**5. Target genes of pharmacogenetics**

About 20 kinds of enzymes are involved in metabolism of drugs. The cytochrome enzyme (CYP450) is regarded as the most important enzyme in drug metabolism. About 15 types of this group have been identified in human beings where they catalyse the biotransformation of many xenobiotics. Other enzymes include thiopurine methyl transferase (TMPT) which metabolizes 6- mercaptopurine and azathioprine, uridyl diphosphate glucuronyl transferase (UDGT) responsible for the conjugation of bilirubin, N – acetyltransferase (NAT2) responsible for metabolism of sulpha containing drugs and caffeine, catachol – o – methyl transferase (COMT) responsible for the metabolism of levodopa and dihydropyrimidine dehydrogenase (DPD) a rate limiting enzyme for the metabolism of 5 – florouracil (5 FU). Genetic polymorphism has been identified in many of these enzymes with varying degrees of drug response (Evans & Relling 1999, Furuta et al 2001, McAlpine et al 2001,Suzuki et al 2011) Of all the enzymes involved in drug metabolism, the cytochrome P450.(CYP450) is regarded as the most important because many drugs are substrates for the enzyme of the

Phase III reactions can be described as a stage of further modification and excretion. Although many authors do not regard this phase as a distinct phase, current knowledge of efflux transporters tend to support the categorization. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates (Boyland and Chassaud 1969). In this scenario, glutamate and glycine residing in the glutathione molecule are removed by gamma-glutamyltranspeptidase and dipeptidases. Finally, the cysteine residue in the conjugate is acetylated .The conjugates and their metabolites can then be excreted from cells in phase III of their metabolism with anionic groups acting as affinity taps for a variety of membrane transporters of the multidrug resistance protein (MRP) family (Homolya et al 2003). These proteins are members of the family of ATP-binding cassette transporters and can facilitate the ATP dependent transport of a large varieties of hydrophobic ions (Konig et al 1999) and thus act to remove phase II products to the extracellular medium, where they may be further metabolized and excreted (Commandeur

Since the discovery of permeability glycoprotein (P – glycoprotein) complex; an initial member of the ATP binding cassette (ABC) family of drug transporters by Juliano and Ling in 1976, (Juliano and Ling 1976) research into this group of proteins has been gaining wide interests. Some of the membrane transporters confer on the cells the ability to be resistant not only to the selective agent but also to a broad spectrum of structurally and functionally distinct antibiotics and alkaloids.This phenotypic character is referred to as multiple drug resistance (MDR). The MDR genotype/phenotype relationship is complex with over 18

In addition to the ABC transporters, other important drug/xenobiotic transporters include the organic cation transporters of the SLC 22A super family and the organic anion transporting peptides of the SLC21 superfamily (Hagenbuch 2010). It is expected that with the growing interests in this phase of drug metabolism, investigations on the transcriptional regulatory control of this important transport system in target organs such as the liver, kidney and

About 20 kinds of enzymes are involved in metabolism of drugs. The cytochrome enzyme (CYP450) is regarded as the most important enzyme in drug metabolism. About 15 types of this group have been identified in human beings where they catalyse the biotransformation of many xenobiotics. Other enzymes include thiopurine methyl transferase (TMPT) which metabolizes 6- mercaptopurine and azathioprine, uridyl diphosphate glucuronyl transferase (UDGT) responsible for the conjugation of bilirubin, N – acetyltransferase (NAT2) responsible for metabolism of sulpha containing drugs and caffeine, catachol – o – methyl transferase (COMT) responsible for the metabolism of levodopa and dihydropyrimidine dehydrogenase (DPD) a rate limiting enzyme for the metabolism of 5 – florouracil (5 FU). Genetic polymorphism has been identified in many of these enzymes with varying degrees of drug response (Evans & Relling 1999, Furuta et al 2001, McAlpine et al 2001,Suzuki et al 2011) Of all the enzymes involved in drug metabolism, the cytochrome P450.(CYP450) is regarded as the most important because many drugs are substrates for the enzyme of the

central nervous system will become intense in the next few decades (Omiecinski 2011).

ABC genes associated with human disease (Dean and Annilo 2005).

**5. Target genes of pharmacogenetics** 

**4.3 Phase III reactions** 

et al 1995).

group. CYP3A4, CYP2D6, CYP2C9, CYP219, CYP2B6 and CYP1A2 play the most critical role and account for more than 90% of drugs metabolized by P450 ( Evans & Relling 1999). These enzymes have proven genetic polymorphism with associated drug responses (Hiratsuka et al 2002, Wong et al 2005, McAlpine et al 2011) and racial variations (Meyer 2004 & Suarez – Kurtz 2005). As discussed earlier, genetic polymorphism can manifest at both pharmacokinetic and pharmacodynamic levels whereby many genetic variants of respective enzymes, membrane transporters, receptors and ion channels have been dectected ( Wiesler et al 2008, Phipps – Green et al 2010 & Bouamar et al 2011)

#### **5.1 Pharmacokinetic related genes**

#### **5.1.1 Genes of phase i reaction enzymes**

Genetic variations have been observed particularly with CYP2D6, CYP2C9 aand CYP2C19 genotypes (Ingelman – Sundberg 1999, Hiratsuka 2006) and therefore will be further elucidated.


Pharmacogenetics: The Scientific Basis 193

genetic variants have been reported (McLeod et al 1998, Collie – Duguid et al 2000). The genetic variant that is responsible for decreased DPD activity has been reported to be DPYD\*2 with a polymorphism at the splicing recognition site. (Wei at al 1996) Administration of 5 – FU to the patients with decreased enzyme activity results in adverse effects such as leukocytopenia, stomatitis, diarrhea, nausea and vomiting (Etienne et al 1994) **Glutathione – S – Tranferase (GST):** GSTs and the human genes encoding these enzymes are highly polymorphic with about 50% and 25% of most populations having a mutation or complete deletion of these gene respectively rendering them deficient or lacking the enzyme. Major racial and ethnic differences exist and GST M and GST T1 are the major genes. Other GSTs include GST P1 and GST\*A which are also subject to genetic polymorphism and have been implicated in resistance to anti cancer drugs. High GST activity has been associated with decreased risk of haematologic relapse, central nervous system response and improved prednisolone response. (Commandeur et.al 1995) Inherited GST – P1 allele encoding for the 11e 105 Val. amino acid substitution, has been associated with improved overall breast cancer survival compared with patients who have at least one wild type GST P1 allele. Conversely in patients with acute myeloid leukaemia treated with high doses of combination therapy, the homozygous GST – T1 deletion is associated with a

**Uridyl Diphosphate Glucuronyl Transferase (UGT):** The UDP – glucuronyl transferase (UGT) belongs to a super family of membrane bound proteins localized in the endoplamic reticulum and are responsible for glucuronidation of many xenobiotics and endobiotics. The UGT genes have been classified into families and sub families based on evolutionary divergence with all known human UGT's being in the UGT1A 2A and 2B sub families. (Mackenzie et al 1997, Randominska – Pandya et al 1999, Tukey & Strassburg 2000). To date, polymorphism in UGTA1 have been more studied extensively and seem to have clinical significance. The anticancer drug irinotecan is metabolized by the enzyme and polymorphism resembling condition seen in Gilbert's syndrome characterised by total lack of UGT enzyme due to deletion of the gene which leads to fifty fold reduction in irinotecan metabolism and such patients can be at risk of toxicity (Huang et al 2002 Desai et al 2003).

Membrane transporters as mentioned earlier are heavily involved in drug clearance and alter drug disposition by actively transporting drugs between organs and tissues. Therefore polymorphisms in the genes encoding these proteins may have significant effects on the absorption, distribution, metabolism and excretion of xenobiotics and may alter the pharmacodynamics of these agents. Uptake transporters are required for the uptake of some drugs into the cell whereas efflux transporters are responsible for pumping some drugs out of cells or preventing them from ever getting in. Transporters are also thought to

The most important families of the transporters include (i**)** ATP binding cassette (ABC) family whose genes include important members like the multi drug resistance gene also classified as ABCB 1 i.e. (ABCB1/MDR1), ABCC1, ABCC2, uric acid transporter (ABCG2), breast cancer resistance protein BCRP also classified ABCG2 i.e. (BCRP/ABCG2).(ii) The solute transporter superfamily (SLC) which include the organic anion transport polypeptide

higher risk of toxic death during remission. ( Arruda et. al 2001)

**5.1.3 Phase III reactions: Transporter genes** 

be involved in drug – drug reactions.

of the population. Conversely, blood concentrations of omeprazole are predicted to be 40% lower in ultrarapid metabolizers than the rest of the population and are therefore at risk of therapeutic failure. (Sum et al 2006)

#### **5.1.2 Genes of phase II reaction enzymes**

**N – Acetyl Transferase:** Activities of human hepatic drug metabolizing enzymes was earlier been recognized as a cause of inter – individual variation in the metabolism of drugs. Therefore acetylation of many drugs like isoniazid caffeine, nitrozepam and sulphonamide exhibit genetic polymorphism. The N – acetyl transferase (NAT) enzyme is controlled by two genes, (NAT 1) and (NAT 2) of which NAT2 A and B are responsible for clinically significant metabolic polymorphism. (Heiss 1988, Grant et al 1990). Three phenotypes have been recognized with activities of NAT2 and these are rapid acetylator (RA), intermediate acetylator (IA) and slow acetylator (SA) status (Cranswick 2005). The frequency of slow acetylator in Caucasians and Negro populations is 50% and 10% in Oriental groups. (.Evans D.A 1989) Slow acetylator phenotype is preponderant among different Arab populations irrespective of geographical location of the country. (Woolhouse et al 1997, At- Moussa et al 2002 & Desoky et al 2005). Three genetic polymorphisms NAT2\*5, NAT2\*6, NAT2\*7 but not NAT2\*4 (wild type allele) are responsible for almost all SAs in the Japanese (Huang et al 2002) Drug induced hepatitis caused by isoniazid occurs often in SA than RA (Ohno et al 2000) and Type II diabetes SA may be predisposed to progression to renal complications than their RA counterparts (Banjoko & Akinlade 2010).

**Thiopurine - S –Methyl Transferase (TPMT:** Catalyses the S – Methylation of the thiopurine agents, azathioprine, mercaptopurine and thiogleamine. These agents are commonly used for a diverse range of medical indications including leukaemia, rheumatic diseases and organ transplant. The principal cytotoxic mechanism of these agents is mediated via incorporation of thioguanine nucleotides (TGN) into DNA. Thiopurines are inactive prodrugs that require metabolism to thioguanine nucleotides to exert cytotoxicity. This activation is catalyzed by a multienzyme pathway which include hypoxanthine phosphoribosyl transeferase (HPRT), oxidation by xanthine oxidase (XO) or methylation by TPMT. During metabolism, hypoxanthine-guanine phosphoribosyl transferase (HPGRT) converts 6-mercaptopurine to cytotoxic6-thioguanine nucleotide analogues, while thiopurine methyl transferase (TPMT) inactivates 6-mercaptopurine through methylation to form 6 –methylmercaptopurine. However, TMPT is the major pathway and it is highly variable and polymorphic. More than 12 TPMT alleles have been identified. The most common ones are TPMT\*2, TPMT\*3A, TPMT\*3C, with all three associated with lower enzyme activity attributable to enhanced rates of proteolysis of the variant proteins (Donnan et al 2011, Haghuid et al 2011, Guengerich 2001). Caucasian infant patients with acute myeloid leukaemia carrying TPMT\*2, TPMT\*3A, TPMT\*3B, TPMT\*3C showed significantly higher concentrations of the thiopurine intermediate metabolite 6-mercaptopurine in their red cells that requires dose reduction or termination of thiopurine administration due to adverse effects such as myelosuppression (Relling et al 1999, Tavadia et al 2001).

**Dihydro Pyrimidine Dehydrogenase (DPD):** Dihydro pyrimidine dehydrogenase (DPD) is a rate limiting enzyme for the metabolism of the anti cancer drug; 5 fluorouracil (5FU). With DPD being responsible for over 50% of its biotransformation. Other substrates for DPD are carmofur, tegafur and doxifluridine. The gene encoding for DPD is DPDY and about 13

**N – Acetyl Transferase:** Activities of human hepatic drug metabolizing enzymes was earlier been recognized as a cause of inter – individual variation in the metabolism of drugs. Therefore acetylation of many drugs like isoniazid caffeine, nitrozepam and sulphonamide exhibit genetic polymorphism. The N – acetyl transferase (NAT) enzyme is controlled by two genes, (NAT 1) and (NAT 2) of which NAT2 A and B are responsible for clinically significant metabolic polymorphism. (Heiss 1988, Grant et al 1990). Three phenotypes have been recognized with activities of NAT2 and these are rapid acetylator (RA), intermediate acetylator (IA) and slow acetylator (SA) status (Cranswick 2005). The frequency of slow acetylator in Caucasians and Negro populations is 50% and 10% in Oriental groups. (.Evans D.A 1989) Slow acetylator phenotype is preponderant among different Arab populations irrespective of geographical location of the country. (Woolhouse et al 1997, At- Moussa et al 2002 & Desoky et al 2005). Three genetic polymorphisms NAT2\*5, NAT2\*6, NAT2\*7 but not NAT2\*4 (wild type allele) are responsible for almost all SAs in the Japanese (Huang et al 2002) Drug induced hepatitis caused by isoniazid occurs often in SA than RA (Ohno et al 2000) and Type II diabetes SA may be predisposed to progression to renal complications

**Thiopurine - S –Methyl Transferase (TPMT:** Catalyses the S – Methylation of the thiopurine agents, azathioprine, mercaptopurine and thiogleamine. These agents are commonly used for a diverse range of medical indications including leukaemia, rheumatic diseases and organ transplant. The principal cytotoxic mechanism of these agents is mediated via incorporation of thioguanine nucleotides (TGN) into DNA. Thiopurines are inactive prodrugs that require metabolism to thioguanine nucleotides to exert cytotoxicity. This activation is catalyzed by a multienzyme pathway which include hypoxanthine phosphoribosyl transeferase (HPRT), oxidation by xanthine oxidase (XO) or methylation by TPMT. During metabolism, hypoxanthine-guanine phosphoribosyl transferase (HPGRT) converts 6-mercaptopurine to cytotoxic6-thioguanine nucleotide analogues, while thiopurine methyl transferase (TPMT) inactivates 6-mercaptopurine through methylation to form 6 –methylmercaptopurine. However, TMPT is the major pathway and it is highly variable and polymorphic. More than 12 TPMT alleles have been identified. The most common ones are TPMT\*2, TPMT\*3A, TPMT\*3C, with all three associated with lower enzyme activity attributable to enhanced rates of proteolysis of the variant proteins (Donnan et al 2011, Haghuid et al 2011, Guengerich 2001). Caucasian infant patients with acute myeloid leukaemia carrying TPMT\*2, TPMT\*3A, TPMT\*3B, TPMT\*3C showed significantly higher concentrations of the thiopurine intermediate metabolite 6-mercaptopurine in their red cells that requires dose reduction or termination of thiopurine administration due to

adverse effects such as myelosuppression (Relling et al 1999, Tavadia et al 2001).

**Dihydro Pyrimidine Dehydrogenase (DPD):** Dihydro pyrimidine dehydrogenase (DPD) is a rate limiting enzyme for the metabolism of the anti cancer drug; 5 fluorouracil (5FU). With DPD being responsible for over 50% of its biotransformation. Other substrates for DPD are carmofur, tegafur and doxifluridine. The gene encoding for DPD is DPDY and about 13

at risk of therapeutic failure. (Sum et al 2006)

than their RA counterparts (Banjoko & Akinlade 2010).

**5.1.2 Genes of phase II reaction enzymes** 

of the population. Conversely, blood concentrations of omeprazole are predicted to be 40% lower in ultrarapid metabolizers than the rest of the population and are therefore genetic variants have been reported (McLeod et al 1998, Collie – Duguid et al 2000). The genetic variant that is responsible for decreased DPD activity has been reported to be DPYD\*2 with a polymorphism at the splicing recognition site. (Wei at al 1996) Administration of 5 – FU to the patients with decreased enzyme activity results in adverse effects such as leukocytopenia, stomatitis, diarrhea, nausea and vomiting (Etienne et al 1994)

**Glutathione – S – Tranferase (GST):** GSTs and the human genes encoding these enzymes are highly polymorphic with about 50% and 25% of most populations having a mutation or complete deletion of these gene respectively rendering them deficient or lacking the enzyme. Major racial and ethnic differences exist and GST M and GST T1 are the major genes. Other GSTs include GST P1 and GST\*A which are also subject to genetic polymorphism and have been implicated in resistance to anti cancer drugs. High GST activity has been associated with decreased risk of haematologic relapse, central nervous system response and improved prednisolone response. (Commandeur et.al 1995) Inherited GST – P1 allele encoding for the 11e 105 Val. amino acid substitution, has been associated with improved overall breast cancer survival compared with patients who have at least one wild type GST P1 allele. Conversely in patients with acute myeloid leukaemia treated with high doses of combination therapy, the homozygous GST – T1 deletion is associated with a higher risk of toxic death during remission. ( Arruda et. al 2001)

**Uridyl Diphosphate Glucuronyl Transferase (UGT):** The UDP – glucuronyl transferase (UGT) belongs to a super family of membrane bound proteins localized in the endoplamic reticulum and are responsible for glucuronidation of many xenobiotics and endobiotics. The UGT genes have been classified into families and sub families based on evolutionary divergence with all known human UGT's being in the UGT1A 2A and 2B sub families. (Mackenzie et al 1997, Randominska – Pandya et al 1999, Tukey & Strassburg 2000). To date, polymorphism in UGTA1 have been more studied extensively and seem to have clinical significance. The anticancer drug irinotecan is metabolized by the enzyme and polymorphism resembling condition seen in Gilbert's syndrome characterised by total lack of UGT enzyme due to deletion of the gene which leads to fifty fold reduction in irinotecan metabolism and such patients can be at risk of toxicity (Huang et al 2002 Desai et al 2003).

#### **5.1.3 Phase III reactions: Transporter genes**

Membrane transporters as mentioned earlier are heavily involved in drug clearance and alter drug disposition by actively transporting drugs between organs and tissues. Therefore polymorphisms in the genes encoding these proteins may have significant effects on the absorption, distribution, metabolism and excretion of xenobiotics and may alter the pharmacodynamics of these agents. Uptake transporters are required for the uptake of some drugs into the cell whereas efflux transporters are responsible for pumping some drugs out of cells or preventing them from ever getting in. Transporters are also thought to be involved in drug – drug reactions.

The most important families of the transporters include (i**)** ATP binding cassette (ABC) family whose genes include important members like the multi drug resistance gene also classified as ABCB 1 i.e. (ABCB1/MDR1), ABCC1, ABCC2, uric acid transporter (ABCG2), breast cancer resistance protein BCRP also classified ABCG2 i.e. (BCRP/ABCG2).(ii) The solute transporter superfamily (SLC) which include the organic anion transport polypeptide

Pharmacogenetics: The Scientific Basis 195

other variants have also been detected (Colombo et al 2005). A haplotype dependent influence on transport capacity of ABCC2 had been observed but seems to be mainly based on post

**TheABCG2 gene** encodes an inhibitor of breast cancer resistance protein (BCRP) (ABCG2) protein, another member of the ABC transporter. The protein confers protection against the development of breast cancers. Evaluation of single nucleotide polymorphism identified 16 variants (Morisaki et al 2005, Colombo et al 2005). Genetic polymorphism in ABCG2 might alter the transport activity of some drugs causing therapy in drugs like irinotecan, to cause severe myelosuppression (Choi et al 2009, Hampras et al 2010). A polymorphism, C421A observed in human placenta is not a genetic variant acting in cis but is considered to influence the translational efficiency (Kobayeshi et al 2005). Another genetic variant (ABCG2) (rs 2231142, Q141K) encoding a uric acid transporter is associated with gout in

The solute carrier (SLC) superfamily of transporters consists of more than 300 members subdivided into 47 families. They are expressed in most tissues but primarily in liver, lungs,

i. **OATP/SLC21:** Organic anion transporter facilitates movement of anion across the cell membrane.OATP1B and OATP1B3 are human hepatocyte transporters that mediate the uptake of various endogenous and exogenous substrates. Genetic variation was observed in the SLCO1B1 and SLCO1B3 genes which encode OATP1B1 and OATP1B3 proteins. Forty nine (49) and 41 nucleotide sequence variants leading to 10 and 9 in SLCO1B1 and SLCO1B3 genes respectively were identified (Bowin et al 2010). Furthermore, in OATPC (SLC21A6) and OATP3 (SLC22A8) genes, polymorphism did not appear to be associated with changes in renal and tubular secretory clearance in the latter but the former was associated with differences in the disposition kinetics of pravastin. Individualswith the OATP – C\*15 allele (ASP 130 Ala 174) had a reduced total and non renal clearance compared with those of OATPC\*15 allele (ASP130Val 174)

ii. **SLC 19A1 (Folate Transporter)member 1:** The SLC19A1 are the proteins responsible for the transport of folate. Transport of folate into the mammalian cells can occur via receptor mediated (folate receptor 1) or carrier mediated (SLC19A1) mechanism. Methotrexate is an antifolate chemotherapeutic agent that is actively transported by the carrier mediated uptake system. Individuals carrying a specific polymorphism of SLC19A1 gene ï.e (C80GG) have lower levels of folate. (Whetsine 2003, Matherly et al 2007) and those carrying the C80AA genotype treated with methrotrexate have higher levels of this antifolate chemotherapeutic agent. This underpins requirements for

iii. **OCT/SLC22:** Most solute carrier transporters are localized at either the basolateral or apical plasma membrane of polarized cells but some are also expressed in mitochondria and other organelles (Wojtal et al 2009). The genes encoding the three organic cation transporter isoforms OCT1, OCT2 and OCT 3 are clustered together on the long arm of chromosome 8 in humans and carry out functions of transport of small organic cations with different molecular structures independent of sodium gradient. These organic

personalized dosing with the drug based on patients genotype

transcriptional modifications rather than transport rates (Laechelt et al 2011).

diverse populations (Phipps – Green et al 2010) **5.1.3.2 Solute Carrier Superfamily: (SLC) Genes** 

kidney and intestine.

(Nishizato Y et al 2003).

(SLC 21/OATP), organic cation transporter SLC 22 OCT), zwitterion/cation transporter (OCTNs), folate transporter(SLC19A1), neurotransmitter transporter(SLC6,SLC17,&SLC18)and serotonin transporter (5HTT).Genetic polymorphism in drug transporter genes have increasingly been recognized as a possible mechanism accounting for variation in drug response because these transporters play important roles in the gastrointestinal absorption, biliary and renal elimination and distribution to target sites of their substrates. (Meier et al 2007, Shu et al 2007, Choi & Song 2008)

#### **5.1.3.1 The ABC family genes**

**ABCB1:** Refers to ATP binding cassette (ABC) sub family B member 1, or MDR 1 also designated cluster of differentiation (CD243) is the permeability glycoprotein (P – glycoprotein).ABC genes are divided into seven distinct sub families (ABC1, MDR/TAP, MRP, ALD, OABP, CaCW 20 andWhite). Members of the MDR/TAP sub- family are involved in multi drug resistance. The protein encoded by this gene is an ATP dependent drug efflux pump of xenobiotics with broad substrate specificity. It is responsible for decreased drug accumulation in multi drug resistant cells and often mediates the development of resistance to cancer cells (Viguie 1998 ). This protein also function as a transporter in the blood brain barrier (Viguie 1998, Phipps – Green et al 2010). It likely evolved as a defense mechanism against harmful substances. Someof the functions of protein encoded by ABCB 1 gene include regulation of distribution and bioavailability of drugs, removal of metabolites and xenobiotics from cells into urine, bile and intestinal lumen, transport of compounds out of the brain across the blood – brain barrier, digoxin uptake, prevention of invermectin entry into the central nervous system and protection of hamatopoietic cells from toxins (Dean 2002.) Mutation of ABCB1 gene will therefore result in disruption of these functions. The activity of the transporter can be determined by both membrane ATPase and cellular calcein assays. Drug resistance had been observed in M89T, L662R, R669 and S1141T variants of the gene and decreased drug efficacy in W1108R variant. In addition, genetic variation in ABCB1 has been associated with both toxicity and drug response in 5Fluoro-uracil (Gonzalez – Haba et al 2011) and pacilitaxel therapy (Henningson et al 2011).

**ABCC 1 genes:** Multidrug resistant protein 1 (MRP1) an ATP bounding cassette transporter encoded by ABCC 1 gene is expressed in many tissues and function as an efflux transporter for glutathione, glycine and sulphate conjugates as well as unconjugated substrates. An evaluation of single nucleotide polymorphism (SNP) revealed 7 mutations in the gene (Colombo et al 2005) while in a Japanese study, 86 genetic variants were identified (Fukushina – Uesaka et al 2007). Mutations in ABC transporters cause or contribute to many different Mendelian and complex disorders including adrenoleukodystrophy, cystic fibrosis and retinal degeneration (Dean & Annilo 2005). There has been no evidence of clinical significance in studies of the variants. (Colombo et al 2005, Pauli Magrus & Kroetz 2005 & Fukushina -Uesaka 2007).

**ABCC2 gene:** ABBCC2 genes codes for the ABCC2 or MRP2 protein. (MRP2) is an export pump expressed at tissue barriers. Genetic variants 24 e>T, 1249Ca>A and 3972 > T had been observed and are thought to cause inter individual differences of bioavailability of various endogenous and exogenous compounds (Colombo et al 2005, Laechelt et al 2011). About 27

(SLC 21/OATP), organic cation transporter SLC 22 OCT), zwitterion/cation transporter (OCTNs), folate transporter(SLC19A1), neurotransmitter transporter(SLC6,SLC17,&SLC18)and serotonin transporter (5HTT).Genetic polymorphism in drug transporter genes have increasingly been recognized as a possible mechanism accounting for variation in drug response because these transporters play important roles in the gastrointestinal absorption, biliary and renal elimination and distribution to target sites

**ABCB1:** Refers to ATP binding cassette (ABC) sub family B member 1, or MDR 1 also designated cluster of differentiation (CD243) is the permeability glycoprotein (P – glycoprotein).ABC genes are divided into seven distinct sub families (ABC1, MDR/TAP, MRP, ALD, OABP, CaCW 20 andWhite). Members of the MDR/TAP sub- family are involved in multi drug resistance. The protein encoded by this gene is an ATP dependent drug efflux pump of xenobiotics with broad substrate specificity. It is responsible for decreased drug accumulation in multi drug resistant cells and often mediates the development of resistance to cancer cells (Viguie 1998 ). This protein also function as a transporter in the blood brain barrier (Viguie 1998, Phipps – Green et al 2010). It likely evolved as a defense mechanism against harmful substances. Someof the functions of protein encoded by ABCB 1 gene include regulation of distribution and bioavailability of drugs, removal of metabolites and xenobiotics from cells into urine, bile and intestinal lumen, transport of compounds out of the brain across the blood – brain barrier, digoxin uptake, prevention of invermectin entry into the central nervous system and protection of hamatopoietic cells from toxins (Dean 2002.) Mutation of ABCB1 gene will therefore result in disruption of these functions. The activity of the transporter can be determined by both membrane ATPase and cellular calcein assays. Drug resistance had been observed in M89T, L662R, R669 and S1141T variants of the gene and decreased drug efficacy in W1108R variant. In addition, genetic variation in ABCB1 has been associated with both toxicity and drug response in 5Fluoro-uracil (Gonzalez – Haba et al 2011) and pacilitaxel therapy

**ABCC 1 genes:** Multidrug resistant protein 1 (MRP1) an ATP bounding cassette transporter encoded by ABCC 1 gene is expressed in many tissues and function as an efflux transporter for glutathione, glycine and sulphate conjugates as well as unconjugated substrates. An evaluation of single nucleotide polymorphism (SNP) revealed 7 mutations in the gene (Colombo et al 2005) while in a Japanese study, 86 genetic variants were identified (Fukushina – Uesaka et al 2007). Mutations in ABC transporters cause or contribute to many different Mendelian and complex disorders including adrenoleukodystrophy, cystic fibrosis and retinal degeneration (Dean & Annilo 2005). There has been no evidence of clinical significance in studies of the variants. (Colombo et al 2005, Pauli Magrus & Kroetz 2005 &

**ABCC2 gene:** ABBCC2 genes codes for the ABCC2 or MRP2 protein. (MRP2) is an export pump expressed at tissue barriers. Genetic variants 24 e>T, 1249Ca>A and 3972 > T had been observed and are thought to cause inter individual differences of bioavailability of various endogenous and exogenous compounds (Colombo et al 2005, Laechelt et al 2011). About 27

of their substrates. (Meier et al 2007, Shu et al 2007, Choi & Song 2008)

**5.1.3.1 The ABC family genes** 

(Henningson et al 2011).

Fukushina -Uesaka 2007).

other variants have also been detected (Colombo et al 2005). A haplotype dependent influence on transport capacity of ABCC2 had been observed but seems to be mainly based on post transcriptional modifications rather than transport rates (Laechelt et al 2011).

**TheABCG2 gene** encodes an inhibitor of breast cancer resistance protein (BCRP) (ABCG2) protein, another member of the ABC transporter. The protein confers protection against the development of breast cancers. Evaluation of single nucleotide polymorphism identified 16 variants (Morisaki et al 2005, Colombo et al 2005). Genetic polymorphism in ABCG2 might alter the transport activity of some drugs causing therapy in drugs like irinotecan, to cause severe myelosuppression (Choi et al 2009, Hampras et al 2010). A polymorphism, C421A observed in human placenta is not a genetic variant acting in cis but is considered to influence the translational efficiency (Kobayeshi et al 2005). Another genetic variant (ABCG2) (rs 2231142, Q141K) encoding a uric acid transporter is associated with gout in diverse populations (Phipps – Green et al 2010)

#### **5.1.3.2 Solute Carrier Superfamily: (SLC) Genes**

The solute carrier (SLC) superfamily of transporters consists of more than 300 members subdivided into 47 families. They are expressed in most tissues but primarily in liver, lungs, kidney and intestine.


Pharmacogenetics: The Scientific Basis 197

variation in those on therapy.

such drugs.

testing.

**6. Pharmacogenetic testing** 

already carry labels addressing such.

Another important enzyme of drug target is vitamin K epoxide reductase complex subunit 1(VKORC1). This enzyme is the drug target for warfarin an anticoagulant with a narrow therapeutic window and with serious consequences of bleeding in the event of an overdose. Variation in maintenance dose of warfarin is largely attributable to genetic variants in the genes that encode the drug target VKORC1 the major metabolizing enzyme. The two genetic polymorphisms explain 30 – 40% of the total

Angiotensin converting enzyme (ACE) genes encode for ACE, a target for ACE inhibitors which improves symptom and survival in cases of heart failure. Genetic polymorphism is suspected to be causing greater effects of the drug in Europeans than Afro-Americans. Pre-treatment genetic screening is therefore apt to improve therapy **iv. Neurotransmitter Transporters:** Neurotransmitter transporters namely SLC6, SLC17 and SLC18 families are primarily expressed in the neurons of the central and peripheral nervous system. These transporters are the sites of action of various drugs of abuse e.g cocaine, amphetamine and other clinically approved drugs like desipramine, reserpine, benztropine and tiagabine. Genetic variation in the SLC6, SLC17 and SLC18 encoding genes may result in altered expression and function of these proteins. In particular, antidepressants and antiepileptic drugs target these neurotransmitters as part of their primary mechanism of action. Therefore genetic variations may affect the efficacy of

A genetic test is the analysis of human DNA, RNA chromosomes, proteins or certain metabolites in order to detect alterations related to a heritable disorder. This can be accomplished by directly examining the DNA or RNA that makes up a gene (Direct testing), looking at markers co-inherited with a disease causing gene (linkage testing), assaying certain metabolites (biochemical testing), or examining the chromosomes (cytogenetic testing). Although genetic testing shares some features common with other kinds of laboratory testing, it is however unique in many ways and therefore requires special consideration .Pharmacogenetic testing can therefore be defined as utilization of aforementioned genetic biomarkers related to drug metabolism and effects. A biomarker can be described as a characteristic that is objectively measured and evaluated as an

indicator of normal biological processes to a therapeutic intervention (EMEA 2006)

Methods of Pharmacogenetic testing depends on the biomarker to be assessed. These vary from simple spectrophotometric estimation of metabolites to DNA sequences, use of PCR and DNA probes, enzymes linked immunosorbent assay, cell culture, gel electrophoresis high performance liquid chromatography and DNA hybridization techniques. It is not uncommon to use combined techniques to study clinical relevance of pharmacogenetic

Because information on pharmacogenetics is still evolving, there is a necessity for guidelines to be adopted for ethical reasons, economic considerations and patient benefit. Overall, the quest for pharmacogenetic information is likely to grow. As a matter of fact some drugs

cation substrate include drugs like metformin, procainamide and cimetidine as well as endogenous compounds like dopamine and norepinephrine and toxic substances like tetraethylammonium bromide (TEA) (Kang et al 2007).

#### **5.2 Pharmacodynamic related genes**

i. **Receptors:** Many receptors are involved with several signaling pathways. Example of which is epidermal growth factor receptor( EGFR). This receptor has been implicated in the oncogenesis and progression of several solid tumours thereby being identified as a suitable target for anticancer treatment. Polymorphism has been observed in the development of cancer on dinucleotide repeats in intron 1 of the EGFR gene and this has correlated with EGFR expression with therapeutic implication for treatment with tyrosinase kinase inhibitor. A higher proportion of Asians do overexpress EGFR that may influence their responses to tyrosine kinase inhibitor (Tan et al 2004).

**G-protein Coupled Receptors (GPCR**):Over 50% of all drug targets have G-protein coupled receptors (GPCR). Genes of GPR has more coding regions than non – GPCR genes making them more important for pharmacological investigations.

**GABAA Receptor** Mutation in GABAA receptor ion channel may be a reason for the diminished protection of anti epileptic drugs.

**Insulin Receptor(INSR):** The receptor is important in the management of diabetes mellitus patients and mutation of the gene encoding the receptor will result in poor response particularly in type 2 diabetes.Mutation of the gene has also been suspected to contribute to genetic susceptibility to the polycystic ovarian syndrome(Siega et al2002)

**B2** Receptor: B2 agonist; albuterol (Proventil) is used to control acute attacks of asthma and are prescribed as needed .Patients with 2 receptor arginine genotype experience poor asthma control with frequent symptoms and a decreasing scores of poor exploratory volume compared with those with glycine genotype (Cowburn et al 1998, de Maat et al 1999). Seventeen (17%) of whites and 20% of blacks carry the arginine genotype (Wechsler et al 2005)

ii. **Ion Channels:** Many genes encode for different ion channels including those of the central nervous system which include KCNJ10, KCNJ3, CLCN2, GABRA1, SCN1B and SCN1A. Some polymorphism of this channel has been linked to idiopathic generalized epilepsy (Lucarini et al 2007)

The 5-HT3 receptor is a ligand-gated ion channel composed of five subunits. To date, five different human subunits are known; 5-HT3A-E, which are encoded by the serotonin receptor genes HTR3A, HTR3B, HTR3C, HTR3D and HTR3E, respectively. Functional receptors are pentameric complexes of diverse composition. Different receptor subtypes seem to be involved in chemotheraphy-induced nausea and vomiting (CINV), irritable bowel syndrome and psychiatric disorders. 5-HTR3A and HTR3B polymorphisms may also contribute to the etiology of psychiatric disorders and serve as predictors in CINV and in the medical treatment of psychiatric patients. (Niesler et al 2008).

iii. **Enzymes:** Polymorphism of pharmacokinetic enzymes no doubt influence the pharmacodynamics of drugs. However there are few enzymes that influence drugs at the point of actions one of these enzymes is the tyrosine kinase which modulate receptor activities. Therefore polymorphism in tyrosine kinase gene will affect drugs at the target point.

i. **Receptors:** Many receptors are involved with several signaling pathways. Example of which is epidermal growth factor receptor( EGFR). This receptor has been implicated in the oncogenesis and progression of several solid tumours thereby being identified as a suitable target for anticancer treatment. Polymorphism has been observed in the development of cancer on dinucleotide repeats in intron 1 of the EGFR gene and this has correlated with EGFR expression with therapeutic implication for treatment with tyrosinase kinase inhibitor. A higher proportion of Asians do overexpress EGFR that

**G-protein Coupled Receptors (GPCR**):Over 50% of all drug targets have G-protein coupled receptors (GPCR). Genes of GPR has more coding regions than non – GPCR

**GABAA Receptor** Mutation in GABAA receptor ion channel may be a reason for the

**Insulin Receptor(INSR):** The receptor is important in the management of diabetes mellitus patients and mutation of the gene encoding the receptor will result in poor response particularly in type 2 diabetes.Mutation of the gene has also been suspected to contribute to genetic susceptibility to the polycystic ovarian syndrome(Siega et al2002) **B2** Receptor: B2 agonist; albuterol (Proventil) is used to control acute attacks of asthma

poor asthma control with frequent symptoms and a decreasing scores of poor exploratory volume compared with those with glycine genotype (Cowburn et al 1998, de Maat et al 1999). Seventeen (17%) of whites and 20% of blacks carry the arginine

ii. **Ion Channels:** Many genes encode for different ion channels including those of the central nervous system which include KCNJ10, KCNJ3, CLCN2, GABRA1, SCN1B and SCN1A. Some polymorphism of this channel has been linked to idiopathic generalized

The 5-HT3 receptor is a ligand-gated ion channel composed of five subunits. To date, five different human subunits are known; 5-HT3A-E, which are encoded by the serotonin receptor genes HTR3A, HTR3B, HTR3C, HTR3D and HTR3E, respectively. Functional receptors are pentameric complexes of diverse composition. Different receptor subtypes seem to be involved in chemotheraphy-induced nausea and vomiting (CINV), irritable bowel syndrome and psychiatric disorders. 5-HTR3A and HTR3B polymorphisms may also contribute to the etiology of psychiatric disorders and serve as predictors in CINV

iii. **Enzymes:** Polymorphism of pharmacokinetic enzymes no doubt influence the pharmacodynamics of drugs. However there are few enzymes that influence drugs at the point of actions one of these enzymes is the tyrosine kinase which modulate receptor activities. Therefore polymorphism in tyrosine kinase gene will affect drugs at

and in the medical treatment of psychiatric patients. (Niesler et al 2008).

2 receptor arginine genotype experience

may influence their responses to tyrosine kinase inhibitor (Tan et al 2004).

genes making them more important for pharmacological investigations.

tetraethylammonium bromide (TEA) (Kang et al 2007).

diminished protection of anti epileptic drugs.

and are prescribed as needed .Patients with

genotype (Wechsler et al 2005)

epilepsy (Lucarini et al 2007)

the target point.

**5.2 Pharmacodynamic related genes** 

cation substrate include drugs like metformin, procainamide and cimetidine as well as endogenous compounds like dopamine and norepinephrine and toxic substances like Another important enzyme of drug target is vitamin K epoxide reductase complex subunit 1(VKORC1). This enzyme is the drug target for warfarin an anticoagulant with a narrow therapeutic window and with serious consequences of bleeding in the event of an overdose. Variation in maintenance dose of warfarin is largely attributable to genetic variants in the genes that encode the drug target VKORC1 the major metabolizing enzyme. The two genetic polymorphisms explain 30 – 40% of the total variation in those on therapy.

Angiotensin converting enzyme (ACE) genes encode for ACE, a target for ACE inhibitors which improves symptom and survival in cases of heart failure. Genetic polymorphism is suspected to be causing greater effects of the drug in Europeans than Afro-Americans. Pre-treatment genetic screening is therefore apt to improve therapy

**iv. Neurotransmitter Transporters:** Neurotransmitter transporters namely SLC6, SLC17 and SLC18 families are primarily expressed in the neurons of the central and peripheral nervous system. These transporters are the sites of action of various drugs of abuse e.g cocaine, amphetamine and other clinically approved drugs like desipramine, reserpine, benztropine and tiagabine. Genetic variation in the SLC6, SLC17 and SLC18 encoding genes may result in altered expression and function of these proteins. In particular, antidepressants and antiepileptic drugs target these neurotransmitters as part of their primary mechanism of action. Therefore genetic variations may affect the efficacy of such drugs.

#### **6. Pharmacogenetic testing**

A genetic test is the analysis of human DNA, RNA chromosomes, proteins or certain metabolites in order to detect alterations related to a heritable disorder. This can be accomplished by directly examining the DNA or RNA that makes up a gene (Direct testing), looking at markers co-inherited with a disease causing gene (linkage testing), assaying certain metabolites (biochemical testing), or examining the chromosomes (cytogenetic testing). Although genetic testing shares some features common with other kinds of laboratory testing, it is however unique in many ways and therefore requires special consideration .Pharmacogenetic testing can therefore be defined as utilization of aforementioned genetic biomarkers related to drug metabolism and effects. A biomarker can be described as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes to a therapeutic intervention (EMEA 2006)

Methods of Pharmacogenetic testing depends on the biomarker to be assessed. These vary from simple spectrophotometric estimation of metabolites to DNA sequences, use of PCR and DNA probes, enzymes linked immunosorbent assay, cell culture, gel electrophoresis high performance liquid chromatography and DNA hybridization techniques. It is not uncommon to use combined techniques to study clinical relevance of pharmacogenetic testing.

Because information on pharmacogenetics is still evolving, there is a necessity for guidelines to be adopted for ethical reasons, economic considerations and patient benefit. Overall, the quest for pharmacogenetic information is likely to grow. As a matter of fact some drugs already carry labels addressing such.

Pharmacogenetics: The Scientific Basis 199

There are currently requirements of pharmacogenetic testing of specific drugs before they can be prescribed and these include cetuximab, trastuzumab, maraviroc and dasatinib. In December 2007, the FDA recommended testing for HLA-B\* 1502 allele in patients with Asian ancestry before initiating carbamazepine therapy because of high risk of developing carbamazepine induced Steven's Johnson syndrome (SSS) or toxic epidermal necrolysis.

Pharmacogenetic testing is also recommended for patients treated with warfarin, thiopurine,

Currently, drug labels contain information on pharmacogenetic tests which are classified as

With the application of molecular biology methods and completion of the human genome projects and establishment of guidelines for pharmacogenetics practices and applications, it is expected that the interwoven field of pharnmacogenetics and pharmacogenomics will revoluntionise personalized medicine. Furthermore the field of predictive medicine is expected to receive a boost from pharmacogenetic information with attendant reduction in morbidity and mortality particularly from adverse drug reactions and therapeutic failure. With more intense researches and genotyping profiling, the challenges of standardization and interpretation of pharmacogenetic testing are apt to be overcome. It is worthy of note that currently some drug labels carry information on pharmacogenetic testing and requirements for therapeutic use. The promise of pharmacogenetics is therefore

Ait Moussa L, Khassouni CE, Hue B, Jana M, Begand B, Soulaymani R (2002). Determination

Arruda VR, Lima CS, Grignoll CR, de Melo MB, Lorrand-Metze I, Alberto FL, Saad ST &

Aquilante CL, Langace TY, Lopez Lm (2006) Influence of coagulation factor, vitamin K

Banjoko, S.O. &Akinlade K.S. (2010) Acetylation Pharmacogenetics and renal function in diabetes mellitus patients. *Indian Journal of Clinical Biochemistry, 25(3) 289* Boivin A A, Cardinal H, Barama A, Pichelte V, Hebert M J, Rocher M (2010) Organic Anion

of the acetylator phenotype in Moroccan tuberculosis patients using isoniazid as

Costa FF (2001) Increased risk of acute myeloid leukaemia in individuals with glutathione-S-transferase MU1(GST M1 and theca 19GST T10 gene defects

epoxide reductase complex subuniit 1 and cytochrome P4502C9 gene polymorphisms on warfarin dose requirements. *Clinical Pharmacology and* 

Transporting Polypeptide 1B1 (OATP1B1) and OATP1B3. genetic variability and haplotype analysis of the white Canadians*. Drug Metabolism and Pharmacokinetics 25* 

valproic acid, irinotecan, abacavir or rasburicase.

**7. Conclusion** 

**8. References** 

test required, test recommended and for information only.

improvement of the overall health being of the patients.

metabolic probe *Critical Care Medicine 30 (2) 107* – 14

.*European Journal of Haematology 66(6) 383-8* 

*Therapeutics 79 (4):291 – 302*

*(5): 508 – 515* 

#### **6.1 European medicines agency guideline for pharmacogenetic testing**

The guidelines for European Medicines Agency (EMEA) was desighned by the Agency's committee for Human Medicinal Products (CHMP). The rationale for this guidelines include standardization, data analysis, interpretation, evaluation of clinical relevance, ethical consideration and setting the stage for technical, scientific and regulatory issues. The guidelines addresses the following among other issues.


\*In the study population e.g. matched groups (responders/non responders, presence/absence of adverse events)


#### **6.2 Pharmacogenetic testing and clinical benefits**

The overall purpose of PG testing is clinical benefits. Pharmacogenetic testing have resulted in some clinical benefit so far, some of which can be life saving. It was observed that roughly about 106,000 deaths and 2.2 million serious events caused by adverse drug reactions were reported yearly (Lazarom 1998) and 5 – 7% of hospital admissions in US and Europe lead to the withdrawal of 4% of new medicines with attendant financial loss. Since such drugs were linked to metabolizing enzymes with known polymorphism,prudence dictates suggestion of pharmacogenetic testing in indicated instances Pharmacogenetics testing is expectedly becoming commonly required particularly with drugs with low therapeutic window (Phillips et al 2001). However, the decision to use pharmacogenetic testing will be influenced by the relative costs of genotyping technologies and the cost of providing a treatment to a patient with an incompatible genotype.

Notable clinical benefits of pharmacogenetic testing have been observed in NAT2 genotyping for isoniazid treatment (Hiratsuka et al 2002, Weishilboum et al 2003, Gardiner and Begg 2006) andCYP2C19 genotyping for omeprazole treatment (Desta et al 2002).

Others are TPMT genotyping for 6-mercaptopurine and azathioprine treatment (Relling et al 1999, Gardener and Begg 2006) mtDNA A155G genotyping for aminoglycoside treatment (Cortopassi and Hatchin 1994, Usami et al 1999) CYP 2D6 genotyping for codeine treatment (Bradford 2002) Hepatitis C genotype for pegylated interferon – alpha – 2a or pegylated – interferon – alpha – 2b treatment. (Ingelman – Sundberg et al 2009, Thomas et al 2009) and Dihydropyrimidine dehydrogenese (DPI) testing for 5-fluoro-Uracil (5FU) treatment (Gionzalez and Fernandez –Salguero, 1995, McMurrough et al 1996, Wei et al 1996, Van Kuilenburg et al 1998).

There are currently requirements of pharmacogenetic testing of specific drugs before they can be prescribed and these include cetuximab, trastuzumab, maraviroc and dasatinib. In December 2007, the FDA recommended testing for HLA-B\* 1502 allele in patients with Asian ancestry before initiating carbamazepine therapy because of high risk of developing carbamazepine induced Steven's Johnson syndrome (SSS) or toxic epidermal necrolysis.

Pharmacogenetic testing is also recommended for patients treated with warfarin, thiopurine, valproic acid, irinotecan, abacavir or rasburicase.

Currently, drug labels contain information on pharmacogenetic tests which are classified as test required, test recommended and for information only.
