from http://www.fda.gov

Table 1. HDAC inhibitors in clinical development

10 day period, SB declined rapidly post infusion with a very short t1/2 (~ 6 min), with high systemic clearance (CL~5 L/h/kg) and low volume of distribution (Vd =0.74 L/kg) (Miller et al., 1987). The amount of unchanged SB in urine was minimal suggesting that SB's clearance was primarily by metabolism. The authors concluded that the lack of efficacy of SB in the leukemic patients was due to its low plasma levels and very short t1/2 (Miller et al., 1987).

## **2.2 Sodium phenyl butyrate (PB)**

104 Drug Development – A Case Study Based Insight into Modern Strategies

**Compound name Structure Class Stage of clinical** 

N H

O Na<sup>+</sup>

<sup>O</sup> <sup>O</sup>

O

H N O

Na O <sup>+</sup>

N O

N H

HN OH

O HN

OH

<sup>O</sup> Na<sup>+</sup> Short chain

O Benzamide Phase 2

NH2 Benzamide Phase 2

fatty acid Phase 2

fatty acid Phase 2

acid Phase 1

acid Phase 1

acid Phase 1

fatty acid Phase 2

Short chain

Short chain

OH Hydroxamic

Hydroxamic

Hydroxamic

O

NH2 <sup>N</sup>

O

O

N N

O

O

H

N N NH

NH

NH

O

N

Table 1. HDAC inhibitors in clinical development

O

N

O N H

O

CI994 (Tacedinaline)

MS-275

Sodium Butyrate

Sodium Phenylbutyrate

CUDC-101

JNJ-26481585

CRA 24781 (PCI-24781)

Sodium Valproate

Reference from http://www.fda.gov

\*

(Entinostat) <sup>N</sup>

**development\***

Sodium phenyl butyrate (PB) is an aromatic fatty acid HDACi, with low potency of 0.5 mM that is in phase 2 trials for cancer. PB (Buphenyl) has already been approved by the FDA for patients with hyperammonemia (Gilbert et al., 2001).

In a phase 1 study in patients with solid tumors, the PK of PB was characterized by rapid absorption (time of peak concentration [tmax] ~1.8 h), dose proportional increase in oral exposures between doses of 9 and 36 g/day, a short t1/2 of 1 h, with mean absolute oral bioavailability (F) of 78% (Gilbert et al., 2001). In the same study, the major circulating metabolites of PB were phenylacetate (PA) and phenyacetylglutamine (PG), the exposures of which were 46-66% and 70-100% respectively of PB, suggesting extensive metabolic clearance of PB in humans. The highest percentage of patients that showed stable disease was from the 36 g/day cohort, in which the time above 0.5 mM was ~ 4.0 h (Gilbert et al., 2001). In another phase 1 study in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML), where PB was dosed as IV infusions, PB showed non-linear PK between 125 and 500 mg/kg/day, with PA and PG being formed as major metabolites (Gore et al., 2001). The low potency of PB requires very high doses in humans, leading to non-linear kinetics, thus making it a less attractive chemotherapeutic agent. In another phase 1 study, where PB was evaluated as continuous IV infusions (120 h) in solid tumors, the PK of PB was best described by saturable elimination, and PG was the major metabolite found in urine which was indicative of extensive metabolic clearance of PB in humans (Carducci et al., 2001). In the same study the plasma clearance (CL) of PB increased during the infusion period in some patients at higher dose levels. In a dose escalation oral study of PB in patients with glioma, who also received anticonvulsants concomitantly, the mean CL of PB was significantly higher than in solid tumor patients, and the possible reason was attributed to the induction of cytochrome P450 (CYP450) enzymes by anticonvulsants (Phuphanich et al., 2005). Thus it appears that the CYP450 metabolism might play a significant role in clearance of PB in humans.

## **2.3 Sodium valproate**

Sodium valproate is a short chain fatty acid that is currently in phase 1 and 2 clinical trials in patients with solid tumors and hematological malignancies (Federico and Bagella, 2011). Sodium valproate (Depakote) has been previously approved for use in epilepsy patients and is in medical use for the last 3 decades (Federico and Bagella, 2011). It is a moderately potent inhibitor of class 1 HDAC enzymes with promising antitumor effects *in vitro* and *in vivo*. The human ADME of sodium valproate is characterized by a) high plasma protein binding (PPB) of 90 % with concentration dependent PPB; b) weak inhibitor of some CYP450, epoxide hydrolase and glucoronosyl transferases; c) entirely metabolized by the liver via glucoronidation and β-oxidation pathways with less than 3% of unchanged parent drug found in the urine; d) minimum drug-drug interaction (DDI) potential with CYP450 inhibitors as CYP450 mediated oxidation is a minor pathway ; e) high absolute oral bioavailability (90%); f) mean terminal half-life of 9-16 h (Depakote prescribing information, http://www.accessdata.fda.gov/drugsatfda\_docs/label).

Histone Deacetylase Inhibitors asTherapeutic

2008).

**4.2 CI994 (N-acetyldinaline)** 

the oral PK of CI-994.

**4.3 Entinostat (MS-275)** 

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 107

Mocetinostat showed moderate Vss (0.35 -0.91 L/kg), moderate to high CL (1.7 to 4.3 L/h/kg), short t1/2 (0.6-1.3 h), with F ranging between low (mice =12%), moderate (rat=47%) and low-high (dogs=1-92%) (Zhou et al., 2008). In preclinical PK and PD studies, where the dihydrobromo salt of Mocetinostat was used, the dosing formulations required acidification

In a phase 1 study in patients with leukemia, the oral PK of Mocetinostat was characterized by rapid absorption (tmax = 0.5-1.2 h), mean elimination t1/2 of 7-11 h, and a dose related increase in peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) between 20 and 60 mg/m2 and tended to plateau at higher doses (Garcia-Manero et al., 2011). Based on the lack of accumulation upon repeated dosing, it was suggested that induction or inhibition of drug elimination was unlikely in humans (Le Tourneau and Siu,

CI994 (N-acetlydinaline), belonging to the benzamide class, is a HDACi with promising antitumor activities in preclinical xenograft models, and subsequently progressed to phase 1 2 clinical trials (Richards et al., 2006). CI994, a small molecule (MW=269.3) and with poor aqueous solubility, was developed as an acetylated analogue of Dinaline (GOE-1734), which, also showed equivalent antitumor activity (LoRusso et al., 1996). CI994 was eventually identified as an active metabolite of Dinaline (LoRusso et al., 1996). Limited data is available on its *in vitro* ADME. It showed low PPB in mice (20%) (Foster et al., 1997). In an oral PK and metabolism study in mice, where CI-994 was dosed once daily at 50 mg/kg for 14 days, it showed moderately rapid absorption (tmax= 30-45 min), 2 compartment disposition with a terminal t1/2 on day 1 (9.4 h) being longer than on day 14 (3.4 h), and oral CL ranging between 0.42 (Day 1) -0.52 (day 14) ml/min (Foster et al., 1997). High amounts of unchanged drug (42-58% of dose) were found in the urine with minimal amounts in fecal samples, suggesting that renal clearance was a major clearance pathway for CI-994. Low amounts of Dinaline were found in urine and feces indicating that *in vivo* conversion of CI-994 to Dinaline were not significant. In rhesus monkeys, the PK of CI-994 was characterized by low volume of distribution (Vd) (0.3 L/kg) and CL (0.05 L/h/kg), a moderate t1/2 (7.4 h), and high brain penetration (Riva et al., 2000). The oral bioavailability of CI-994 in preclinical species was 100% (Riva et al., 2000). In a phase 1 study in cancer patients following oral dosing (5-15 mg/m2), CI-994 showed rapid absorption (tmax 0.7-1.6 h), oral CL ranging between ~30-48 ml/min/m2), dose proportional increases in Cmax and AUC, and moderately long t1/2 (7.4-14 h) (Prakash et al., 2001). In the same study, no food effects were observed on

Entinostat (MS-275) is a small molecule, synthetic benzamide that is currently in phase 2 trials (Mercurio et al., 2010). It is moderately lipophilic (LogD= 1.79), with moderate plasma protein binding (fraction unbound [fu] ranged between 0.375 to 0.439 in preclinical species, and 0.188 in humans) (Hooker et al., 2010; Acharya et al., 2006). In preclinical pharmacology studies, the tmax of Entinostat ranged between 30-40 minutes with a t1/2 of ~ 1 h in rats, mice and dogs, and the oral bioavailability was high (F~ 85%) (Ryan et al., 2005). In a radiolabeled tissue distribution and brain penetration study in baboons, radioactivity was cleared both by renal and biliary systems, and showed poor brain penetration (Hooker et al,

and cosolvent addition indicating solubility issues (Zhou et al, 2008).

## **3. Cyclic tetrapeptides**

## **3.1 Romidepsin (FK228, depsipeptide, ISTODAXTM)**

Romidepsin is a bicyclic peptide that was isolated as a secondary metabolite from a naturally occurring soil bacterium, and found to be a potent anti-tumor agent *in vitro* and *in vivo* (Ueda et al., 1994) and subsequently found to be a potent HDACi. It was approved by the FDA for treatment of patients with refractory CTCL (Mercurio et al., 2010). Romidepsin is a high molecular weight drug (Mw ~ 541), highly lipophilic, and insoluble in water, necessitating intraperitoneal and subcutaneous administrations in pharmacology studies (Ueda et al., 1994). The *in vitro* PPB of Romidepsin to human plasma was 92-94 % over a concentration of 50-1000 ng/mL, indicating high binding (http://www.accessdata.fda). Romidepsin is a substrate of PgP and MRP1 (Xiao et al., 2005). Depsipeptide was extensively metabolized by human liver microsomes, leading to the formation of at least 10 different metabolites, and was found to be primarily metabolized by CYP3A4 *in vitro* (Shiraga et al., 2005). Among the metabolites formed, mono-oxidation, di-oxidation, reduction of disulfide metabolites and two unidentified metabolites were the major metabolites in humans (http://www.accessdata.fda). It did not seem to inhibit any of the major human CYP450 enzymes *in vitro*, and there are no reports on its effect on the induction of human CYP450s (http://www.accessdata.fda). The preclinical PK of depsipeptide was characterized by high systemic CL and long t1/2 (~ 6.0 h) in mice (Graham et al., 2006). In rats, the volume of distribution at steady state (Vss) was very high (100 L/kg) and systemic CL was high (~ 49 L/h/kg), t1/2 was short (18 min), and had poor oral bioavailability (F= ~ 2-11%) (Li and Chan, 2000). The low F in rats may be could be due to high first-pass effect, poor solubility and PgP efflux. Systemic CL (~1.8 L/h/kg) and t1/2 (205 min) were moderate in nonhuman primates (Berg et al., 2004). In a radiolabelled mass-balance study in rats with FK228, approximately 98% of the dose was recovered in excreta with ~ 79% of the dose in the feces, and biliary clearance appeared to be the main clearance mechanism (http://www.accessdata.fda; Shiraga et al., 2005). Unchanged FK228 accounted for 3% of the dose, with > 30 metabolites detected in bile, indicating extensive metabolism of FK228 (Shiraga et al., 2005). The clinical PK of Romidepsin was characterized by low Vss (54 L), low CL (20 L/h), and a short t1/2 (~ 3.5 h) (http://www.accessdata.fda; Woo et al., 2009). The intra-patient variability was moderate to high (30-80%) and the inter-patient variability was high (50-70%) (http://www.accessdata.fda;). Despite the high inter-patient variability the AUC and Cmax increased dose proportionally (http://www.accessdata.fda).

Romidepsin is the only HDACi that seems to be a PgP substrate. Romidepsin induced PgP expression in the HCT15 tumor cell line and conferred resistance to its action (Xiao et al., 2005). A possibility of correlation between PgP induction and the poor response rate of Romidepsin in cancer patients has been proposed (Xiao et al., 2005).

## **4. Benzamides**

## **4.1 Mocetinostat (MGCD0103)**

Mocetinostat (MGCD0103), an aminophenyl benzamide, is a potent inhibitor of HDAC 1, 2, and 3 enzymes and has recently completed Phase 2 clinical trials (Mercurio et al., 2010). It is a small molecule (Mw~396) and moderately lipophilic (LogP=2.6). There is no information available on its permeability, microsomal stability, metabolism, plasma protein binding, CYP450 inhibition and induction. In preclinical PK studies in mice, rat and dog, Mocetinostat showed moderate Vss (0.35 -0.91 L/kg), moderate to high CL (1.7 to 4.3 L/h/kg), short t1/2 (0.6-1.3 h), with F ranging between low (mice =12%), moderate (rat=47%) and low-high (dogs=1-92%) (Zhou et al., 2008). In preclinical PK and PD studies, where the dihydrobromo salt of Mocetinostat was used, the dosing formulations required acidification and cosolvent addition indicating solubility issues (Zhou et al, 2008).

In a phase 1 study in patients with leukemia, the oral PK of Mocetinostat was characterized by rapid absorption (tmax = 0.5-1.2 h), mean elimination t1/2 of 7-11 h, and a dose related increase in peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) between 20 and 60 mg/m2 and tended to plateau at higher doses (Garcia-Manero et al., 2011). Based on the lack of accumulation upon repeated dosing, it was suggested that induction or inhibition of drug elimination was unlikely in humans (Le Tourneau and Siu, 2008).

## **4.2 CI994 (N-acetyldinaline)**

106 Drug Development – A Case Study Based Insight into Modern Strategies

Romidepsin is a bicyclic peptide that was isolated as a secondary metabolite from a naturally occurring soil bacterium, and found to be a potent anti-tumor agent *in vitro* and *in vivo* (Ueda et al., 1994) and subsequently found to be a potent HDACi. It was approved by the FDA for treatment of patients with refractory CTCL (Mercurio et al., 2010). Romidepsin is a high molecular weight drug (Mw ~ 541), highly lipophilic, and insoluble in water, necessitating intraperitoneal and subcutaneous administrations in pharmacology studies (Ueda et al., 1994). The *in vitro* PPB of Romidepsin to human plasma was 92-94 % over a concentration of 50-1000 ng/mL, indicating high binding (http://www.accessdata.fda). Romidepsin is a substrate of PgP and MRP1 (Xiao et al., 2005). Depsipeptide was extensively metabolized by human liver microsomes, leading to the formation of at least 10 different metabolites, and was found to be primarily metabolized by CYP3A4 *in vitro* (Shiraga et al., 2005). Among the metabolites formed, mono-oxidation, di-oxidation, reduction of disulfide metabolites and two unidentified metabolites were the major metabolites in humans (http://www.accessdata.fda). It did not seem to inhibit any of the major human CYP450 enzymes *in vitro*, and there are no reports on its effect on the induction of human CYP450s (http://www.accessdata.fda). The preclinical PK of depsipeptide was characterized by high systemic CL and long t1/2 (~ 6.0 h) in mice (Graham et al., 2006). In rats, the volume of distribution at steady state (Vss) was very high (100 L/kg) and systemic CL was high (~ 49 L/h/kg), t1/2 was short (18 min), and had poor oral bioavailability (F= ~ 2-11%) (Li and Chan, 2000). The low F in rats may be could be due to high first-pass effect, poor solubility and PgP efflux. Systemic CL (~1.8 L/h/kg) and t1/2 (205 min) were moderate in nonhuman primates (Berg et al., 2004). In a radiolabelled mass-balance study in rats with FK228, approximately 98% of the dose was recovered in excreta with ~ 79% of the dose in the feces, and biliary clearance appeared to be the main clearance mechanism (http://www.accessdata.fda; Shiraga et al., 2005). Unchanged FK228 accounted for 3% of the dose, with > 30 metabolites detected in bile, indicating extensive metabolism of FK228 (Shiraga et al., 2005). The clinical PK of Romidepsin was characterized by low Vss (54 L), low CL (20 L/h), and a short t1/2 (~ 3.5 h) (http://www.accessdata.fda; Woo et al., 2009). The intra-patient variability was moderate to high (30-80%) and the inter-patient variability was high (50-70%) (http://www.accessdata.fda;). Despite the high inter-patient variability the

AUC and Cmax increased dose proportionally (http://www.accessdata.fda).

Romidepsin in cancer patients has been proposed (Xiao et al., 2005).

**4. Benzamides** 

**4.1 Mocetinostat (MGCD0103)** 

Romidepsin is the only HDACi that seems to be a PgP substrate. Romidepsin induced PgP expression in the HCT15 tumor cell line and conferred resistance to its action (Xiao et al., 2005). A possibility of correlation between PgP induction and the poor response rate of

Mocetinostat (MGCD0103), an aminophenyl benzamide, is a potent inhibitor of HDAC 1, 2, and 3 enzymes and has recently completed Phase 2 clinical trials (Mercurio et al., 2010). It is a small molecule (Mw~396) and moderately lipophilic (LogP=2.6). There is no information available on its permeability, microsomal stability, metabolism, plasma protein binding, CYP450 inhibition and induction. In preclinical PK studies in mice, rat and dog,

**3. Cyclic tetrapeptides** 

**3.1 Romidepsin (FK228, depsipeptide, ISTODAXTM)** 

CI994 (N-acetlydinaline), belonging to the benzamide class, is a HDACi with promising antitumor activities in preclinical xenograft models, and subsequently progressed to phase 1 2 clinical trials (Richards et al., 2006). CI994, a small molecule (MW=269.3) and with poor aqueous solubility, was developed as an acetylated analogue of Dinaline (GOE-1734), which, also showed equivalent antitumor activity (LoRusso et al., 1996). CI994 was eventually identified as an active metabolite of Dinaline (LoRusso et al., 1996). Limited data is available on its *in vitro* ADME. It showed low PPB in mice (20%) (Foster et al., 1997). In an oral PK and metabolism study in mice, where CI-994 was dosed once daily at 50 mg/kg for 14 days, it showed moderately rapid absorption (tmax= 30-45 min), 2 compartment disposition with a terminal t1/2 on day 1 (9.4 h) being longer than on day 14 (3.4 h), and oral CL ranging between 0.42 (Day 1) -0.52 (day 14) ml/min (Foster et al., 1997). High amounts of unchanged drug (42-58% of dose) were found in the urine with minimal amounts in fecal samples, suggesting that renal clearance was a major clearance pathway for CI-994. Low amounts of Dinaline were found in urine and feces indicating that *in vivo* conversion of CI-994 to Dinaline were not significant. In rhesus monkeys, the PK of CI-994 was characterized by low volume of distribution (Vd) (0.3 L/kg) and CL (0.05 L/h/kg), a moderate t1/2 (7.4 h), and high brain penetration (Riva et al., 2000). The oral bioavailability of CI-994 in preclinical species was 100% (Riva et al., 2000). In a phase 1 study in cancer patients following oral dosing (5-15 mg/m2), CI-994 showed rapid absorption (tmax 0.7-1.6 h), oral CL ranging between ~30-48 ml/min/m2), dose proportional increases in Cmax and AUC, and moderately long t1/2 (7.4-14 h) (Prakash et al., 2001). In the same study, no food effects were observed on the oral PK of CI-994.

## **4.3 Entinostat (MS-275)**

Entinostat (MS-275) is a small molecule, synthetic benzamide that is currently in phase 2 trials (Mercurio et al., 2010). It is moderately lipophilic (LogD= 1.79), with moderate plasma protein binding (fraction unbound [fu] ranged between 0.375 to 0.439 in preclinical species, and 0.188 in humans) (Hooker et al., 2010; Acharya et al., 2006). In preclinical pharmacology studies, the tmax of Entinostat ranged between 30-40 minutes with a t1/2 of ~ 1 h in rats, mice and dogs, and the oral bioavailability was high (F~ 85%) (Ryan et al., 2005). In a radiolabeled tissue distribution and brain penetration study in baboons, radioactivity was cleared both by renal and biliary systems, and showed poor brain penetration (Hooker et al,

Histone Deacetylase Inhibitors asTherapeutic

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 109

6-anilino-oxohexanoic acid (6-AOB) (~10-14%), O-glucoronide in trace amounts, and the parent accounting for 0.7- 5%. In dog urine, the major metabolites found were 4-AOB (31- 34%), ortho-hydroxyaniline O-sulfate (17-21%), with minor amounts of the O-glucoronide and carnitine esters of 6-AOH and 8-AOO. Thus, Vorinostat was primarily cleared by metabolism and renally excreted in rat and dog. The data suggest that the low bioavailability of Vorinostat in rat and dog was due to a high first-pass effect and not due to absorption since the > 90% of the dose was recovered in urine, indicative of high intestinal

Vorinostat did not inhibit any of the major human CYP450 enzymes (http://www.accessdata.fda). It did not significantly induce CYP1A2, 2B6, 2C9, 2C19 and 3A4 in freshly cultured human hepatocytes, although the induction activity of 2C9 and 2C19

In the first clinical trial in cancer patients Vorinostat was administered intravenously as a 2 h infusion (Kelly et al., 2003). The intravenous route was chosen due to predictions of poor oral bioavailability based on its preclinical ADME properties (Kelly et al., 2003). In a subsequent phase 1 trial, Vorinostat was dosed orally in patients with advanced cancer in which the oral PK was also characterized (Kelly et al., 2005). Vorinostat showed dose proportional increase in Cmax and AUC following single oral doses of 100, 400 and 600 mg, with the average terminal t1/2 ranging between ~ 92 to 127 minutes, median tmax ranging between 53 to 150 minutes, and an absolute oral bioavailability of 43%. No apparent changes were observed in PK following multiple oral dosing. The t1/2 following oral dosing was longer than the t1/2 observed after i.v. dosing (range of ~35-42 min), suggesting that the elimination of Vorinostat was absorption rate limited (Kelly et al., 2005). In another study investigating the PK of Vorinostat, at 400 mg, and its major metabolites in cancer patients, the mean serum exposures of the O-glucoronide and 4-AOB were 3-4 fold and 10-to-13 fold higher, respectively, than that of Vorinostat (Rubin et al., 2006). In the same study, up to 18% and 36% of the O-glucoronide and 4-AOB, respectively, were recovered in urine, with the parent accounting for < 1 % of the total dose, clearly indicating that Vorinostat was cleared primarily by metabolism in humans, and that the O-glucoronide and 4-AOB were the major metabolites. The main enzymes responsible for the formation of the Oglucoronide were identified as the UDP-glucoronosyltransferases (UGTs), such as the UGTs 2B17 and 1A9, which are expressed in the liver, and the extrahepatic UGTs 1A8 and 1A10 (Balliet et al.,2009). UGT2B17 was one of the major enzymes contributing to the formation of the O-glucoronide of Vorinostat in humans (Balliet et al., 2009). Since UGTs are known to show extensive polymorphism, including UGT2B17, they have been associated with the

absorption (fraction of dose absorbed [Fa]=0.8-1.0) (Sandhu et al., 2007).

variable PK and response of Vorinostat in patients (Balliet et al., 2009).

rodents (F=6% in rats) and moderate F in dogs (33-50%) (Konsoula et al, 2009).

preclinical ADME data so far.

**5.2 Panabinostat (LBH589)** 

There have been no reports on allometric scaling or the predictions of human PK based on

Panabinostat (LBH589) is a cinnamic hydroxamic acid and a potent pan HDAC inhibitor that is currently in phase 2 clinical trials (Mercurio et al., 2010). Very little information is available on its preclinical ADME characteristics. It showed poor oral bioavailability in

Like SAHA, Panabinostat was first tried as an intravenous formulation in the phase 1 clinical trials (Giles et al., 2006). In that study, LBH589 showed dose proportional increase in

were suppressed at the highest concentration (http://www.accessdata.fda).

2010). The authors concluded that PgP mediated efflux was probably not the main mechanism for the poor brain penetration.

The clinical PK of Entinostat, in cancer patients, was characterized by variable absorption rates (tmax ranged between 0.5 to 60 h), a mean terminal elimination half-life of ~ 52 h, low oral clearance (CL/F=17.4 L/h/m2), nearly dose proportional increase in exposures with dose (range 2-12 mg/m2), and with substantial interpatient variability (Ryan et al., 2005). The nearly 50 fold longer t1/2 in humans was not predicted based on the preclinical PK (Ryan et al., 2005). The possible reasons for the extended t1/2 in humans were attributed to entero-hepatic recirculation and higher binding to human plasma proteins to some extent (Ryan et al., 2005). In an *in vitro* study, no metabolites could be detected after incubation of MS-275 in human liver microsomes, indicating that hepatic metabolism was a minor pathway of elimination in humans (Acharya et al., 2006).

## **5. Hydroxamic acids**

### **5.1 Vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZATM)**

Vorinostat (SAHA, ZOLINZATM), belonging to the hydroxamic acid class, was the first HDACi to be clinically approved for the treatment of refractory cutaneous T-cell lymphoma (Mann et al., 2007). Vorinostat (Mw=264) is poorly soluble in aqueous solutions ~ 191 µg/mL [~0.7 mM] (Cai et al., 2010), has a pKa of 9.2 and a LogP ~1.0 (http://www.accessdata.fda). It was moderately permeable in Caco-2 cell permeability assays (~ 2 X 10-6 cm/sec), based on which, and its poor solubility, it was classified as a Biopharmaceutical Classification System (BCS) class 4 drug (http://www.accessdata.fda). It displayed low to moderate binding to plasma proteins, with mean PPB of 71.3, 62.5, 43.6, 32.4, and 31.1 % in human, rabbit, dog, rat and mouse plasma, respectively (http://www.accessdata.fda). The mean blood-to-plasma partition ratio was 1.2, 0.7, and 2.0 in rat, dog and human blood, respectively (http://www.accessdata.fda). In *in vitro* metabolism studies, using S9 and liver microsomal fractions from rat, dog and humans, the major metabolic pathway was *O*glucoronidation of Vorinostat in all the 3 species, and a minor pathway was the hydrolysis of parent to 8-anilino-8-oxooctanoic acid (8-AOO) (http://www.accessdata.fda). In metabolism studies with hepatocytes from rat, dog and humans, the major metabolites formed in all the 3 species were 4-anilino-4-oxobutanoic acid (4-AOB, β-oxidation product) and 8-AOO (hydrolysis). In dog hepatocytes, the *O*-glucoronide was also a major metabolite, with human hepatocytes generating small amounts of it (http://www.accessdata.fda). The CYP450 enzymes were not responsible for the biotransformation of Vorinostat (http://www.accessdata.fda).

In preclinical studies in rats and dogs (Sandhu et al., 2007), the PK of Vorinostat was characterized by high systemic CL (7.8 and 3.3 L/h/kg in dog (> liver blood flow of ~ 1.9 L/h/kg) and rat (=liver blood flow of 3.3 L/h/kg), respectively), low to moderate Vss (1.6 and 0.6 L/kg in dog and rat respectively), short half-lives (12 min in dog and rat), and poor oral bioavailability (11 % and ~ 2% in dog and rat, respectively). The *O*-glucoronide and 4- AOB metabolites of Vorinostat were detected in significant levels in both the species following oral dosing (AUC ratio of *O*-glucoronide to Vorinostat was ~ 1.0 and 2.3 in dog and rat, respectively; and the AUC ratio of 4-AOB to Vorinostat was 10 and 23 in dog and rat, respectively). In excretion studies with radiolabeled Vorinostat, 89-91% and 68-81% of the total dose was recovered in urine of rat and dog, respectively. The major metabolites in rat urine (over a period of 24 h) were acetaminophen-O-sulfate (~16-19%), 4-AOB (47-48%),

2010). The authors concluded that PgP mediated efflux was probably not the main

The clinical PK of Entinostat, in cancer patients, was characterized by variable absorption rates (tmax ranged between 0.5 to 60 h), a mean terminal elimination half-life of ~ 52 h, low oral clearance (CL/F=17.4 L/h/m2), nearly dose proportional increase in exposures with dose (range 2-12 mg/m2), and with substantial interpatient variability (Ryan et al., 2005). The nearly 50 fold longer t1/2 in humans was not predicted based on the preclinical PK (Ryan et al., 2005). The possible reasons for the extended t1/2 in humans were attributed to entero-hepatic recirculation and higher binding to human plasma proteins to some extent (Ryan et al., 2005). In an *in vitro* study, no metabolites could be detected after incubation of MS-275 in human liver microsomes, indicating that hepatic metabolism was a minor

Vorinostat (SAHA, ZOLINZATM), belonging to the hydroxamic acid class, was the first HDACi to be clinically approved for the treatment of refractory cutaneous T-cell lymphoma (Mann et al., 2007). Vorinostat (Mw=264) is poorly soluble in aqueous solutions ~ 191 µg/mL [~0.7 mM] (Cai et al., 2010), has a pKa of 9.2 and a LogP ~1.0 (http://www.accessdata.fda). It was moderately permeable in Caco-2 cell permeability assays (~ 2 X 10-6 cm/sec), based on which, and its poor solubility, it was classified as a Biopharmaceutical Classification System (BCS) class 4 drug (http://www.accessdata.fda). It displayed low to moderate binding to plasma proteins, with mean PPB of 71.3, 62.5, 43.6, 32.4, and 31.1 % in human, rabbit, dog, rat and mouse plasma, respectively (http://www.accessdata.fda). The mean blood-to-plasma partition ratio was 1.2, 0.7, and 2.0 in rat, dog and human blood, respectively (http://www.accessdata.fda). In *in vitro* metabolism studies, using S9 and liver microsomal fractions from rat, dog and humans, the major metabolic pathway was *O*glucoronidation of Vorinostat in all the 3 species, and a minor pathway was the hydrolysis of parent to 8-anilino-8-oxooctanoic acid (8-AOO) (http://www.accessdata.fda). In metabolism studies with hepatocytes from rat, dog and humans, the major metabolites formed in all the 3 species were 4-anilino-4-oxobutanoic acid (4-AOB, β-oxidation product) and 8-AOO (hydrolysis). In dog hepatocytes, the *O*-glucoronide was also a major metabolite, with human hepatocytes generating small amounts of it (http://www.accessdata.fda). The CYP450 enzymes were not responsible for the

In preclinical studies in rats and dogs (Sandhu et al., 2007), the PK of Vorinostat was characterized by high systemic CL (7.8 and 3.3 L/h/kg in dog (> liver blood flow of ~ 1.9 L/h/kg) and rat (=liver blood flow of 3.3 L/h/kg), respectively), low to moderate Vss (1.6 and 0.6 L/kg in dog and rat respectively), short half-lives (12 min in dog and rat), and poor oral bioavailability (11 % and ~ 2% in dog and rat, respectively). The *O*-glucoronide and 4- AOB metabolites of Vorinostat were detected in significant levels in both the species following oral dosing (AUC ratio of *O*-glucoronide to Vorinostat was ~ 1.0 and 2.3 in dog and rat, respectively; and the AUC ratio of 4-AOB to Vorinostat was 10 and 23 in dog and rat, respectively). In excretion studies with radiolabeled Vorinostat, 89-91% and 68-81% of the total dose was recovered in urine of rat and dog, respectively. The major metabolites in rat urine (over a period of 24 h) were acetaminophen-O-sulfate (~16-19%), 4-AOB (47-48%),

mechanism for the poor brain penetration.

**5. Hydroxamic acids** 

pathway of elimination in humans (Acharya et al., 2006).

biotransformation of Vorinostat (http://www.accessdata.fda).

**5.1 Vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZATM)** 

6-anilino-oxohexanoic acid (6-AOB) (~10-14%), O-glucoronide in trace amounts, and the parent accounting for 0.7- 5%. In dog urine, the major metabolites found were 4-AOB (31- 34%), ortho-hydroxyaniline O-sulfate (17-21%), with minor amounts of the O-glucoronide and carnitine esters of 6-AOH and 8-AOO. Thus, Vorinostat was primarily cleared by metabolism and renally excreted in rat and dog. The data suggest that the low bioavailability of Vorinostat in rat and dog was due to a high first-pass effect and not due to absorption since the > 90% of the dose was recovered in urine, indicative of high intestinal absorption (fraction of dose absorbed [Fa]=0.8-1.0) (Sandhu et al., 2007).

Vorinostat did not inhibit any of the major human CYP450 enzymes (http://www.accessdata.fda). It did not significantly induce CYP1A2, 2B6, 2C9, 2C19 and 3A4 in freshly cultured human hepatocytes, although the induction activity of 2C9 and 2C19 were suppressed at the highest concentration (http://www.accessdata.fda).

In the first clinical trial in cancer patients Vorinostat was administered intravenously as a 2 h infusion (Kelly et al., 2003). The intravenous route was chosen due to predictions of poor oral bioavailability based on its preclinical ADME properties (Kelly et al., 2003). In a subsequent phase 1 trial, Vorinostat was dosed orally in patients with advanced cancer in which the oral PK was also characterized (Kelly et al., 2005). Vorinostat showed dose proportional increase in Cmax and AUC following single oral doses of 100, 400 and 600 mg, with the average terminal t1/2 ranging between ~ 92 to 127 minutes, median tmax ranging between 53 to 150 minutes, and an absolute oral bioavailability of 43%. No apparent changes were observed in PK following multiple oral dosing. The t1/2 following oral dosing was longer than the t1/2 observed after i.v. dosing (range of ~35-42 min), suggesting that the elimination of Vorinostat was absorption rate limited (Kelly et al., 2005). In another study investigating the PK of Vorinostat, at 400 mg, and its major metabolites in cancer patients, the mean serum exposures of the O-glucoronide and 4-AOB were 3-4 fold and 10-to-13 fold higher, respectively, than that of Vorinostat (Rubin et al., 2006). In the same study, up to 18% and 36% of the O-glucoronide and 4-AOB, respectively, were recovered in urine, with the parent accounting for < 1 % of the total dose, clearly indicating that Vorinostat was cleared primarily by metabolism in humans, and that the O-glucoronide and 4-AOB were the major metabolites. The main enzymes responsible for the formation of the Oglucoronide were identified as the UDP-glucoronosyltransferases (UGTs), such as the UGTs 2B17 and 1A9, which are expressed in the liver, and the extrahepatic UGTs 1A8 and 1A10 (Balliet et al.,2009). UGT2B17 was one of the major enzymes contributing to the formation of the O-glucoronide of Vorinostat in humans (Balliet et al., 2009). Since UGTs are known to show extensive polymorphism, including UGT2B17, they have been associated with the variable PK and response of Vorinostat in patients (Balliet et al., 2009).

There have been no reports on allometric scaling or the predictions of human PK based on preclinical ADME data so far.

## **5.2 Panabinostat (LBH589)**

Panabinostat (LBH589) is a cinnamic hydroxamic acid and a potent pan HDAC inhibitor that is currently in phase 2 clinical trials (Mercurio et al., 2010). Very little information is available on its preclinical ADME characteristics. It showed poor oral bioavailability in rodents (F=6% in rats) and moderate F in dogs (33-50%) (Konsoula et al, 2009).

Like SAHA, Panabinostat was first tried as an intravenous formulation in the phase 1 clinical trials (Giles et al., 2006). In that study, LBH589 showed dose proportional increase in

Histone Deacetylase Inhibitors asTherapeutic

UGT1A1, in humans.

**5.5 CUDC-101** 

(Cai et al., 2010)

**5.6 JNJ-26481585** 

exposures were achieved in humans.

at ~ 60 % of the parent (Undevia et al., 2008).

**5.7 CRA-024781(PCI-24781)** 

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 111

t1/2 of 1.5 h (Steele et al, 2011). High variability was observed in oral clearance (39-71%) due to which dose proportionality analysis was not attempted. The oral t1/2 was longer than that of the IV, which was attributed to a slow absorption rate (Steele et al., 2011). Oral bioavailability ranged between low to moderate (20-50%) in patients with advanced solid tumors (Kelly et al., 2007). Although a correlation between H4 acetylation and concentrations was observed following oral dosing at 1000 mg/m2 (Steele et al., 2011), recent phase 2 trials have employed IV dosing of Belinostat (Cashen et al., 2011). In another Phase 1 study, where the metabolism of Belinostat was studied in patients with hepatocellular carcinoma, five metabolites were identified (Wang et al, 2010). Glucoronidation was the most significant pathway of metabolism, and the methylated and amide (reduction of hydroxamic acid) products were also detected. The acid and N-glucoside forms of Belinostat were found as minor metabolites. In an *in vitro* assay using 12 isoforms forms of human UGTs, Belinostat was mainly cleared by UGT1A1 (Wang et al., 2010). The data taken together suggest that Belinostat was primarily cleared by phase 2 metabolism, involving

CUDC-101 is a small molecule (Mw 434.5) hydroxamic acid HDACi, synthesized by incorporating the hydroxamic acid group into the epidermal growth factor receptor (EGFR) pharmacophore, that exhibited antiproliferative effects *in vitro* and *in vivo* (Cai et al., 2010; Lai et al, 2010). The preclinical ADME of CUDC-101 is not available (Cai et al., 2010). The fact that CUDC-101 was dosed IV in the preclinical efficacy studies suggests that it may have had poor oral bioavailability (Cai et al., 2010). CUDC-101 is currently in phase 1 trials

JNJ-26481585 is a second-generation, small molecule hydroxamic acid based potent pan-HDACi that is currently in phase 1 trials (Mercurio et al., 2010). The preclinical ADME information for this compound is minimal. JNJ-26481585 has been shown to undergo extensive first-pass metabolism resulting in poor oral bioavailability in rodents, due to which it had to be dosed intraperitoneally (IP) in xenograft models (Arts et al., 2009). In a phase 1 oral PK/PD study in solid tumor patients, the exposures of JNJ-26481585 (dosed *q.d*. in 3 weekly cycles) increased dose proportionally between 2 and 12 mg (Postel-Vinay et al., 2009)**.** In the same study promising antitumor activity was observed indicating orally active

CRA-024781(PCI-24781) is a small molecule, hydroxamic based pan HDACi that is currently in phase 1 trials (Mercurio et al., 2010). In preclinical murine models of efficacy, its PK was characterized by a very short t1/2 (~ 7 min), very high CL (~ 18 L/h/kg) and high Vss (~ 9 l/kg) (Buggy et al., 2006). It was administered intravenously at high doses of up to 200 mg/kg in the efficacy models, most probably owing to poor oral bioavailability and high CL (Buggy et al, 2006). In a phase 1 study in patients with solid tumors, where PCI-24781was dosed as a 2 h IV infusion, the mean elimination t1/2 was ~ 6 h, high CL and moderately high Vss, low oral bioavailability of 28%, with the carboxylic acid and amide metabolites formed

Cmax and AUC between 4.8 and 14 mg/m2, with the terminal half-life ranging between 8-16 h. The Vss and CL were not reported. The oral PK of Panabinostat was characterized by rapid absorption (tmax =1-1.5 h), linear increase in dose between 20 and 80 mg and the terminal t1/2 ranged between 16-17 h (Prince et al, 2009). In an oral mass-balance study in patients with advanced cancer, following a single oral dose of 20 mg of 14C radioactively labeled Panabinostat, 87% of the administered dose was recovered in the excreta, with unchanged drug accounting for <3% of the administered dose in the feces, suggesting good oral absorption and extensive metabolism (Clive et al, 2006). The major circulating metabolites were glucoronidation products of Panabinostat, in addition to hydrolysis and reduction products. Thus, it appears that there is no single major metabolic pathway for the elimination of Panabinostat in humans. CYP3A4 does not significantly contribute to the elimination of Panabinostat in humans (DeJonge et al, 2009). Human PK data suggest that Panabinostat is a permeable drug and the poor bioavailability in preclinical rodents could be due high first-pass and poor solubility.

#### **5.3 Givinostat (ITF2357)**

Givinostat (ITF2357) is a pan HDAC inhibitor, belonging to the hydroxamic acid class that is currently in phase 2 trials for many hematological malignancies (Mercurio et al., 2010). Preclinical ADME information is either limited or qualitative for Givinostat. Metabolism was the primary clearance mechanism in preclinical species like rats, dogs, rabbits and monkeys, with excretion being biliary or renal (Furlan et al, 2011). In a phase 1 study in healthy volunteers, the oral PK of Givinostat was characterized by rapid absorption, dose proportional increases in Cmax and AUC upon single and multiple oral dosing, and the terminal half-life ranged between 5-7 h (Furlan et al, 2011). Two major circulating metabolites of Givinostat, a carboxylate and an amide formed due to oxidation and reduction of the hydroxamic acid group, were detected at significant levels in plasma.

#### **5.4 Belinostat (PXD101)**

Belinostat (PXD101) is a hydroxamic acid class potent pan HDAC inhibitor that is currently in phase 2 clinical trials (Mercurio et al., 2010). It is a small molecule (Mw 318) and sparingly soluble in aqueous solutions (Urbinati et al., 2010). Preclinical ADME information on Belinostat is limited. Preclinical pharmacodynamic studies in mice (Plumb et al., 2003) and PK studies in non-human primates (Warren et al 2008) have been performed using IV administrations, suggesting that Belinostat may have poor solubility and bioavailability issues. However, in dogs an oral bioavailability of 30-35% was reported (Steele et al, 2011). In rhesus monkeys, clearance was rapid (425 mL/min/m2) with a t1/2 of 1.0 h (Warren et al 2008). In a PK/PD study in mice following IV dosing at 200 mg/kg, Belinostat declined rapidly in plasma (ca t1/2 ~ 0.4 h), suggesting high systemic clearance (Marquard et al 2008). In the same study a correlation was observed between tumor concentrations and histone 4 acetylation levels indicating that Belinostat penetrated solid tumors.

In a phase 1 clinical study in patients with solid tumors, where Belinostat was administered as a 30 min IV infusion, its PK was characterized by dose proportional increase in AUC and Cmax, and a short t1/2 (0.45 to 0.79 h) (Steele et al., 2008). The oral PK of Belinostat following a 1000 mg/m2 dose in patients with solid tumors, was characterized by mean tmax of 1.9 h (although the oral concentration-time profile showed a flat absorption phase), with a mean t1/2 of 1.5 h (Steele et al, 2011). High variability was observed in oral clearance (39-71%) due to which dose proportionality analysis was not attempted. The oral t1/2 was longer than that of the IV, which was attributed to a slow absorption rate (Steele et al., 2011). Oral bioavailability ranged between low to moderate (20-50%) in patients with advanced solid tumors (Kelly et al., 2007). Although a correlation between H4 acetylation and concentrations was observed following oral dosing at 1000 mg/m2 (Steele et al., 2011), recent phase 2 trials have employed IV dosing of Belinostat (Cashen et al., 2011). In another Phase 1 study, where the metabolism of Belinostat was studied in patients with hepatocellular carcinoma, five metabolites were identified (Wang et al, 2010). Glucoronidation was the most significant pathway of metabolism, and the methylated and amide (reduction of hydroxamic acid) products were also detected. The acid and N-glucoside forms of Belinostat were found as minor metabolites. In an *in vitro* assay using 12 isoforms forms of human UGTs, Belinostat was mainly cleared by UGT1A1 (Wang et al., 2010). The data taken together suggest that Belinostat was primarily cleared by phase 2 metabolism, involving UGT1A1, in humans.

## **5.5 CUDC-101**

110 Drug Development – A Case Study Based Insight into Modern Strategies

Cmax and AUC between 4.8 and 14 mg/m2, with the terminal half-life ranging between 8-16 h. The Vss and CL were not reported. The oral PK of Panabinostat was characterized by rapid absorption (tmax =1-1.5 h), linear increase in dose between 20 and 80 mg and the terminal t1/2 ranged between 16-17 h (Prince et al, 2009). In an oral mass-balance study in patients with advanced cancer, following a single oral dose of 20 mg of 14C radioactively labeled Panabinostat, 87% of the administered dose was recovered in the excreta, with unchanged drug accounting for <3% of the administered dose in the feces, suggesting good oral absorption and extensive metabolism (Clive et al, 2006). The major circulating metabolites were glucoronidation products of Panabinostat, in addition to hydrolysis and reduction products. Thus, it appears that there is no single major metabolic pathway for the elimination of Panabinostat in humans. CYP3A4 does not significantly contribute to the elimination of Panabinostat in humans (DeJonge et al, 2009). Human PK data suggest that Panabinostat is a permeable drug and the poor bioavailability in preclinical rodents could be

Givinostat (ITF2357) is a pan HDAC inhibitor, belonging to the hydroxamic acid class that is currently in phase 2 trials for many hematological malignancies (Mercurio et al., 2010). Preclinical ADME information is either limited or qualitative for Givinostat. Metabolism was the primary clearance mechanism in preclinical species like rats, dogs, rabbits and monkeys, with excretion being biliary or renal (Furlan et al, 2011). In a phase 1 study in healthy volunteers, the oral PK of Givinostat was characterized by rapid absorption, dose proportional increases in Cmax and AUC upon single and multiple oral dosing, and the terminal half-life ranged between 5-7 h (Furlan et al, 2011). Two major circulating metabolites of Givinostat, a carboxylate and an amide formed due to oxidation and reduction of the hydroxamic acid group, were detected at significant levels in plasma.

Belinostat (PXD101) is a hydroxamic acid class potent pan HDAC inhibitor that is currently in phase 2 clinical trials (Mercurio et al., 2010). It is a small molecule (Mw 318) and sparingly soluble in aqueous solutions (Urbinati et al., 2010). Preclinical ADME information on Belinostat is limited. Preclinical pharmacodynamic studies in mice (Plumb et al., 2003) and PK studies in non-human primates (Warren et al 2008) have been performed using IV administrations, suggesting that Belinostat may have poor solubility and bioavailability issues. However, in dogs an oral bioavailability of 30-35% was reported (Steele et al, 2011). In rhesus monkeys, clearance was rapid (425 mL/min/m2) with a t1/2 of 1.0 h (Warren et al 2008). In a PK/PD study in mice following IV dosing at 200 mg/kg, Belinostat declined rapidly in plasma (ca t1/2 ~ 0.4 h), suggesting high systemic clearance (Marquard et al 2008). In the same study a correlation was observed between tumor concentrations and histone 4

In a phase 1 clinical study in patients with solid tumors, where Belinostat was administered as a 30 min IV infusion, its PK was characterized by dose proportional increase in AUC and Cmax, and a short t1/2 (0.45 to 0.79 h) (Steele et al., 2008). The oral PK of Belinostat following a 1000 mg/m2 dose in patients with solid tumors, was characterized by mean tmax of 1.9 h (although the oral concentration-time profile showed a flat absorption phase), with a mean

acetylation levels indicating that Belinostat penetrated solid tumors.

due high first-pass and poor solubility.

**5.3 Givinostat (ITF2357)** 

**5.4 Belinostat (PXD101)** 

CUDC-101 is a small molecule (Mw 434.5) hydroxamic acid HDACi, synthesized by incorporating the hydroxamic acid group into the epidermal growth factor receptor (EGFR) pharmacophore, that exhibited antiproliferative effects *in vitro* and *in vivo* (Cai et al., 2010; Lai et al, 2010). The preclinical ADME of CUDC-101 is not available (Cai et al., 2010). The fact that CUDC-101 was dosed IV in the preclinical efficacy studies suggests that it may have had poor oral bioavailability (Cai et al., 2010). CUDC-101 is currently in phase 1 trials (Cai et al., 2010)

#### **5.6 JNJ-26481585**

JNJ-26481585 is a second-generation, small molecule hydroxamic acid based potent pan-HDACi that is currently in phase 1 trials (Mercurio et al., 2010). The preclinical ADME information for this compound is minimal. JNJ-26481585 has been shown to undergo extensive first-pass metabolism resulting in poor oral bioavailability in rodents, due to which it had to be dosed intraperitoneally (IP) in xenograft models (Arts et al., 2009). In a phase 1 oral PK/PD study in solid tumor patients, the exposures of JNJ-26481585 (dosed *q.d*. in 3 weekly cycles) increased dose proportionally between 2 and 12 mg (Postel-Vinay et al., 2009)**.** In the same study promising antitumor activity was observed indicating orally active exposures were achieved in humans.

## **5.7 CRA-024781(PCI-24781)**

CRA-024781(PCI-24781) is a small molecule, hydroxamic based pan HDACi that is currently in phase 1 trials (Mercurio et al., 2010). In preclinical murine models of efficacy, its PK was characterized by a very short t1/2 (~ 7 min), very high CL (~ 18 L/h/kg) and high Vss (~ 9 l/kg) (Buggy et al., 2006). It was administered intravenously at high doses of up to 200 mg/kg in the efficacy models, most probably owing to poor oral bioavailability and high CL (Buggy et al, 2006). In a phase 1 study in patients with solid tumors, where PCI-24781was dosed as a 2 h IV infusion, the mean elimination t1/2 was ~ 6 h, high CL and moderately high Vss, low oral bioavailability of 28%, with the carboxylic acid and amide metabolites formed at ~ 60 % of the parent (Undevia et al., 2008).

Histone Deacetylase Inhibitors asTherapeutic

b) Cmax/ IC50, HCT116 ; c) time above IC50, HCT116.

r 2=0.93 p<0.05

Pracinostat (SB939)

advanced hydroxamic acid HDACi.

Vorinostat (SAHA)

**0**

**20**

**40**

**60**

**%TGI**

**80**

**100**

**b**

that of Zolinza and Belinostat, and shorter than Panabinostat.

Parameter

**0**

**20**

**40**

**60**

**% TGI**

**80**

**100**

**a**

**1 10**

**PXD101 (100mg/kg)**

**AUC/IC50,HCT116**

**LBH589**

**SB869**

**SB939 (100mg/kg)**

**PXD101 (50mg/kg)**

**SAHA**

**SB207 SB939 (50mg/kg)**

AUC0-inf

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 113

Fig. 1. The relationship between tumor growth inhibition (%TGI) and PK/PD parameters for HDACi in the murine HCT116 xenograft model (Jayaraman et al., 2009). a) AUC/IC50, HCT116;

**Cmax/IC50HCT116**

**PXD101 (50mg/kg)**

**PXD101 (100mg/kg)**

**SB939 (50mg/kg) SB869**

**LBH589**

**SB939 (100mg/kg)**

r 2=0.744 p<0.05

**1 10**

**SAHA**

**SB207**

Belinostat (PXD101)

Cmax(ng/mL) 2632 501 489 116 1537 35

tmax(h) 0.17 0.5 0.17 0.17 0.8 0.7

t1/2(h) 2.4 0.75 1.3 2.9 4.1 0.2

In the first phase 1 study in patients with solid tumors, Pracinostat showed rapid absorption (tmax = 0.9-2 h), dose proportional increase in Cmax and AUC between 10 and 60 mg doses, a mean terminal t1/2 of ~ 7 h, and lack of significant accumulation on repeated dosing (Yong et al, 2011). In the same study, pharmacologically active concentrations were achieved at the starting dose of 10 mg, and a dose dependent increase in histone acetylation was observed. At the 60 mg dose high acetylation levels was observed in all patients indicating sustained target inhibition, and two of the patients experienced prolonged disease stabilization. The clinical PK of Pracinostat was superior to the other hydroxamic acid HDACi in the clinic (table 3). The high aqueous solubility, permeability, good oral bioavailability and predictable human PK of Pracinostat contributed to obtaining active exposures in the clinic when dosed orally, which was in contrast to the intravenous dosing of Zolinza, Panabinostat and Belinostat in the initial clinical trials. The terminal t1/2 of Pracinostat was longer than

(ng.h/mL) 1841 619 287 126 4481 55 F (%) 34 8.3 6.7 4.6 65 2

Table 2. Comparison of preclinical pharmacokinetics of Pracinostat with that of other

mice dog

**0**

**20**

**40**

**60**

**%TGI**

**80**

**100**

**c**

Pracinostat (SB939)

**10 100**

**Time >HCT116,IC50 (min)**

**PXD101 (50mg/kg)**

**PXD101 (100mg/kg) SAHA**

Vorinostat (SAHA)

r 2=0.895 p<0.05

**SB869**

**LBH589 SB207**

**SB939 (50mg/kg)**

**SB939 (100mg/kg)**

Panabinostat (LBH589)

#### **5.8 Pracinostat (SB939)**

Pracinostat (SB939) is a hydroxamic acid based potent HDACi that is in multiple phase 2 clinical trials (http://clinicaltrials.gov/ct2/results?term=Sb939) in patients with solid tumors and hematological malignancies. Since the clinically advanced hydroxamic acid HDACi (Zolinza, Panabinostat and Belinostat) had ADME liabilities, such as poor solubility and oral bioavailability, we sought to identify a candidate that would achieve pharmacologically active exposures in humans when dosed orally. Pracinostat is a small molecule (Mw 359) moderately lipophilic base (LogD7.4 =2.1) with high aqueous solubility (>100 mg/mL in water for the HCl salt of SB939) and high permeability with low efflux which indicated that Pracinostat would show high intestinal absorption *in vivo* (Wang et al., 2011). Based on its solubility and permeability Pracinostat was categorized as a BCS class 1 compound (S\*BIO Data files). In preclinical PK studies Pracinostat showed higher oral bioavailability in mice (F=34%) and dogs (F=65%), than Zolinza, Panabinostat and Belinostat (table 2). The superior efficacy of Pracinostat, over Zolinza and Belinostat, when dosed orally in murine xenograft models was consistent its improved PK profile (Novotny-Diermayr et al., 2011). Pracinostat was found to selectively accumulate in tumors which correlated well with increased and prolonged acetylation levels in tumor which, in turn correlated with high tumor growth inhibition in mice (Novotny-Diermayr et al., 2011).

Preclinical ADME of Pracinostat was characterized by: a) in *in vitro* liver microsomal stability studies, Pracinostat was most stable in human and dog, moderate in mouse, and least stable in rat; b) uniform PPB of 84-94% in preclinical species and humans; c) was metabolized mainly by human CYP3A4 and 1A2; d) did not inhibit the major human CYPs except moderate inhibition of 2C19 (~ 6 µM); e) lack of significant induction of human CYP3A4 and 1A2 *in vitro*; f) metabolite identification studies using liver microsomes showed the formation of *N*-deethylation and bis-*N*-deethylation as major metabolites in addition to minor oxidative products; g) a glucoronidation product of SB939 was found as the major metabolite in rat urine following oral dosing; h) PK: high systemic clearance of 9.2, 4.5 and 1.5 L/h/kg in mice, rat and dog, respectively and high volume of distribution (Vss ranged between 1.7 to 4.2 L/kg) in preclinical species; i) moderate F in mice and dogs and poor in rats (Jayaraman et al., 2011). In PK/PD studies in HCT116 xenograft models, studying the relationship between tumor growth inhibition and the PK/PD indices such as AUC/IC50,HCT116, Cmax/ IC50,HCT116, and time above IC50,HCT116, Pracinostat was found to have the highest PK/PD ratios for all the three PK/PD parameters when compared to Vorinostat, Panabinostat and Belinostat (figure 1) (Jayaraman et al., 2009).

Pracinostat showed linear allometric relationships for Vss and CL in preclinical species. Prediction of human PK parameters using allometry indicated oral exposures would be achieved in humans with an acceptable t1/2 which, was subsequently found to be consistent with the observed data from cancer patients (Jayaraman et al., 2011). The human PK of Pracinostat was simulated with the Simcyp ADME simulator (Jamei et al., 2009) using the physico-chemical and *in vitro* ADME data. The simulated PK profiles were in good agreement with the observed mean data, and the mean oral clearance and AUCs were predicted reasonably well (within 2 fold of observed data) (Jayaraman et al., 2011). Furthermore, simulations of drug-drug interactions (DDI) of Pracinostat in humans with the potent CYP3A inhibitor and inducers, ketoconazole and rifampicin, respectively, and with omeprazole (substrate of 2C19) showed lack of potential DDI at the clinically relevant dose of 60 mg (Jayaraman et al., 2011).

Pracinostat (SB939) is a hydroxamic acid based potent HDACi that is in multiple phase 2 clinical trials (http://clinicaltrials.gov/ct2/results?term=Sb939) in patients with solid tumors and hematological malignancies. Since the clinically advanced hydroxamic acid HDACi (Zolinza, Panabinostat and Belinostat) had ADME liabilities, such as poor solubility and oral bioavailability, we sought to identify a candidate that would achieve pharmacologically active exposures in humans when dosed orally. Pracinostat is a small molecule (Mw 359) moderately lipophilic base (LogD7.4 =2.1) with high aqueous solubility (>100 mg/mL in water for the HCl salt of SB939) and high permeability with low efflux which indicated that Pracinostat would show high intestinal absorption *in vivo* (Wang et al., 2011). Based on its solubility and permeability Pracinostat was categorized as a BCS class 1 compound (S\*BIO Data files). In preclinical PK studies Pracinostat showed higher oral bioavailability in mice (F=34%) and dogs (F=65%), than Zolinza, Panabinostat and Belinostat (table 2). The superior efficacy of Pracinostat, over Zolinza and Belinostat, when dosed orally in murine xenograft models was consistent its improved PK profile (Novotny-Diermayr et al., 2011). Pracinostat was found to selectively accumulate in tumors which correlated well with increased and prolonged acetylation levels in tumor which, in turn correlated with high tumor growth inhibition in mice (Novotny-Diermayr et al., 2011). Preclinical ADME of Pracinostat was characterized by: a) in *in vitro* liver microsomal stability studies, Pracinostat was most stable in human and dog, moderate in mouse, and least stable in rat; b) uniform PPB of 84-94% in preclinical species and humans; c) was metabolized mainly by human CYP3A4 and 1A2; d) did not inhibit the major human CYPs except moderate inhibition of 2C19 (~ 6 µM); e) lack of significant induction of human CYP3A4 and 1A2 *in vitro*; f) metabolite identification studies using liver microsomes showed the formation of *N*-deethylation and bis-*N*-deethylation as major metabolites in addition to minor oxidative products; g) a glucoronidation product of SB939 was found as the major metabolite in rat urine following oral dosing; h) PK: high systemic clearance of 9.2, 4.5 and 1.5 L/h/kg in mice, rat and dog, respectively and high volume of distribution (Vss ranged between 1.7 to 4.2 L/kg) in preclinical species; i) moderate F in mice and dogs and poor in rats (Jayaraman et al., 2011). In PK/PD studies in HCT116 xenograft models, studying the relationship between tumor growth inhibition and the PK/PD indices such as AUC/IC50,HCT116, Cmax/ IC50,HCT116, and time above IC50,HCT116, Pracinostat was found to have the highest PK/PD ratios for all the three PK/PD parameters when compared to Vorinostat,

Panabinostat and Belinostat (figure 1) (Jayaraman et al., 2009).

of 60 mg (Jayaraman et al., 2011).

Pracinostat showed linear allometric relationships for Vss and CL in preclinical species. Prediction of human PK parameters using allometry indicated oral exposures would be achieved in humans with an acceptable t1/2 which, was subsequently found to be consistent with the observed data from cancer patients (Jayaraman et al., 2011). The human PK of Pracinostat was simulated with the Simcyp ADME simulator (Jamei et al., 2009) using the physico-chemical and *in vitro* ADME data. The simulated PK profiles were in good agreement with the observed mean data, and the mean oral clearance and AUCs were predicted reasonably well (within 2 fold of observed data) (Jayaraman et al., 2011). Furthermore, simulations of drug-drug interactions (DDI) of Pracinostat in humans with the potent CYP3A inhibitor and inducers, ketoconazole and rifampicin, respectively, and with omeprazole (substrate of 2C19) showed lack of potential DDI at the clinically relevant dose

**5.8 Pracinostat (SB939)** 

Fig. 1. The relationship between tumor growth inhibition (%TGI) and PK/PD parameters for HDACi in the murine HCT116 xenograft model (Jayaraman et al., 2009). a) AUC/IC50, HCT116; b) Cmax/ IC50, HCT116 ; c) time above IC50, HCT116.


Table 2. Comparison of preclinical pharmacokinetics of Pracinostat with that of other advanced hydroxamic acid HDACi.

In the first phase 1 study in patients with solid tumors, Pracinostat showed rapid absorption (tmax = 0.9-2 h), dose proportional increase in Cmax and AUC between 10 and 60 mg doses, a mean terminal t1/2 of ~ 7 h, and lack of significant accumulation on repeated dosing (Yong et al, 2011). In the same study, pharmacologically active concentrations were achieved at the starting dose of 10 mg, and a dose dependent increase in histone acetylation was observed. At the 60 mg dose high acetylation levels was observed in all patients indicating sustained target inhibition, and two of the patients experienced prolonged disease stabilization. The clinical PK of Pracinostat was superior to the other hydroxamic acid HDACi in the clinic (table 3). The high aqueous solubility, permeability, good oral bioavailability and predictable human PK of Pracinostat contributed to obtaining active exposures in the clinic when dosed orally, which was in contrast to the intravenous dosing of Zolinza, Panabinostat and Belinostat in the initial clinical trials. The terminal t1/2 of Pracinostat was longer than that of Zolinza and Belinostat, and shorter than Panabinostat.

Histone Deacetylase Inhibitors asTherapeutic

*Metab*. 4(5):423-59.

*Pharm Pharmacol* 59:905-916

*Oncologist.* 12(10):1247-52.

59

5(2):211-223

302.

Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties 115

[2] Prentis RA, Lis Y and Walker SR. 1988 Pharmaceutical innovation by the seven UKowned pharmaceutical companies (1964-1985). *Br. J. Clin. Pharmacol*. 25: 387-396 [3] Yengi LG, Leung L, and Kao J. 2007. The evolving role of drug metabolism in drug

[4] Smith DA., Jones BC, and Walker DK.1996. Design of drugs involving the concepts and theories of drug metabolism and pharmacokinetics. *Med Res Rev*. 16(3):243-266 [5] Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ, Wastall

[6] Venkatakrishnan K, von Moltke LL, Obach RS, Greenblatt DJ. 2003. Drug metabolism

[7] Pelkonen O and Raunio H. 2005. In vitro screening of drug metabolism during drug

[8] Thompson TN. 2000. Early ADME in support of drug discovery: the role of metabolic

[9] Fagerholm U .2007. Prediction of human pharmacokinetics-gastrointestinal absorption. *J* 

[10] Lipinski CA. 2000. Drug-like properties and the causes of poor solubility and poor

[11] Jamei M, Marciniak S, Feng K, Barnett A, Tucker G, Rostami-Hodjegan A.2009. The

[12] Minucci S and Pelicci PG. 2006. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. *Nat. Rev. Cancer* 6(1):38-51 [13] Mercurio C, Minucci S, and Pelicci PG. 2010. Histone deacetylases and epigenetic therapies of hematological malignancies. *Pharmacol Res*. 62(1):18-34. [14] Stimson L, Wood V, Khan O, Fotheringham S, and La Thangue NB. 2009. HDAC

[15] Christensen DP, Dahllöf M, Lundh M, Rasmussen DN, Nielsen MD, Billestrup N,

[16] Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R. 2007. FDA approval summary:

[17] Grant C, Rahman F, Piekarz R, Peer C, Frye R, Robey RW, Gardner ER, Figg WD, Bates

diabetes mellitus. *Mol Med*. doi: 10.2119/molmed.2011.00021

Simcyp population-based ADME simulator. *Expert Opin Drug Metab Toxicol*

inhibitor-based therapies and haematological malignancy. *Ann Oncol*. 20(8):1293-

Grunnet LG, Mandrup-Poulsen T. 2011. HDAC inhibition as a novel treatment for

vorinostat for treatment of advanced primary cutaneous T-cell lymphoma.

SE. 2010. Romidepsin: a new therapy for cutaneous T-cell lymphoma and a potential therapy for solid tumors. *Expert Rev Anticancer Ther*. 10(7):997-1008. [18] Wang H, Yu N, Chen D, Lee KC, Lye PL, Chang JW, Deng W, Ng MC, Lu T, Khoo ML,

Poulsen A, Sangthongpitag K, Wu X, Hu C, Goh KC, Wang X, Fang L, Goh KL, Khng HH, Goh SK, Yeo P, Liu X, Bonday Z, Wood JM, Dymock BW, Kantharaj E, Sun ET. 2011. Discovery of (2E)-3-{2-Butyl-1-[2- (diethylamino)ethyl]-1Hbenzimidazol-5-yl}-Nhydroxyacrylamide (SB939), an Orally Active Histone Deacetylase Inhibitor with a Superior Preclinical Profile. *J Med Chem* in press. [19] Novotny-Diermayr V, Sangthongpitag K, Hu CY, Wu X, Sausgruber N, Yeo P, Greicius

G, Pettersson S, Liang AL, Loh YK, Bonday Z, Goh KC, Hentze H, Hart S, Wang H,

P.1997. The prediction of human pharmacokinetic parameters from preclinical and

and drug interactions: application and clinical value of in vitro models. *Curr Drug* 

development: can we trust the predictions? *Expert Opin Drug Metab Toxicol* 1(1):49-

discovery and development. *Pharm Res*. 24(5): 842-858

in vitro metabolism data. *J Pharmacol Exp Ther* 283(1):46-58.

stability studies. *Curr Drug Metab* 1(3): 215-241

permeability. *J Pharmacol and Toxicol Meth* 44: 235- 249


In summary, the superior preclinical ADME of Pracinostat over Zolinza, Panabinostat and Belinostat was translated into the clinic.

\* Yong et al., 2011
