**3.1 Antisense oligonucleotides: An overview**

Oligonucleotides (ODN) are short polymers of nucleotides that come in various forms, lengths and modifications which can be distinguished into two main groups based on two distinct mechanisms of action; ODN in the first group target RNA and those from the second group target proteins.

RNA-targeting ODN drugs are designed to bind to a specific sequence of a messenger RNA (mRNA) through Watson-Crick base-pairing interactions. Therefore, the site of action of this class of drug is not the protein itself, but rather "upstream" of it, the RNA coding for the protein. The principle of RNA-based therapy is the reduction in the level of a protein through hindrance of its translation. Archetypes of this class of ODN are AON and small interfering RNA (siRNA). AON drugs are single stranded, usually only approximately 20 bases long, which prevent translation of the target RNA via one of two mechanisms. The first mechanism involves the activation of the enzyme RNAse H, which cleaves the RNA moiety of the duplex formed by the binding of the AON drug to its target RNA leading to subsequent reduction in protein synthesis (Stein &Hausen 1969). The second mechanism involves a steric interaction of the AON with the target mRNA that prevents key maturation

A Multi-Targeted Antisense Oligonucleoitde-Based

and does not require any specialized carriers.

**Potential advantages of application in lung** 

**disease**

candidates.

overall improved lung tolerability.

**3.2 Drug design** 

Therapy Directed at Phosphodiesterases 4 and 7 for COPD 441

changes in the lung following administration of the modified AON demonstrating an

The drug candidate, PXS TPI1100, is a 1:1 mixture of two AON, one which targets two isoforms of PDE4 (4B and 4D) and the second AON targeting PDE7A (Fortin et al. 2008). The rationale for developing these new specific and multi-targeted AON is to provide a new class of anti-inflammatory agents that act more broadly on the underlying inflammatorytriad - recruitment, activation and potentiation of processes in chronic respiratory diseases and that is more potent than selective PDE4 inhibitors. Delivery directly to the site of action, the lung, will ensure local deposition of the drug and limited systemic exposure thus reducing potential side effects associated with the systemic delivery (e.g. oral delivery) of PDE4 inhibitors. Lastly, PXS TPI1100 consists of aerosolization of a simple aqueous solution,

AON drugs, while still early in development, possess properties that could be advantageous over classical small molecule drugs (Table 2). First, as a single mRNA strand can be translated into multiple copies of proteins (~5000 copies), there is a clear advantage of "upstream" targeting, that is targeting the mRNA rather than the protein (Popescu 2005). The "upstream" targeting approaching with AON can be achieved irrespectively of the location of the target protein, whether it is inside the cell or outside the cell. AON have the potential to amplify

**Potential advantages**  High degree of specificity (primarily for RNA-targeted

Broad range of potential targets

**Challenges**  Cellular uptake and intracellular release for larger

Potential immunostimulatory effects

Specific systemic toxicological findings

Direct delivery to the site of action in the lungs

Cellular uptake and release without additional carrier or

oligonucleotides

Oligonucleotide stability

Multi-targeting feasible

formulation technologies Low systemic exposure

Table 2. Advantages and challenges in the development of antisense oligonucleotides drug

through chemical modification

 Absence of hypersensitivity reactions Relatively short development timelines

Ability to screen efficiently for off-target effects

 Relative ease of formulation for inhaled delivery Relative ease of formulation of combination products Relative stability of drug compound and product

Ability to modify the properties of the oligonucleotide

drugs)

steps processes such as splicing and thus preventing translation (Crooke 2008). siRNA drugs share the same mechanism of action to AON, degradation of the protein encoding RNA. However, these drugs are distinct from AON molecules, as they comprise double stranded RNA (varying from 19 to 27 base pair long) (Wu et al. 1998) and induces silencing via the RNA-induced silencing complex (RISC), which is composed of several proteins, including specific RNA-degrading enzymes (Holen et al. 2003).

Similar to conventional small molecules drugs or biologics, the second group of ODN comprises molecules that target proteins directly. Two examples of this group include aptamers and immunostimulatory sequences (ISS). Aptamers comprise either DNA or RNA and typically have a longer chain length (ie, approximately 40 nucleotides) than other ODN. These agents have a specific 3D structure (Ellington &Szostak 1992; Jayasena 1999) that determines their ability to bind specifically to their protein target acting in a similar manner to conventional antibody therapies (Lee et al. 2006). ISS molecules are single stranded, which sequence is enriched with unmethylated cysteine and guanine motifs (CpG) motifs (Vollmer et al. 2004). ISS can mediate immunostimulatory effects following binding to TLR9, a key member of the innate immune system (Agrawal &Kandimalla 2007).

ODN drugs share a relatively common chemical composition that is based on naturally occurring RNA and DNA, and comprises the three elements of nucleotide bases, pentose sugars and linking phosphate groups. In the past decade, medicinal chemistry has allowed significant improvements in the drug-like properties of ODN including the potential to optimize the stability as well as the pharmacologic, pharmacokinetic and toxicologic properties of these molecules. In general, three types of modifications of ODN can be distinguished. The first type of modification, and the one most commonly used, is the replacement of the oxygen atoms of the naturally occurring phosphodiester bond by sulfur groups (phosphorothioate (PS) linkages) to confer stability to the drug molecule. Nucleotide analogs have also been incorporated. For example, adenosine has been replaced with 2 amino-2'-deoxydenosine, which improves binding of the drug to the target and minimizes the potential for bronchospasm and inflammation induced by adenosine (Vollmer et al. 2004). Finally, the sugar moiety can be modified; for example, the addition of a 2'-*O*methoxyethyl group to the pentose sugar confers stability to the ODN and enhances binding affinity to the target mRNA (Ward 2010).

The AON constituents comprising PXS TPI1100 incorporate two modifications: a modified phosphate backbone and the incorporation of 2-amino-2'-deozyadenosine. These modifications were aimed at improving the binding affinity of the drug to its mRNA target, reduce the immunostimulatory effect of this class of drug, and improve the lung tolerability after administration by the pulmonary route. *In vivo* testing of these molecules by multiple dosing via intratracheal (i.t.) administration in mice demonstrated that the modified chemistry contained in the PXS TPI1100 sequences was far less immunostimulatory than the typical PS-containing AON. Repeated daily i.t. delivery of PS-containing AON at a dose of 2.5 mg/kg induced a 4-fold increase in the recruitment of total cells in bronchoalveolar lavage (BAL) compared to control mice (treated with vehicle) and lung tissue changes as assessed by the presence of moderate (grade 3) perivascular mixed cell infiltrate and severe (grade 4) alveolar inflammation. In contrast, in mice treated with the same dose of AON bearing the modified chemistry no difference in BAL cells (total cells as well as differential cells) as compared to the vehicle group were observed, nor were there any histopathological changes in the lung following administration of the modified AON demonstrating an overall improved lung tolerability.

#### **3.2 Drug design**

440 Chronic Obstructive Pulmonary Disease – Current Concepts and Practice

steps processes such as splicing and thus preventing translation (Crooke 2008). siRNA drugs share the same mechanism of action to AON, degradation of the protein encoding RNA. However, these drugs are distinct from AON molecules, as they comprise double stranded RNA (varying from 19 to 27 base pair long) (Wu et al. 1998) and induces silencing via the RNA-induced silencing complex (RISC), which is composed of several proteins, including

Similar to conventional small molecules drugs or biologics, the second group of ODN comprises molecules that target proteins directly. Two examples of this group include aptamers and immunostimulatory sequences (ISS). Aptamers comprise either DNA or RNA and typically have a longer chain length (ie, approximately 40 nucleotides) than other ODN. These agents have a specific 3D structure (Ellington &Szostak 1992; Jayasena 1999) that determines their ability to bind specifically to their protein target acting in a similar manner to conventional antibody therapies (Lee et al. 2006). ISS molecules are single stranded, which sequence is enriched with unmethylated cysteine and guanine motifs (CpG) motifs (Vollmer et al. 2004). ISS can mediate immunostimulatory effects following binding to TLR9,

ODN drugs share a relatively common chemical composition that is based on naturally occurring RNA and DNA, and comprises the three elements of nucleotide bases, pentose sugars and linking phosphate groups. In the past decade, medicinal chemistry has allowed significant improvements in the drug-like properties of ODN including the potential to optimize the stability as well as the pharmacologic, pharmacokinetic and toxicologic properties of these molecules. In general, three types of modifications of ODN can be distinguished. The first type of modification, and the one most commonly used, is the replacement of the oxygen atoms of the naturally occurring phosphodiester bond by sulfur groups (phosphorothioate (PS) linkages) to confer stability to the drug molecule. Nucleotide analogs have also been incorporated. For example, adenosine has been replaced with 2 amino-2'-deoxydenosine, which improves binding of the drug to the target and minimizes the potential for bronchospasm and inflammation induced by adenosine (Vollmer et al. 2004). Finally, the sugar moiety can be modified; for example, the addition of a 2'-*O*methoxyethyl group to the pentose sugar confers stability to the ODN and enhances binding

The AON constituents comprising PXS TPI1100 incorporate two modifications: a modified phosphate backbone and the incorporation of 2-amino-2'-deozyadenosine. These modifications were aimed at improving the binding affinity of the drug to its mRNA target, reduce the immunostimulatory effect of this class of drug, and improve the lung tolerability after administration by the pulmonary route. *In vivo* testing of these molecules by multiple dosing via intratracheal (i.t.) administration in mice demonstrated that the modified chemistry contained in the PXS TPI1100 sequences was far less immunostimulatory than the typical PS-containing AON. Repeated daily i.t. delivery of PS-containing AON at a dose of 2.5 mg/kg induced a 4-fold increase in the recruitment of total cells in bronchoalveolar lavage (BAL) compared to control mice (treated with vehicle) and lung tissue changes as assessed by the presence of moderate (grade 3) perivascular mixed cell infiltrate and severe (grade 4) alveolar inflammation. In contrast, in mice treated with the same dose of AON bearing the modified chemistry no difference in BAL cells (total cells as well as differential cells) as compared to the vehicle group were observed, nor were there any histopathological

a key member of the innate immune system (Agrawal &Kandimalla 2007).

specific RNA-degrading enzymes (Holen et al. 2003).

affinity to the target mRNA (Ward 2010).

The drug candidate, PXS TPI1100, is a 1:1 mixture of two AON, one which targets two isoforms of PDE4 (4B and 4D) and the second AON targeting PDE7A (Fortin et al. 2008). The rationale for developing these new specific and multi-targeted AON is to provide a new class of anti-inflammatory agents that act more broadly on the underlying inflammatorytriad - recruitment, activation and potentiation of processes in chronic respiratory diseases and that is more potent than selective PDE4 inhibitors. Delivery directly to the site of action, the lung, will ensure local deposition of the drug and limited systemic exposure thus reducing potential side effects associated with the systemic delivery (e.g. oral delivery) of PDE4 inhibitors. Lastly, PXS TPI1100 consists of aerosolization of a simple aqueous solution, and does not require any specialized carriers.

AON drugs, while still early in development, possess properties that could be advantageous over classical small molecule drugs (Table 2). First, as a single mRNA strand can be translated into multiple copies of proteins (~5000 copies), there is a clear advantage of "upstream" targeting, that is targeting the mRNA rather than the protein (Popescu 2005). The "upstream" targeting approaching with AON can be achieved irrespectively of the location of the target protein, whether it is inside the cell or outside the cell. AON have the potential to amplify


Table 2. Advantages and challenges in the development of antisense oligonucleotides drug candidates.

A Multi-Targeted Antisense Oligonucleoitde-Based

better results in fighting this disease.

need for AON against multiple genes in one product.

reach and enter the target cells (Figure 2).

**3.3 Preclinical pharmacology** 

response to IL-1β was observed.

Therapy Directed at Phosphodiesterases 4 and 7 for COPD 443

mediators such as cytokines, chemokines or their receptors. It is believed that in order to treat chronic inflammation a single drug directed against multiple targets and pathways would be better at arresting the progression of these respiratory diseases. However, to date there has been limited success with therapies targeting either a single cytokine, chemokine or their receptor highlighting the challenge in treating these complex inflammatory diseases by focusing on a single component or aspect of the inflammation process. Drugs acting on individual molecular targets usually exert unsatisfying therapeutic effects or have severe toxicity or undesired side effects when used in diseases of complicated causes such as in oncology or in inflammatory diseases. One approach to address such limited efficacy and toxicity has been by the development of novel therapies using a mixture of molecules. In oncology for example, a prevailing idea is that inhibiting both cancer cells and cells of the stroma supporting the tumor or blood vessels would gain

There is a fine balance between specificity and reduced toxicity that can be obtained by targeting more than one cytokine or chemokine or receptor in the immune response without the overwhelming suppression observed with corticosteroids. The era of designing "one target for one disease" has evolved such that the single-target therapy is fading in favor of a multi-targeted approach and the new generation therapies are selected on the basis of their ability to simultaneously inhibit or affect several targets. Through combining two or more molecules which individually have their own target into a single therapeutic product, it may be possible to generate a drug that is potentially more effective, in particular in those patients non-responding to the conventional therapies. In addition, the lower doses could results in less side effects than with broader therapies like corticosteroids. This approach is especially important because of the redundancy of inflammatory pathways indicates the

Lastly, PXS TPI1100 consists of aerosolization of a simple aqueous solution, and does not require any specialized carriers unlike many other AON therapies. Indeed, direct administration of low doses to the site of action by inhalation permits AON to efficiently

*In vitro* pharmacology studies of the AON candidates of PXS TPI1100 were conducted in both human and animal cell cultures. Results in normal human bronchoepithelial (NHBE) primary cells and a lung epithelial cell line (A549) confirmed the efficacy of PXS TPI1100 at reducing PDE mRNA target knockdown, which is the proposed mechanism of action of the drug. Moreover, in NHBE cells, inhibition of the PDE4B, PDE4D and PDE7A with PXS TPI 1100 resulted with a synergistic effect on the inhibition of IL-8 secretion in response to a stimulus (a mixture of cytokines TNF-α, IL-1β and IFN-γ) compared to when cells were treated with each AON alone (Figure 3). These results and the lack of efficacy of rolipram (small molecule PDE4 inhibitor) on IL-8 confirmed the benefit of PDE4 and PDE7 inhibition. Besides IL-8, cells treated with PXS TPI1100 had an inhibition of the expression and release of other inflammatory mediators (e.g. MCP-1, MMPs). A second model used the lung epithelial cell line, A549, stimulated with the cytokine IL-1β, and again the inhibitory effect of PXS TPI1100 upon the induction of key inflammatory mediators (IL-8, MCP-1) in

potency as compared to small molecule drugs which target the protein directly. Furthermore, by targeting the mRNA this method avoids the complications of protein interactions and effects of phosphorylation which can be of concern for PDE inhibitors.

By its very nature, AON are designed to target a specific RNA sequence and this specificity lends an advantage over ICS. As comparison, corticosteroids which are believed to directly regulate between 10 to 100 genes per cell, with a further estimation of many other genes indirectly regulated through interaction of other transcription factors and coactivators by yet unclarified mechanisms (Barnes 2006). In contrast, the inherent specificity of AON for its target avoids the non-selective inhibition nature of steroids. However, AON, as all drugs, have the potential of causing unwanted toxicities or side effects, of which some of these unwanted toxicities can arise because of the inherent capacity of AON to hybridize to RNA. Such toxicities are termed hybridization-dependent and can be subdivided into effects caused by exaggerated pharmacology, i.e. inhibition of the intended target to a degree that produces deleterious effects, and hybridization-dependent effects on unintended RNA targets (off-targets*)* that happen to be completely or partially complementary to the AON sequence. For the former, with recent advances for the modifications of the AON chemistry to improve binding affinity, as well as improvements for more effective delivery systems, there could be potential risk in designing AON that are too effective. Correct dosing assessment would be imperative. With regard to off-target effects, the use of genomic information databases allows for identification of possible off targets early in the drug discovery process. Any potential off targets can then be monitored both during the preclinical development and safety assessment stage as well as in clinical studies if needed. Along with the hybridization-dependent toxicities, there are also hybridization-independent which are due to interactions between the AON and proteins. The majority of toxicities observed for AON tested to date are hybridization independent and result from AON chemistry or composition of the delivery system and such potential is assessed in animal toxicology studies (Levin et al. 2001).

A further advantage of AON is the common composition and chemical nature of AON allow for an ease in combining two or more AON for a multi-targeted drug, unlike typical combination therapies. Historically, combination therapies have resulted from combining two marketed drugs into a single drug product. In the respiratory space, the combination of a corticosteroid with a long acting β2-adrenergic receptor agonist has been effective at producing billion dollar drugs like Advair (fluticasone/salmeterol), and Symbicort (budesonide/formoterol). Each of the individual components of these drugs had undergone the development process as single entities which were then combined later for a final product. However, the current understanding of various disease systems would suggest the selection and development of drugs that contain at least 2 molecules directed against at least 2 targets from the beginning of the development process.

The rationale for developing these new specific and multi-targeted ODN inhibitors is to provide a new class of anti-inflammatory agents that act more broadly on the underlying inflammatory-triad - recruitment, activation and potentiation of processes in chronic respiratory diseases. Complex diseases require multiple approaches to circumvent the cellular signaling redundancy underlying inflammatory conditions. In an attempt to improve bronchoconstriction and airway hyperresponsiveness in respiratory diseases, drugs have been designed to modulate the immune response by targeting immune

potency as compared to small molecule drugs which target the protein directly. Furthermore, by targeting the mRNA this method avoids the complications of protein interactions and effects of phosphorylation which can be of concern for PDE inhibitors.

By its very nature, AON are designed to target a specific RNA sequence and this specificity lends an advantage over ICS. As comparison, corticosteroids which are believed to directly regulate between 10 to 100 genes per cell, with a further estimation of many other genes indirectly regulated through interaction of other transcription factors and coactivators by yet unclarified mechanisms (Barnes 2006). In contrast, the inherent specificity of AON for its target avoids the non-selective inhibition nature of steroids. However, AON, as all drugs, have the potential of causing unwanted toxicities or side effects, of which some of these unwanted toxicities can arise because of the inherent capacity of AON to hybridize to RNA. Such toxicities are termed hybridization-dependent and can be subdivided into effects caused by exaggerated pharmacology, i.e. inhibition of the intended target to a degree that produces deleterious effects, and hybridization-dependent effects on unintended RNA targets (off-targets*)* that happen to be completely or partially complementary to the AON sequence. For the former, with recent advances for the modifications of the AON chemistry to improve binding affinity, as well as improvements for more effective delivery systems, there could be potential risk in designing AON that are too effective. Correct dosing assessment would be imperative. With regard to off-target effects, the use of genomic information databases allows for identification of possible off targets early in the drug discovery process. Any potential off targets can then be monitored both during the preclinical development and safety assessment stage as well as in clinical studies if needed. Along with the hybridization-dependent toxicities, there are also hybridization-independent which are due to interactions between the AON and proteins. The majority of toxicities observed for AON tested to date are hybridization independent and result from AON chemistry or composition of the delivery system and such potential is assessed in animal

A further advantage of AON is the common composition and chemical nature of AON allow for an ease in combining two or more AON for a multi-targeted drug, unlike typical combination therapies. Historically, combination therapies have resulted from combining two marketed drugs into a single drug product. In the respiratory space, the combination of a corticosteroid with a long acting β2-adrenergic receptor agonist has been effective at producing billion dollar drugs like Advair (fluticasone/salmeterol), and Symbicort (budesonide/formoterol). Each of the individual components of these drugs had undergone the development process as single entities which were then combined later for a final product. However, the current understanding of various disease systems would suggest the selection and development of drugs that contain at least 2 molecules directed against at least

The rationale for developing these new specific and multi-targeted ODN inhibitors is to provide a new class of anti-inflammatory agents that act more broadly on the underlying inflammatory-triad - recruitment, activation and potentiation of processes in chronic respiratory diseases. Complex diseases require multiple approaches to circumvent the cellular signaling redundancy underlying inflammatory conditions. In an attempt to improve bronchoconstriction and airway hyperresponsiveness in respiratory diseases, drugs have been designed to modulate the immune response by targeting immune

toxicology studies (Levin et al. 2001).

2 targets from the beginning of the development process.

mediators such as cytokines, chemokines or their receptors. It is believed that in order to treat chronic inflammation a single drug directed against multiple targets and pathways would be better at arresting the progression of these respiratory diseases. However, to date there has been limited success with therapies targeting either a single cytokine, chemokine or their receptor highlighting the challenge in treating these complex inflammatory diseases by focusing on a single component or aspect of the inflammation process. Drugs acting on individual molecular targets usually exert unsatisfying therapeutic effects or have severe toxicity or undesired side effects when used in diseases of complicated causes such as in oncology or in inflammatory diseases. One approach to address such limited efficacy and toxicity has been by the development of novel therapies using a mixture of molecules. In oncology for example, a prevailing idea is that inhibiting both cancer cells and cells of the stroma supporting the tumor or blood vessels would gain better results in fighting this disease.

There is a fine balance between specificity and reduced toxicity that can be obtained by targeting more than one cytokine or chemokine or receptor in the immune response without the overwhelming suppression observed with corticosteroids. The era of designing "one target for one disease" has evolved such that the single-target therapy is fading in favor of a multi-targeted approach and the new generation therapies are selected on the basis of their ability to simultaneously inhibit or affect several targets. Through combining two or more molecules which individually have their own target into a single therapeutic product, it may be possible to generate a drug that is potentially more effective, in particular in those patients non-responding to the conventional therapies. In addition, the lower doses could results in less side effects than with broader therapies like corticosteroids. This approach is especially important because of the redundancy of inflammatory pathways indicates the need for AON against multiple genes in one product.

Lastly, PXS TPI1100 consists of aerosolization of a simple aqueous solution, and does not require any specialized carriers unlike many other AON therapies. Indeed, direct administration of low doses to the site of action by inhalation permits AON to efficiently reach and enter the target cells (Figure 2).

#### **3.3 Preclinical pharmacology**

*In vitro* pharmacology studies of the AON candidates of PXS TPI1100 were conducted in both human and animal cell cultures. Results in normal human bronchoepithelial (NHBE) primary cells and a lung epithelial cell line (A549) confirmed the efficacy of PXS TPI1100 at reducing PDE mRNA target knockdown, which is the proposed mechanism of action of the drug. Moreover, in NHBE cells, inhibition of the PDE4B, PDE4D and PDE7A with PXS TPI 1100 resulted with a synergistic effect on the inhibition of IL-8 secretion in response to a stimulus (a mixture of cytokines TNF-α, IL-1β and IFN-γ) compared to when cells were treated with each AON alone (Figure 3). These results and the lack of efficacy of rolipram (small molecule PDE4 inhibitor) on IL-8 confirmed the benefit of PDE4 and PDE7 inhibition. Besides IL-8, cells treated with PXS TPI1100 had an inhibition of the expression and release of other inflammatory mediators (e.g. MCP-1, MMPs). A second model used the lung epithelial cell line, A549, stimulated with the cytokine IL-1β, and again the inhibitory effect of PXS TPI1100 upon the induction of key inflammatory mediators (IL-8, MCP-1) in response to IL-1β was observed.

A Multi-Targeted Antisense Oligonucleoitde-Based

treated with vehicle or a comparable dose of a control AON.

continuing its pre-clinical development as a treatment for COPD.

had no effect.

Therapy Directed at Phosphodiesterases 4 and 7 for COPD 445

In two different *in vivo* models, PXS TPI1100 was shown to reduce the neutrophil influx in the BAL of mice either in response to cigarette smoke or to LPS challenge. Cigarette smoke exposure of laboratory animals reproduces many of the anatomic/physiologic lesions (neutrophilic inflammation, emphysema, small-airway remodeling and pulmonary hypertension) of human COPD (Wright et al. 2008) and has been used for the preclinical assessment of Daxas/Deliresp (Martorana et al. 2005). In this model, mice were exposed to cigarette smoke for 4 consecutive days and treated with PXS TPI1100 every other day (two treatments only) 3 h prior to cigarette smoke exposure. Following repeated smoke exposure, a significant increase (180-fold) recruitment of neutrophils in BAL collected the day after the last smoke exposure was observed compared to mice not exposed to smoke. The percentage of neutrophils in BAL also increased with smoke from 0.8% to 35%. When mice were treated with PXS TPI1100 at 0.1 or 0.4 mg/kg every other day, the smoke-induced neutrophil recruitment was significantly reduced (up to 52% inhibition p<0.01) when compared to mice

In the second model of acute lung inflammation, mice exposed to LPS (nasal instillation) had a strong inflammatory response with significant increase in neutrophils in BAL. PXS TPI1100 treatment at 1.2 mg/kg (1 h prior to LPS challenge) resulted in a 33% reduction of neutrophil recruitment induced by LPS (p<0.05) whereas treatment with the control AON

The potency of PXS TPI1100 at reducing the smoke-induced or LPS-induced lung inflammation was compared to the PDE4 inhibitor roflumilast (Daxas). Roflumilast (5 mg/kg, p.o.) given daily 1 h prior to cigarette smoke exposure reduced neutrophil recruitment by only 25% (Fortin et al., 2009). In the LPS model, roflumilast, given once at a dose of 10 mg/kg (~10-fold more than PXS TPI1100) had no effect on the neutrophil influx, whereas at a higher dose of 100 mg/kg (~100-fold that of PXS TPI1100) it reduced neutrophil recruitment by 46% (p<0.05). This effective dose of roflumilast exceeds the current clinical dose for Daxas of 500 microgram per adult per day. PXS TPI1100 is

PXS TPI1100 has not yet performed nonclinical drug depositions studies however, from tests with different AON that recognize the same PDE targets yet lacked the modified chemistry backbone we can extrapolate how PXS TPI1100 will behave following pulmonary delivery. In CD-1 mice, following 14 days of daily dosing with AON by inhalation, AON plasma concentrations were not detectable (< LLOQ of 5-10 ng/mL) at all time points for all dose levels. In the lungs, the AON concentrations were dose-related, and there was evidence of accumulation in lungs over the 14 days, based on the higher levels at 24 h after the last dose *vs.* 24 h after the first dose. The systemic exposure was extremely low with only small amounts of AON detectable in the kidneys and liver of high-dose mice (2.5 mg/kg/day), and the levels were similar following the first and last doses. In monkeys, following 14 days of inhalation of AON there were detectable levels of AON in plasma only in a few high-dose animals up to 1 h post dosing on Day 1 while samples from Day 14 were all <LLOQ (Guimond et al. 2008). In the lung of animals on the day after last drug exposure, the AON levels were approximately dose proportional. In kidney and liver, low levels of AON were quantified one day after the last dose and only in high dose animals, demonstrating that similar to mice, the systemic exposure was low. When AON were delivered by slow bolus intravenous (IV) administration in monkey, the highest plasma levels were measured immediately at the first time point after IV

Fig. 2. Intracellular localization of PXS TPI1100 AON constituents in the lung of mice following cigarette smoke exposure. Mice exposed to cigarette smoke were treated intratracheally with a single dose of labelled PXS TPI1100 (a FITC-labeled AON against PDE4B/4D) and a Cy3-labeled AON against PDE7A. Images obtained using a confocal microscope (FITC in green, Cy3 in red and DAPI in blue). Magnification of 200X (left panel) and insert shown at 630X (right panel).

Fig. 3. Activity of PXS TPI1100 in NHBE cells. NHBE cells were treated with the PDE7A or the PDE4B/4D AON alone at indicated concentration or in combination prior to stimulation of the cells (mix of TNF-α, IL-1β and IFN-γ). Inhibition of the three PDE isoforms resulted with a synergistic effect (\*\*p<0.05) on IL-8 secretion compared to each AON alone, and a more potent effect than rolipram or dexamethasone (DEX).

Fig. 2. Intracellular localization of PXS TPI1100 AON constituents in the lung of mice following cigarette smoke exposure. Mice exposed to cigarette smoke were treated intratracheally with a single dose of labelled PXS TPI1100 (a FITC-labeled AON against PDE4B/4D) and a Cy3-labeled AON against PDE7A. Images obtained using a confocal microscope (FITC in green, Cy3 in red and DAPI in blue). Magnification of 200X (left panel)

Fig. 3. Activity of PXS TPI1100 in NHBE cells. NHBE cells were treated with the PDE7A or the PDE4B/4D AON alone at indicated concentration or in combination prior to stimulation of the cells (mix of TNF-α, IL-1β and IFN-γ). Inhibition of the three PDE isoforms resulted with a synergistic effect (\*\*p<0.05) on IL-8 secretion compared to each AON alone, and a

more potent effect than rolipram or dexamethasone (DEX).

and insert shown at 630X (right panel).

In two different *in vivo* models, PXS TPI1100 was shown to reduce the neutrophil influx in the BAL of mice either in response to cigarette smoke or to LPS challenge. Cigarette smoke exposure of laboratory animals reproduces many of the anatomic/physiologic lesions (neutrophilic inflammation, emphysema, small-airway remodeling and pulmonary hypertension) of human COPD (Wright et al. 2008) and has been used for the preclinical assessment of Daxas/Deliresp (Martorana et al. 2005). In this model, mice were exposed to cigarette smoke for 4 consecutive days and treated with PXS TPI1100 every other day (two treatments only) 3 h prior to cigarette smoke exposure. Following repeated smoke exposure, a significant increase (180-fold) recruitment of neutrophils in BAL collected the day after the last smoke exposure was observed compared to mice not exposed to smoke. The percentage of neutrophils in BAL also increased with smoke from 0.8% to 35%. When mice were treated with PXS TPI1100 at 0.1 or 0.4 mg/kg every other day, the smoke-induced neutrophil recruitment was significantly reduced (up to 52% inhibition p<0.01) when compared to mice treated with vehicle or a comparable dose of a control AON.

In the second model of acute lung inflammation, mice exposed to LPS (nasal instillation) had a strong inflammatory response with significant increase in neutrophils in BAL. PXS TPI1100 treatment at 1.2 mg/kg (1 h prior to LPS challenge) resulted in a 33% reduction of neutrophil recruitment induced by LPS (p<0.05) whereas treatment with the control AON had no effect.

The potency of PXS TPI1100 at reducing the smoke-induced or LPS-induced lung inflammation was compared to the PDE4 inhibitor roflumilast (Daxas). Roflumilast (5 mg/kg, p.o.) given daily 1 h prior to cigarette smoke exposure reduced neutrophil recruitment by only 25% (Fortin et al., 2009). In the LPS model, roflumilast, given once at a dose of 10 mg/kg (~10-fold more than PXS TPI1100) had no effect on the neutrophil influx, whereas at a higher dose of 100 mg/kg (~100-fold that of PXS TPI1100) it reduced neutrophil recruitment by 46% (p<0.05). This effective dose of roflumilast exceeds the current clinical dose for Daxas of 500 microgram per adult per day. PXS TPI1100 is continuing its pre-clinical development as a treatment for COPD.

PXS TPI1100 has not yet performed nonclinical drug depositions studies however, from tests with different AON that recognize the same PDE targets yet lacked the modified chemistry backbone we can extrapolate how PXS TPI1100 will behave following pulmonary delivery. In CD-1 mice, following 14 days of daily dosing with AON by inhalation, AON plasma concentrations were not detectable (< LLOQ of 5-10 ng/mL) at all time points for all dose levels. In the lungs, the AON concentrations were dose-related, and there was evidence of accumulation in lungs over the 14 days, based on the higher levels at 24 h after the last dose *vs.* 24 h after the first dose. The systemic exposure was extremely low with only small amounts of AON detectable in the kidneys and liver of high-dose mice (2.5 mg/kg/day), and the levels were similar following the first and last doses. In monkeys, following 14 days of inhalation of AON there were detectable levels of AON in plasma only in a few high-dose animals up to 1 h post dosing on Day 1 while samples from Day 14 were all <LLOQ (Guimond et al. 2008). In the lung of animals on the day after last drug exposure, the AON levels were approximately dose proportional. In kidney and liver, low levels of AON were quantified one day after the last dose and only in high dose animals, demonstrating that similar to mice, the systemic exposure was low. When AON were delivered by slow bolus intravenous (IV) administration in monkey, the highest plasma levels were measured immediately at the first time point after IV

A Multi-Targeted Antisense Oligonucleoitde-Based

little validity in using them in face of the current evidence.

allows for other inflammatory indicators to be measured.

Therapy Directed at Phosphodiesterases 4 and 7 for COPD 447

measures and the relationship to health status as defined by the use of the St. Georges Respiratory Questionnaire (SGRQ) (Singh et al. 2010). Furthermore sputum neutrophil measures in the stable state were shown not be predictive of the future rate of exacerbations (Singh et al. 2010). Lastly, no association between sputum neutrophils measures and emphysema or systemic inflammation as measured by serum levels of IL-6, IL-8, C-reactive protein (CRP) and surfactant protein D was observed (Singh et al. 2010). In short, although there is a plausible assumption for the use of sputum neutrophils as a biomarker, there is

In lieu of the identification and validation of a biomarker that could predict the rate of lung function decline in COPD, most COPD clinical trials attempt to measure relevant changes in exacerbations. Exacerbation frequency has been considered to be an important outcome parameter in COPD as it is associated with increase in mortality (Patil et al. 2003) (Fuso et al. 1995). Measuring exacerbations is not without its challenges. It is difficult among studies to find consensus on what is defined as an exacerbation and to gauge the severity of the exacerbation. Symptom-based definitions include use of diaries, while event-based definitions may refer to hospitalizations or use of antibiotics and/or steroids (Miravitlles et al. 2004). Although a systematic literature review of studies reporting exacerbation frequency in COPD patients showed the relationship between increased exacerbation frequency with decreasing lung function to be borderline significance (p=0.053) (Spencer et al. 2004), exacerbations are still considered to be an important parameter in COPD. Exacerbations are more likely to occur in winter and according to current recommendations (Cazzola et al. 2008) studies need to have at least a 12 month follow up to give reliable estimate of exacerbation frequency, which requires the planning of lengthy clinical trials.

The clinical program of PXS TPI1100 has not been initiated yet we expect its design can follow that of other PDE4 inhibitors. An initial Phase 2 study design does not test in COPD patients but rather in allergic asthmatic patients following inhaled allergen challenge (2009). Another AON drug, ASM8 designed specifically for asthma and as such has targets different from PXS TPI1100, has demonstrated clinical efficacy in this allergen challenge model (Gauvreau et al. 2008; Gauvreau 2010) clearly showing the potential for the AON approach. An advantage of this allergen challenge model is that the studies are generally brief in duration and the fall in FEV1 is a well-recognized response as well as the incorporation of monitoring induced sputum

PXS TPI1100 has an advantage in that the clinical studies performed by Daxas/Daliresp can be used as a guide, as the two drugs share a common target. As Daxas/Daliresp was breaking new ground many studies had to be performed and it is plausible to conclude that for other drugs in the same class fewer studies may be required. In all, six phase 3 clinical trials were undertaken with Daxas/Daliresp which have been excellently reviewed by Giembycz and Field (Giembycz &Field 2010). Key aspects of these trials that can be used for PXS TPI1100's clinical development include criteria for patient selection and parameters selected for primary and secondary outcomes. In the phase 3 study named RECORD, patients with moderate-to-severe COPD (postbronchodilator FEV1 of 30% to 80% predicted and a FEV1/FVC ratio of less than 70%) were randomized to receive either Daxas/Daliresp at 250 µg or 500 µg or placebo (2:2:1 ratio) for 24 weeks (Rabe et al. 2005). Results showed treated patients experienced improvement in postbronchodilator FEV1 (Rabe et al. 2005) and a change in SGRQ but this change did not reach clinical significant threshold. Although direct

injection (approximately 5 min) and these levels were greatly reduced by 4 h post-dose and near LLOQ by 24 h demonstrating the clearance of AON from the system.

The pharmacokinetics properties following pulmonary delivery has been well characterized (Templin et al. 2000; Ali et al. 2001; Guimond et al. 2008) and confers a significant advantage of AON over small molecule drugs. For example, orally-delivered Daxas/Daliresp has a bioavailabilty of 79%(David 2004) and with an elimination half-life of 14-18 h there is a greater opportunity for this drug to act upon PDE4 outside of the lung and for a long period of time. In comparison, PXS TPI1100 has reduced systemic bioavailability and based on results in mouse lung, the half-life of PXS TPI 1100 has been shown to be relatively short (<5h) suggesting a potentially safer drug that would work locally at the site of action in the lung.
