Pharmacogenetics and drug response

Pharmacogenetics and Drug Response

Author & Course Teacher

MAIZBHA UDDIN AHMEDAssistant Professor

Department of Clinical Pharmacy and PharmacologyFaculty of PharmacyUniversity of Dhaka

Contents

1 Pharmacogenetics 51.1 Pharmacogenetics and Drug Response . . . . . . . . . . . . . . . . . . . . . . 61.2 Pharmacogenetics and Drug safety: Pharmacogenetics of Irinotecan . . . . . . 61.3 Genotype Phenotype correlation: CYP3A4 . . . . . . . . . . . . . . . . . . . . 8

1.3.1 Genotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.2 Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.3 Phenotypic diversity due to heterogeneity of mutations . . . . . . . . . 9

1.4 Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.5 Classification of Metabolizer groups . . . . . . . . . . . . . . . . . . . . . . . 111.6 CYP3A4 genotype and Phenotype . . . . . . . . . . . . . . . . . . . . . . . . 12

1.6.1 CYP3A4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.6.2 CYP3A4 genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.6.3 CYP3A4 phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2 Pharmacogenetics of Warfarin 172.1 Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.1.1 Warfarin Pharmacgology . . . . . . . . . . . . . . . . . . . . . . . . . 172.1.2 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.2 Metabolism of Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.3 Pharmacogenetics of Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.3.1 CYP2C9 and Warfarin dosing . . . . . . . . . . . . . . . . . . . . . . 212.3.2 VKORC1 and Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 212.3.3 CYP4F2 and Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.4 CPIC dosing guideline for Warfarin . . . . . . . . . . . . . . . . . . . . . . . 212.5 References: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Pharmacogenetics of Clopidogrel 233.1 Clopidogrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.1.1 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.1.2 Why it is important to study CYP2C19 polymorphism . . . . . . . . . 24

3.2 Bioactivation of Clopidogrel and Prasugrel . . . . . . . . . . . . . . . . . . . . 243.3 Clopidogrel and CYP2C19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.4 Genetic variation in CYP2C19 influences individual response to clopidogrel . . 253.5 Choosing the best option: Clopidogrel? Prasugrel? or Ticagrelor? . . . . . . . 263.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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CONTENTS 2

4 Pharmacogenetics of NAT2 304.1 NAT localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304.2 Human NAT2 Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304.3 NAT and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314.4 NAT2 and Drug Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4.4.1 NAT2 and Isoniazid . . . . . . . . . . . . . . . . . . . . . . . . . . . 314.4.2 NAT2 and Environmental carcinogen . . . . . . . . . . . . . . . . . . 324.4.3 NAT2 and Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

5 Pharmacogenetics of CYP2D6 355.1 CYP2D6 allelic variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355.2 CYP2D6 and Antipsychotics Pharmacology . . . . . . . . . . . . . . . . . . . 37

5.2.1 Metabolism of Amytriptyline . . . . . . . . . . . . . . . . . . . . . . 385.2.2 Dosing recommendations for amitriptyline/nortriptyline based on CYP2D6

phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385.2.3 Recommended dosing of codeine by CYP2D6 Phenotype . . . . . . . . 40

5.3 CYP2D6 and Tamoxifen: Why endoxifen is a better option than Tamoxifen . . 415.4 Why Endoxifen? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.4.1 Why Endoxifen Treatment may have better therapeutic outcome . . . . 425.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

6 Pharmacogenetics of cancer 466.1 Interindividual variation in Chemotherapeutic drug response . . . . . . . . . . 466.2 Genetic variations affecting drug response and toxicity with cancer chemotherapy 476.3 Polymorphism in Drug-Metabolizing Enzymes . . . . . . . . . . . . . . . . . 48

6.3.1 Thiopurine methyltransferase and 6-Mercaptopurine . . . . . . . . . . 486.3.2 UDP-glucuronosyltransferase 1A1 and Irinotecan . . . . . . . . . . . . 496.3.3 Dihydropyrimidine dehydrogenase and 5-FU . . . . . . . . . . . . . . 506.3.4 MTHFR and Methotrexate . . . . . . . . . . . . . . . . . . . . . . . . 50

6.4 Polymorphisms in Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . 536.4.1 Clinical Annotation for ABCB1 (rs1045642) and 5-fluorouracil . . . . 546.4.2 Clinical Annotation for rs1045642 and paclitaxel . . . . . . . . . . . . 55

6.5 Polymorphisms in Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . 556.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

List of Figures

1.1 Principle of Pharmacogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . 61.2 Pharmacogenetics based Dosing/Diagnostics . . . . . . . . . . . . . . . . . . . 71.3 Metabolism of Irinotecan. Abbreviations: APC, 7-ethyl-10[4-N-(5-aminopentanoic

acid)-1-piperidino]-carbonyloxycamptothecin; CYP3A4, cytochrome P450 3A4enzyme; CYP3A5, cytochrome P450, family 3, subfamily A, polypeptide 5;NPC, 7-ethyl-10[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin; SN-38,7-ethyl-10-hydroxy-camptothecin; SN-38G, glucuronidated SN-38; Topo 1, DNAtopoisomerase I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.4 Irinotecan Metabolizer and Toxicities . . . . . . . . . . . . . . . . . . . . . . 81.5 Genetics of Sickle cell anaemia . . . . . . . . . . . . . . . . . . . . . . . . . . 91.6 Overview of Drug Metabolism and Excretion . . . . . . . . . . . . . . . . . . 101.7 Genotype-Phenotype Association of Drug Metabolism and Pharmacology . . . 111.8 Genotyping protocol of CYP3A4 . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1 Warfarin Metabolism and Mechanism of Action . . . . . . . . . . . . . . . . . 182.2 Warfarin Metabolic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3.1 Comparative metabolic pattern of Clopidogrel and Prasugrel . . . . . . . . . . 253.2 Comparative Pharmacokinetic pathway of Clopidogrel, Prasugrel and Ticagrelor 27

4.1 Metabolism of Isoniazid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

5.1 Metabolism of Amytriptyline . . . . . . . . . . . . . . . . . . . . . . . . . . . 395.2 Tamoxifen metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435.3 Tamoxifen: Interaction with other drugs . . . . . . . . . . . . . . . . . . . . . 44

6.1 Metabolism of 6-Mercaptopurine . . . . . . . . . . . . . . . . . . . . . . . . . 486.2 6-Mercaptopurine dosing according to TPMT genotype . . . . . . . . . . . . . 496.3 5-FU metabolism and Rate limiting role of DPYD enzyme . . . . . . . . . . . 506.4 Illustrates some of the key enzymes involved in metabolism of MTX: RFC,

reduced folate carrier; MTX-PGs, polyglutamated MTX; FPGS, folylpolyglu-tamate synthase; GGH, gamma-glutamyl hydrolase; DHF, dihydrofolate; THF,tetrahydrofolate; TYMS, thymidylate synthase; DHFR, dihydrofolate reduc-tase; MTHFR, methylenetetrahydrofolate reductase; ATIC, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. . . . . . . . . . . . . . . . . . . 52

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List of Tables

1.1 Metabolizer’s Genotype and Phenotype in different CYP450 isoenzymes . . . . 121.2 PCR mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3 PCR condition and PCR size . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.4 Primers for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.1 Enzymatic activity of different CYP2C9 allelic variants . . . . . . . . . . . . . 202.2 Genotype based Dosing guideline by Clinical Pharmacogenetics Implementa-

tion Consortium (CPIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1 Common polymorphisms in the CYP2C19 gene that influence anti-platelet re-sponse to clopidogrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.2 Allelic Frequency of common CYP2C19 variants by ethnicity . . . . . . . . . 26

4.1 Common NAT2 alleles and their phenotype . . . . . . . . . . . . . . . . . . . 34

5.1 CYP2D6 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355.2 CYP2D6 Allelic variants with enzyme function in different ethnic groups . . . 365.3 Genotype and Phenotype of CYP2D6 Alleles . . . . . . . . . . . . . . . . . . 375.4 Metabolizer classification of CYP2D6 . . . . . . . . . . . . . . . . . . . . . . 38

6.1 Examples of genetic variations in major Phase-I and Phase-II enzymes as-sociated with changes in patient responses to chemotherapy agents. Ab-breviations: CYP = cytochrome P450; DPD = dihydropyrimidine dehydroge-nase; GST = glutathione S-transferase; MDR1 = multidrug resistance 1; MRP =multidrug-resistancerelated protein; MTHFR = 5,10-methylenetetrahydrofolatereductase; NAT = N-acetyl transferase; TPMT = thiopurine methyltransferase;TS = thymidylate synthase; UGT1A1 = UDP-glucuronosyltransferase 1A1; 5-FU = 5-fluorouracil; 6-MP = 6-mercaptopurine. . . . . . . . . . . . . . . . . . 47

6.2 Frequency of MTHFR allelic variation among different ethnic groups . . . . . 516.3 Clinical Annotation for Methotrexate in different MTHFR alleles . . . . . . . . 536.4 Clinical Annotation of ABCB1 rs1045642 and 5-FU . . . . . . . . . . . . . . 546.5 Clinical Annotation of ABCB1 rs1045642 and Paclitaxel . . . . . . . . . . . . 55

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Chapter 1

Pharmacogenetics

Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathwayswhich can affect individual responses to drugs, both in terms of therapeutic effect as well asadverse effects.The term pharmacogenetics is often used interchangeably with the term phar-macogenomics which also investigates the role of acquired and inherited genetic differences inrelation to drug response and drug behavior through a systematic examination of genes, geneproducts, and inter- and intra-individual variation in gene expression and function.

In oncology, pharmacogenetics historically is the study of germline mutations (e.g., single-nucleotide polymorphisms affecting genes coding for liver enzymes responsible for drug de-position and pharmacokinetics), whereas pharmacogenomics refers to somatic mutations in tu-moral DNA leading to alteration in drug response (e.g., KRAS mutations in patients treatedwith anti-Her1 biologics).

Pharmacogenetics is a rising concern in clinical oncology, because the therapeutic win-dow of most anticancer drugs is narrow and patients with impaired ability to detoxify drugswill undergo life-threatenting toxicities. In particular, genetic deregulations affecting genescoding for DPD, UGT1A1, TPMT, CDA and Cyp2D6 are now considered as critical issuesfor patients treated with 5-FU/capecitabine, irinotecan, mercaptopurine/azathioprine, gemc-itabine/capecitabine/AraC and tamoxifen, respectively. The decision to use pharmacogenetictechniques is influenced by the relative costs of genotyping technologies and the cost of provid-ing a treatment to a patient with an incompatible genotype. When available, phenotype-basedapproaches proved their usefulness while being cost-effective.

Pharmacogenetics has several advantages:

• The genotype of an individual is essentially invariable and remains unaffected by thetreatment itself.

• Molecular biology techniques provide an accurate assessment of the genotype of an indi-vidual.

• There has been a dramatic increase in the amount of genomic information that is available.This information provides the necessary data for comprehensive studies of individualgenes and broad investigation of genome-wide variation.

• The ease of accessibility to genotype information through peripheral blood or saliva sam-pling and advances in molecular techniques has increased the feasibility of DNA collec-tion and genotyping in large-scale clinical trials.

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CHAPTER 1. PHARMACOGENETICS 6

1.1 Pharmacogenetics and Drug Response1. Dose adaptation:Variability in drug pharmacokinetics resulting from polymorphisms af-

fecting transport and metabolism might be alleviated, in part, by appropriate dose adjust-ments. Dose adjustment according to Pharmacogenetics based Diagnostics (PGDx) fromfields of drug therapy where compensation for differences in individual drug exposurecan be expected to have a beneficial effect on the therapeutic outcome. [Figure 1.1]

Figure 1.1: Principle of Pharmacogenetics

2. Drug selection using PGDx: Polymorphisms in receptors or other targets of a drug canaffect the probability of obtaining efficacy for that drug. It is unlikely that any physicianwould restrict a patient from treatment with a drug because the chance of response mightbe only 40% in carriers of a certain PGx variant, when the average chance is 70% incarriers of the respective wild-type genotype. However, such PGx information might behelpful if alternative medications are available or if a better response might be achievedby a change in dose or by a specific additive medication. [Figure 1.2]

1.2 Pharmacogenetics and Drug safety: Pharmacogenetics ofIrinotecan

Irinotecan is another example of a drug that is metabolized differently in some patients; thesedifferences can predispose a subset of patients to increased drug toxicity. The addition ofirinotecan to the combination of fluorouracil and leucovorin in 1996 represented the first majoradvance in the treatment of colorectal cancer since the late 1950s, when fluorouracil originallybecame the standard of care. Regimens that contained irinotecan resulted in a near-doublingof response rates, from around 20% to 40%, and prolongation of overall survival by up to 3months. These advances, however, came with the risk of significant and unpredictable toxicity.The most common toxicities of irinotecan are acute and delayed diarrhea and neutropenia.


Figure 1.2: Pharmacogenetics based Dosing/Diagnostics

The drug-associated mortality from bolus fluorouracil and leucovorin regimens that con-tained irinotecan was threefold higher than that of regimens that did not contain the drug. Deathoften resulted from drug-related gastrointestinal and vascular syndromes. Differences betweenindividuals with respect to the drug’s metabolism were a rational route of exploration becauseof the noted correlation between active drug concentration and toxicity.

Irinotecan is primarily metabolized by carboxylesterase to the active metabolite, SN-38,which is responsible for inhibition of DNA topoisomerase I. SN-38 is glucuronidated by UGT1A1to the inactive metabolite SN-38G, which is subsequently excreted. Other irinotecan inactiva-tion pathways include oxidation by CYP3A4 and CYP3A5 into APC and NPC, which can alsobe metabolized by carboxylesterase to SN-38. [Figure 1.3]

Patients with the UGT1A1*28 allele have lower glucuronidating ability than patients withwild-type alleles. In the Caucasian population, 917% of individuals are homozygous (7/7)and 2836% are heterozygous (6/7) for the UGT1A1*28 allele. In African-Americans, 1733%and 3850% are homozygous and heterozygous, respectively. This variant allele is seen to alesser extent in the Asian population; only 14% of individuals are homozygous and 1531% areheterozygous for the UGT1A1*28 allele. [Figure 1.4]


Figure 1.3: Metabolism of Irinotecan. Abbreviations: APC, 7-ethyl-10[4-N-(5-aminopentanoicacid)-1-piperidino]-carbonyloxycamptothecin; CYP3A4, cytochrome P450 3A4 enzyme;CYP3A5, cytochrome P450, family 3, subfamily A, polypeptide 5; NPC, 7-ethyl-10[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin; SN-38, 7-ethyl-10-hydroxy-camptothecin;SN-38G, glucuronidated SN-38; Topo 1, DNA topoisomerase I.

Figure 1.4: Irinotecan Metabolizer and Toxicities

1.3 Genotype Phenotype correlation: CYP3A4An organisms genotype is the set of genes that it carries. An organisms phenotype is all of itsobservable characteristicswhich are influenced both by its genotype and by the environment.So in defining evolution, we are really concerned with changes in the genotypes that make upa population from generation to generation. However, since an organisms genotype generallyaffects its phenotype, the phenotypes that make up the population are also likely to change.


1.3.1 GenotypeThis is the ”internally coded, inheritable information” carried by all living organisms. Thisstored information is used as a ”blueprint” or set of instructions for building and maintaining aliving creature. These instructions are found within almost all cells (the ”internal” part), theyare written in a coded language (the genetic code), they are copied at the time of cell divisionor reproduction and are passed from one generation to the next (”inheritable”). These instruc-tions are intimately involved with all aspects of the life of a cell or an organism. They controleverything from the formation of protein macromolecules, to the regulation of metabolism andsynthesis.

1.3.2 PhenotypeThis is the ”outward, physical manifestation” of the organism. These are the physical parts, thesum of the atoms, molecules, macromolecules, cells, structures, metabolism, energy utilization,tissues, organs, reflexes and behaviors; anything that is part of the observable structure, functionor behavior of a living organism.

1.3.3 Phenotypic diversity due to heterogeneity of mutationsMutations that alter the structure of gene products, missense mutations, may give rise to aremarkable diversity of phenotypes associated with mutations at the same gene locus. One ofthe best examples comes from the inherited disorders of haemoglobin.

Figure 1.5: Genetics of Sickle cell anaemia

The structure of human haemoglobin (Hb) changes during embryonic, fetal and adult life.All normal haemoglobins are tetramers of two pairs of dissimilar globin chains. Adult and fetalhaemoglobins have α chains combined with β (HbA α2β2) or γ chains (HbF α2γ2). Over 400structural haemoglobin variants have been identified, many of which cause no clinical disabil-ity. However, some, because the amino acid substitution alters the stability or function of thehaemoglobin molecule, result in a disease phenotype. For example, the substitution of glutamic


acid for valine in the sixth position in the β chain causes the haemoglobin molecules to formlinear stacks in the deoxy configuration which, in turn, causes the red cells to assume a sickledconfiguration. The resulting disease in homozygotes, sickle cell anaemia, is characterized bychronic anaemia and tissue damage due to blockage of the micro-circulation with aggregatesof sickled red cells. Other amino acid substitutions result in instability of the haemoglobinmolecule, which precipitates in the red cells during their life in the circulation. This results indamage to their membranes and increased rigidity, and hence in a variable degree of anaemiadue to their premature destruction.

1.4 Drug MetabolismDrug metabolism also known as xenobiotic metabolism is the biochemical modification of phar-maceutical substances or xenobiotics respectively by living organisms, usually through special-ized enzymatic systems. Drug metabolism often converts lipophilic chemical compounds intomore readily excreted hydrophilic products.

Figure 1.6: Overview of Drug Metabolism and Excretion

In the process of metabolism, drugs will be more water soluble, therefore more accessiblefor renal excretion. During drug metabolism, sometimes toxic compounds are formed. In manycases, metabolism is responsible for the activation of the prodrug. This process can be dividedinto two different types of reaction. Phase-I reactions are: oxydation, reduction and hydrolysis.Phase-II (conjugation) reactions include sulfation, methylation, glucuronidation, acetylation.Both reactions-whose names do not indicate the succession of the reactions - normally make theoriginally lipophilic compound more hydrophilic. The genes coding the proteins responsible forthese processes are usually highly polymorphic. This means that in some cases, the individualresponse after the administration of a specific drug can be attributed to the genetic backgroundof the patient. It also means that in some cases, the knowledge of the patients genetic status


makes individualized therapy possible. Individualized therapy has two major goals: it mighthelp not only in quickly establishing the correct dose of certain drug, but also in avoiding thedangerous, sometimes life-threatening side effects.

1.5 Classification of Metabolizer groupsDrug or other xenobiotics metabolizers can be classified according to their metabolic capacitiesand their genetic make up.

1. Ultra Rapid Metabolizer/Ultra Metabolizer: Ultra-rapid metabolizers have one ormore alleles which result in increased enzyme activity compared to extensive metabo-lizers.

2. Rapid/Extensive Metabolizer: Extensive metabolizers have 2 normally functioning al-leles and therefore have normal enzyme activity.

3. Intermediate Metabolizer: Intermediate metabolizers have one non-functional alleleand one normally functioning allele, and therefore have decreased enzyme activity.

4. Poor Metabolizer: Poor metabolizers have two non-functional alleles and therefore havelittle to no enzyme activity.

Figure 1.7: Genotype-Phenotype Association of Drug Metabolism and Pharmacology


Table 1.1: Metabolizer’s Genotype and Phenotype in different CYP450 isoenzymes

1.6 CYP3A4 genotype and Phenotype

1.6.1 CYP3A4Cytochrome P450 3A4 (abbreviated CYP3A4) (EC 1.14.13.97), is an important enzyme in thebody, mainly found in the liver and in the intestine. Its purpose is to oxidize small foreignorganic molecules (xenobiotics), such as toxins or drugs, so that they can be removed fromthe body. While many drugs are deactivated by CYP3A4, there are also some drugs which areactivated by the enzyme. Some substances, such as grapefruit juice and some drugs, interferewith the action of CYP3A4. These substances will therefore either amplify or weaken the actionof those drugs that are modified by CYP3A4.

CYP3A4 is a member of the cytochrome P450 family of oxidizing enzymes. Several othermembers of this family are also involved in drug metabolism, but CYP3A4 is the most commonand the most versatile one. Like all members of this family, it is a hemoprotein, i.e. a proteincontaining a heme group with an iron atom. In humans, the CYP3A4 protein is encoded bythe CYP3A4 gene. This gene is part of a cluster of cytochrome P450 genes on chromosome7q21.1.

Cytochrome P450 (CYP) enzymes metabolize more than 70% of drugs for clinical use.Among them, CYP3A4 is quantitatively the most important P450 enzyme in adults. It is ex-pressed to a major extent not only in the human liver (95%) but also in the small intestine, thuscontributing to the presystemic and systemic metabolism of 30% of all drugs.

1.6.2 CYP3A4 genotypingGenotyping is normally done by two step gel electrophoresis: a) one after PCR and b) Anotherafter restriction enzyme digestion. A typical PCR mixture contains: forward primer, reverseprimer, dNTP, Taq DNA polymerase, Buffer and a template DNA. PCR condition will be initialdenaturation of 2min at 94o, 35 cycles of 30 sec at 94o, anneling of 30sec at 60o, chain elon-


gation of 30 sec at 72o and final chain elongation for 5min and cooling forever until machinestops. A flowchart of CYP3A4 genotyping is given below:

Figure 1.8: Genotyping protocol of CYP3A4

A typical PCR mixture contains the following reagents:

Table 1.2: PCR mixture

1.6.3 CYP3A4 phenotypingCYP3A is involved in the metabolism of endogenous cortisol to 6β -hydroxy-cortisol. Theurinary excretion of 6β -hydroxycortisol and its ratio to free cortisol reflected its activities. It


Table 1.3: PCR condition and PCR size

Table 1.4: Primers for PCR

has been reported that measuring the urinary morning spot of 6β -hydroxy-cortisol/cortisol ra-tio is a good indication of CYP3A activity. Hsieh et al. reported that, from the 6β -hydroxy-cortisol/cortisol ratio, those withCYP3A4*4, *5and*6 alleles have shown below average CYP3A4activity implying reduced catalytic activity for the corresponding protein variants compared tothose with no mutations shown.

Phenotyping is normally done by measuring the ratio of 6β -hydroxycortisol (6β -OH-CS)and cortisol (CS) in urine by HPLC method. Extensive class group of individual will be repre-sented by higher cortisol metabolite and poor metabolizer will be phenotyped by lower cortisolmetabolite concentration.


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9. The Creative Destruction of Medicine: How the Digital Revolution Will Create BetterHealth Care. New York: Basic Books. 2012. ISBN 0-465-02550-1.

10. Farbstein D, Blum S, Pollak M, Asaf R, Viener HL, Lache O, Asleh R, Miller-LotanR, Barkay I, Star M, Schwartz A, Kalet-Littman S, Ozeri D, Vaya J, Tavori H, VardiM, Laor A, Bucher SE, Anbinder Y, Moskovich D, Abbas N, Perry N, Levy Y, LevyAP (November 2011). ”Vitamin E therapy results in a reduction in HDL function inindividuals with diabetes and the haptoglobin 2-1 genotype”. Atherosclerosis 219 (1):2404. doi:10.1016/j.atherosclerosis.2011.06.005. PMC 3200506. PMID 21722898.

11. Yang CG, Ciccolini J, Blesius A, Dahan L, Bagarry-Liegey D, Brunet C, Varoquaux A,Frances N, Marouani H, Giovanni A, Ferri-Dessens RM, Chefrour M, Favre R, Duffaud F,Seitz JF, Zanaret M, Lacarelle B, Mercier C (January 2011). ”DPD-based adaptive dosingof 5-FU in patients with head and neck cancer: impact on treatment efficacy and toxicity”.Cancer Chemother. Pharmacol. 67 (1): 4956. doi:10.1007/s00280-010-1282-4. PMID20204365.

12. Malhotra AK (2010). ”The state of pharmacogenetics”. Psychiatry Times 27 (4): 3841,62.


13. Gardiner SJ, Begg EJ (September 2006). ”Pharmacogenetics, drug-metabolizing en-zymes, and clinical practice”. Pharmacol. Rev. 58 (3): 52190. doi:10.1124/pr.58.3.6.PMID 16968950.

14. Genetic Science Learning Center. ”Your Doctor’s New Genetic Tools.”. Lern.Genetics.Retrieved 15 April 2012.

15. ”Pharmacogenomics: Personalizing Medicine”. Discovery’s Edge. Mayo Clinic. Re-trieved April 15, 2012.

16. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Heinzen EL, Qiu P, Ber-telsen AH, Muir AJ, Sulkowski M, McHutchison JG, Goldstein DB (September 2009).”Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance”. Na-ture 461 (7262): 399401. doi:10.1038/nature08309. PMID 19684573.

17. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O’Huigin C, Kidd J, Kidd K, KhakooSI, Alexander G, Goedert JJ, Kirk GD, Donfield SM, Rosen HR, Tobler LH, Busch MP,McHutchison JG, Goldstein DB, Carrington M (October 2009). ”Genetic variation inIL28B and spontaneous clearance of hepatitis C virus”. Nature 461 (7265): 798801.doi:10.1038/nature08463. PMC 3172006. PMID 19759533.

18. Frueh FW, Amur S, Mummaneni P, Epstein RS, Aubert RE, DeLuca TM, Verbrugge RR,Burckart GJ, Lesko LJ (August 2008). ”Pharmacogenomic biomarker information in druglabels approved by the United States food and drug administration: prevalence of relateddrug use”. Pharmacotherapy 28 (8): 9928. doi:10.1592/phco.28.8.992. PMID 18657016.

19. Corrigan OP (2011). ”Personalized Medicine in a Consumer Age”. Current Pharmacoge-nomics and Personalized Medicine 9: 168176. doi:10.2174/187569211796957566.

20. Paul NW, Fangerau H (December 2006). ”Why should we bother? Ethical and social is-sues in individualized medicine”. Curr Drug Targets 7 (12): 17217. doi:10.2174/138945006779025428.PMID 17168846.

21. Payne K, Shabaruddin FH (May 2010). ”Cost-effectiveness analysis in pharmacoge-nomics”. Pharmacogenomics 11 (5): 6436. doi:10.2217/pgs.10.45. PMID 20415553.

22. Oxford Nanopore Technologies. 2012. Press releases - News - Oxford Nanopore Tech-nologies. Available from: http://www.nanoporetech.com/news/press-releases/view/39

23. Breckenridge A, Lindpaintner K, Lipton P, McLeod H, Rothstein M, Wallace H (Septem-ber 2004). ”Pharmacogenetics: ethical problems and solutions”. Nat. Rev. Genet. 5 (9):67680. doi:10.1038/nrg1431. PMID 15372090.

24. Corrigan OP (March 2005). ”Pharmacogenetics, ethical issues: review of the NuffieldCouncil on Bioethics Report”. J. Med. Ethics 31 (3): 1448. doi:10.1136/jme.2004.007229.PMC 1734105. PMID 15738433.

Chapter 2

Pharmacogenetics of Warfarin

2.1 WarfarinWarfarin (also known by the brand names Coumadin, Jantoven, Marevan, Uniwarfin) is ananticoagulant normally used in the prevention of thrombosis and thromboembolism, the forma-tion of blood clots in the blood vessels and their migration elsewhere in the body, respectively.Warfarin is commonly but incorrectly referred to as a blood thinner.

Despite its effectiveness, treatment with warfarin has several shortcomings. Many com-monly used medications interact with warfarin, as do some foods (particularly leaf vegetablefoods or ”greens,” since these typically contain large amounts of vitamin K1) and its activityhas to be monitored by blood testing for the international normalized ratio (INR) to ensure anadequate yet safe dose is taken. A high INR predisposes to a high risk of bleeding, while anINR below the therapeutic target indicates the dose of warfarin is insufficient to protect againstthromboembolic events.

2.1.1 Warfarin PharmacgologyWarfarin and related 4-hydroxycoumarin-containing molecules decrease blood coagulation byinhibiting vitamin K epoxide reductase, an enzyme that recycles oxidized vitamin K1 to itsreduced form after it has participated in the carboxylation of several blood coagulation proteins,mainly prothrombin and factor VII. Despite being labeled a vitamin K antagonist, warfarin doesnot antagonize the action of vitamin K1, but rather antagonizes vitamin K1 recycling, depletingactive vitamin K1.

Warfarin is used to decrease the tendency for thrombosis or as secondary prophylaxis (pre-vention of further episodes) in those individuals who have already formed a blood clot (throm-bus). Warfarin treatment can help prevent formation of future blood clots and help reduce therisk of embolism (migration of a thrombus to a spot where it blocks blood supply to a vitalorgan).

Warfarin is best suited for anticoagulation (clot formation inhibition) in areas of slowly run-ning blood (such as in veins and the pooled blood behind artificial and natural valves) and inblood pooled in dysfunctional cardiac atria. Thus, common clinical indications for warfarinuse are atrial fibrillation, the presence of artificial heart valves, deep venous thrombosis, andpulmonary embolism (where the embolized clots first form in veins). Warfarin is also usedin antiphospholipid syndrome. It has been used occasionally after heart attacks (myocardialinfarctions), but is far less effective at preventing new thromboses in coronary arteries. Preven-tion of clotting in arteries is usually undertaken with antiplatelet drugs, which act by a different

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CHAPTER 2. PHARMACOGENETICS OF WARFARIN 18

mechanism from warfarin (which normally has no effect on platelet function).

2.1.2 Mechanism of ActionWarfarin inhibits the vitamin K-dependent synthesis of biologically active forms of the calcium-dependent clotting factors II, VII, IX and X, as well as the regulatory factors protein C, proteinS, and protein Z. Other proteins not involved in blood clotting, such as osteocalcin, or matrixGla protein, may also be affected. The precursors of these factors require carboxylation of their

Figure 2.1: Warfarin Metabolism and Mechanism of Action

glutamic acid residues to allow the coagulation factors to bind to phospholipid surfaces insideblood vessels, on the vascular endothelium. The enzyme that carries out the carboxylation ofglutamic acid is gamma-glutamyl carboxylase. The carboxylation reaction will proceed onlyif the carboxylase enzyme is able to convert a reduced form of vitamin K (vitamin K hydro-quinone) to vitamin K epoxide at the same time. The vitamin K epoxide is in turn recycled backto vitamin K and vitamin K hydroquinone by another enzyme, the vitamin K epoxide reduc-tase (VKOR). Warfarin inhibits epoxide reductase (specifically the VKORC1 subunit), therebydiminishing available vitamin K and vitamin K hydroquinone in the tissues, which inhibits thecarboxylation activity of the glutamyl carboxylase. When this occurs, the coagulation factorsare no longer carboxylated at certain glutamic acid residues, and are incapable of binding tothe endothelial surface of blood vessels, and are thus biologically inactive. As the body’s stores


of previously produced active factors degrade (over several days) and are replaced by inactivefactors, the anticoagulation effect becomes apparent. The coagulation factors are produced,but have decreased functionality due to undercarboxylation; they are collectively referred to asPIVKAs (proteins induced [by] vitamin K absence/antagonism), and individual coagulation fac-tors as PIVKA-number (e.g.PIVKA-II). The end result of warfarin use, therefore, is to diminishblood clotting in the patient.

2.2 Metabolism of WarfarinWarfarin is a natural product and given as racemic mixture of the R and S stereoisomers ofthe drug. S-warfarin is 3-5 times more potent an inhibitor of the vitamin K epoxide reductasecomplex, the target of action, than R-warfarin. The stereoisomers are metabolized by differ-ent phase 1 enzymes; the predominant metabolism of the S isomer is via CYP2C9 whereasmetabolism of R-warfarin is mainly via CYP3A4 with involvement of CYP1A1, CYP1A2,CYP2C8, CYP2C9, CYP2C18 and CYP2C19 as depicted in the Warfarin PharmacokineticsPathway [Figure 2.2]. Phase 2 metabolism of warfarin has not been well studied and is notdepicted in this pathway representation, although it is known that sulfated and glucuronyl con-jugates can be formed. Elimination is predominantly renal however warfarin has been shown tointeract with the ABCB1 transporter in liver.

Figure 2.2: Warfarin Metabolic Pathway

2.3 Pharmacogenetics of WarfarinThe association between warfarin dose and the CYP2C9 and VKORC1genes is now unquestion-able, as evidenced by numerous studies. Single nucleotide polymorphisms in the cytochromeP450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKOR) genes have been shown to have


a significant effect on warfarin dose requirement. Other genes mediating the action of warfarinmake either little or no contribution to dose requirement. Although the polymorphisms in-CYP2C9andVKORC1explain a significant proportion of the interindividual variability in war-farin dose requirement, currently available evidence based on a few small studies relating tothe use of pharmacogenetics-guided dosing in the initiation of warfarin therapy has not shownimproved outcomes in either safety or efficacy of therapy. Better clinical evidence of beneficialeffects on patient outcome, particularly at the extremes of the dose requirements in geographi-cally and ethnically diverse patient populations, is needed before the role of a pharmacogenomicapproach to oral anticoagulation therapy in clinical practice can be established.

Patients carrying CYP2C9 variant alleles had a higher rate of above-range INR values, tooklonger to reach stable dosing, and had a higher risk of serious and life-threatening bleedingevents than patients with the wild-type allele. Similar results were noted in a study of warfarin-treated patients whereCYP2C92 or 3 compound heterozygotes and homozygotes had low war-farin requirements and increased rates of excessive (INR>6.0) anticoagulation and bleedingcom-pared with wild-type patients.

Table 2.1: Enzymatic activity of different CYP2C9 allelic variants


2.3.1 CYP2C9 and Warfarin dosingCYP2C9*1 metabolizes warfarin normally, CYP2C9*2 reduces warfarin metabolism by 30%,and CYP2C9*3 reduces warfarin metabolism by 90%. Because warfarin given to patients with*2 or *3 variants will be metabolized less efficiently, the drug will remain in circulation longer,so lower warfarin doses will be needed to achieve anticoagulation.

2.3.2 VKORC1 and WarfarinIn the VKORC1 1639 (or 3673) SNP, the common G allele is replaced by the A allele. Becausepeople with an A allele (or the ”A haplotype”) produce less VKORC1 than do those with the Gallele (or the ”non-A haplotype”), lower warfarin doses are needed to inhibit VKORC1 and toproduce an anticoagulant effect in carriers of the A allele. The prevalence of these variants alsovaries by race, with 37% of Caucasians and 14% of Africans carrying the A allele.

2.3.3 CYP4F2 and WarfarinRecent genome wide association studies have not only confirmed these observations but alsoidentified a novel association between rs2108622 in CYP4F2 and reduced hepatic CYP4F2,higher levels of hepatic vitamin K, and higher warfarin dose requirements.

2.4 CPIC dosing guideline for WarfarinApproach to pharmacogenetic-based warfarin dosing without access to dosing algorithms: In2007, the FDA modified the warfarin label, stating that CYP2C9 and VKORC1 genotypes maybe useful in determining the optimal initial dose of warfarin. The label was further updated in2010 to include a table describing recommendations for initial dosing ranges for patients withdifferent combinations of CYP2C9 and VKORC1 genotypes. Genetics-based algorithms alsobetter predict warfarin dose than the FDA-approved warfarin label table. Therefore, the use ofpharmacogenetic algorithm-based dosing is recommended when possible, although if electronicmeans for such dosing are not available, the table-based dosing approaches are suggested.

Table 2.2: Genotype based Dosing guideline by Clinical Pharmacogenetics ImplementationConsortium (CPIC)

2.5 References:1. Johnson JA, Gong L, Whirl-Carrillo M, Gage BF, Scott SA, Stein CM, Anderson JL,

Kimmel SE, Lee MT, Pirmohamed M, Wadelius M, Klein TE, Altman RB; Clinical Phar-


macogenetics Implementation Consortium. Clinical Pharmacogenetics ImplementationConsortium Guidelines for CYP2C9 and VKORC1 genotypes and warfarin dosing. ClinPharmacol Ther. 2011 Oct;90(4):625-9. doi: 10.1038/clpt.2011.185. Epub 2011 Sep 7.

2. Holford, NH (December 1986). ”Clinical Pharmacokinetics and Pharmacodynamics ofWarfarin Understanding the Dose-Effect Relationship”. Clinical Pharamacokinetics (SpringerInternational Publishing) 11 (6): 483504. doi:10.2165/00003088-198611060-00005. PMID3542339

3. Holbrook AM, Pereira JA, Labiris R, McDonald H, Douketis JD, Crowther M, Wells PS(May 2005). ”Systematic overview of warfarin and its drug and food interactions”. Arch.Intern. Med. 165 (10): 1095106. doi:10.1001/archinte.165.10.1095. PMID 15911722

4. Ansell J, Hirsh J, Hylek E, et al. (2008). ”Pharmacology and management of the vitaminK antagonists: American College of Chest Physicians evidence-based clinical practiceguidelines (8th Edition)”. Chest 133 (6 Suppl): 160S198S. doi:10.1378/chest.08-0670.PMID 18574265

5. Hirsh J, Fuster V, Ansell J, Halperin JL (2003). ”American Heart Association/AmericanCollege of Cardiology Foundation guide to warfarin therapy”. J. Am. Coll. Cardiol. 41(9): 163352. doi:10.1016/S0735-1097(03)00416-9. PMID 12742309

6. Ansell J, Jacobson A, Levy J, Vller H, Hasenkam JM (March 2005). ”Guidelines for im-plementation of patient self-testing and patient self-management of oral anticoagulation.International consensus guidelines prepared by International Self-Monitoring Associationfor Oral Anticoagulation”. Int. J. Cardiol. 99 (1): 3745. doi:10.1016/j.ijcard.2003.11.008.PMID 15721497

7. Aithal GP, Day CP, Kesteven PJ, Daly AK. 1999. Association of polymorphism in thecytochrome P450 CYP2C9 with warfarin dose requirements and risk of bleeding compli-cations.Lancet353:71719

8. Daly AK, King BP. 2003. Pharmacogenetics of oral anticoagulants.Pharmacogenetics13:24752

9. DAndrea G, DAmbrosio RL, Di Perna P, et al. 2005. A polymorphism in the VKORC1gene is associated with an interindividual variability in the dose-anticoagulant effect ofwarfarin.Blood105:64549

10. Scordo MG, Aklillu E, Yasar U, et al. 2001. Genetic polymorphism of cytochrome P4502C9 in a Caucasian and a black African population.Br. J. Clin. Pharmacol.52:44750

Chapter 3

Pharmacogenetics of Clopidogrel

3.1 ClopidogrelClopidogrel (INN) is an oral, thienopyridine-class antiplatelet agent used to inhibit blood clotsin coronary artery disease, peripheral vascular disease, cerebrovascular disease, and to preventmyocardial infarction (heart attack). It is marketed by Bristol-Myers Squibb and Sanofi underthe trade name Plavix. The drug works by irreversibly inhibiting a receptor called P2Y12,an adenosine diphosphate (ADP) chemoreceptor on platelet cell membranes. Adverse effectsinclude hemorrhage, severe neutropenia, and thrombotic thrombocytopenic purpura.

Clopidogrel is used to prevent myocardial infarction (heart attack) and stroke in people whoare at high risk of these events, including those with a history of myocardial infarction and otherforms of acute coronary syndrome, stroke and those with peripheral artery disease. Treatmentwith clopidogrel or a related drug is recommended by the American Heart Association and theAmerican College of Cardiology for people who:

1. Present for treatment with a myocardial infarction with ST-elevation, including

(a) A loading dose given in advance of percutaneous coronary intervention (PCI), fol-lowed by a full year of treatment for those receiving a vascular stent

(b) A loading dose given in advance of fibrinolytic therapy, continued for at least 14days

2. Present for treatment of a non-ST elevation myocardial infarction (NSTEMI) or unstableangina.

(a) Including a loading dose and maintenance therapy in those receiving PCI and unableto tolerate aspirin therapy

(b) Maintenance therapy for up to 12 months in those at medium to high risk for whicha non-invasive treatment strategy is chosen

3. In those with stable ischemic heart disease,[4] treatment with clopidogrel is described as a”reasonable” option for monotherapy in those who cannot tolerate aspirin, as is treatmentwith clopidogrel in combination with aspirin in certain high risk patients.

3.1.1 PharmacologyClopidogrel is a prodrug, which requires CYP2C19 for its activation. It acts on the ADP recep-tor on platelet cell membranes. The drug specifically and irreversibly inhibits the P2Y12 subtype

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CHAPTER 3. PHARMACOGENETICS OF CLOPIDOGREL 24

of ADP receptor, which is important in activation of platelets and eventual cross-linking by theprotein fibrin. Platelet inhibition can be demonstrated two hours after a single dose of oral clopi-dogrel, but the onset of action is slow, so a loading dose of either 600 or 300 mg is administeredwhen a rapid effect is needed.

3.1.2 Why it is important to study CYP2C19 polymorphism1. Antiplatelet drugs reduce the risk of heart attack, unstable angina, stroke, and cardiovas-

cular death in patients with coronary heart disease by making platelets less likely to formblood clots.

2. Antiplatelet drug does not have its antiplatelet effects until it is metabolized into its activeform by CYP2C19.

3. Study by De Morais et al, 1994 detects the *17 allele as an ultra fast metabolizer ofantiplatelet drugs. A recent report indicates the increased risk for bleeding in individualstreated with clopidogrel who carry the *17 allele.

4. People who have reduced functioning of their CYP2C19 liver enzyme due to inactivatingpolymorphisms in their CYP2C19 gene cannot effectively convert Plavix to its activeform. As a result, Plavix may be less effective in altering platelet activity in those people.These poor metabolizers may not receive the full benefit of Plavix treatment and mayremain at risk for heart attack, stroke, and cardiovascular death.

5. It is estimated that 1.5% to 19.2% of the Asian population are ultra fast metabolizers.

3.2 Bioactivation of Clopidogrel and PrasugrelClopidogrel is a prodrug, and its activation is complex (Figure 3.1). Some 85% of the doseadministered is hydrolyzed in the liver, where it is converted into an inactive metabolite (SR26334); the remainder is metabolized by CYP2C19 and to a lesser degree by CYP1A2 andCYP2B6 to 2-oxo-clopidogrel. Around half of this metabolite, which is also inactive, is hy-drolyzed and converted into an inactive thiolactone, while the other half is metabolized bythe 3A4, 3A5, 2B6, 2C9 and 2C19 isoenzymes, finally producing the active metabolite (R-130964). The fact that a significant proportion of the absorbed drug is wasted (converted toinactive metabolites), the complex activation of the prodrug requiring two oxidation steps, andits heavy dependence on the function of the CYP2C19 isoenzyme (which is involved in the me-tabolization of numerous drugs and whose reduced-function genetic variants have a prevalenceof around 25%), are the main pharmacokinetic factors affecting the variability of response toclopidogrel.

3.3 Clopidogrel and CYP2C19One of the main causes of this clopidogrel nonresponsiveness is interindividual variability in themetabolic activation of clopidogrel. Polymorphisms in the CYP2C19 gene affect clopidogrelpharmacokinetics, causing the CYP2C19 enzyme that converts Plavix to its active form to bemore or less effective. The most common CYP2C19 polymorphisms,*2, *3, and *17, resultin highly variable enzyme activity, and are used to classify individuals as to metabolizer type:


Figure 3.1: Comparative metabolic pattern of Clopidogrel and Prasugrel

poor, intermediate, extensive (normal), or ultrarapid. These polymorphisms may be useful toidentify clopidogrel nonresponders (carrying the *2 or *3 alleles) who may benefit by taking analternative antiplatelet agent, such as prasugrel and ticagrelor, or individuals who may be at riskof bleeding due to clopidogrel overactivity (*17 allele carriers).

A loss-of-function polymorphism in the CYP2C19 gene, known as the CYP2C19*2 allelicvariant, has been associated with higher levels of ADP-induced platelet aggregation values inclopidogrel-treated patients and consequently a higher risk of major adverse cardio vascularevents, including the occurrence of stent thrombosis (ST).

A novel allelic variant, CYP2C19*17, results in an increased enzyme function of CYP2C19because of a mutation (806C>T) in the 5-flanking region of the gene that causes an increasedtranscription of CYP2C19. Such increased transcriptional activity of CYP2C19 may confera rapid metabolization of CYP2C19 substrates, which may lead to an enhanced response toantiplatelet treatment. Although this may improve the prevention of thrombotic events, it alsomay increase the risk of bleeding.

3.4 Genetic variation in CYP2C19 influences individual re-sponse to clopidogrel

The CYP2C19 gene is highly polymorphic with over 25 known variants. Functional polymor-phisms in the CYP2C19 gene, the most common of which are *2, *3, and *17, result in highlyvariable enzyme activity, influencing an individuals ability to activate clopidogrel. These vari-ants cause the CYP2C19 enzyme that converts Plavix to its active form to be more or lesseffective and are used to classify individuals as to metabolizer type: extensive metabolizers


(normal conversion to the active metabolite), intermediate/poor metabolizers (incomplete con-version to the active metabolite), and ultrarapid metabolizers (increased conversion to the activemetabolite).

Table 3.1: Common polymorphisms in the CYP2C19 gene that influence anti-platelet responseto clopidogrel

Approximately 2-4% of Caucasian, 4% of African or African/American, and 14-20% ofAsian populations are poor metabolizers (PM) because these individuals have two loss-of-function alleles and very low CYP2C19 activity. Intermediate metabolizers have one CYP2C19loss-of-function allele. Approximately 30-50% of the population are intermediate metabolizers( 30% of Caucasians, 40% of African-Americans, and more than 55% of Asians).

Table 3.2: Allelic Frequency of common CYP2C19 variants by ethnicity

3.5 Choosing the best option: Clopidogrel? Prasugrel? orTicagrelor?

Prasugrel and ticagrelor are newer antiplatelet drugs that could be an alternative to clopidogrel.Their effect is less variable and fewer ischemic events are associated with their use; however,


more bleeding is related to the use of these drugs. Pharmacogenetic studies are currently con-sidering these drugs. So far, no association has been found.

Prasugrel are found to be a risk factor for intracranial bleeding resulting in strokes in around2.5% population. CYP2C19 variants shows differences in pharmacodynamic and Pharmacoki-netic properties variation of Clopidogrel.

Figure 3.2: Comparative Pharmacokinetic pathway of Clopidogrel, Prasugrel and Ticagrelor

Clopidogrel is activated in a 2-step process mediated by oxidative biotransformation in theliver, in which CYP2C19 and CYP3A have particularly important roles. The parent com-pound clopidogrel, and to a lesser extent 2-oxo-clopidogrel, are both substrates and inhibitorsof CYP1A2, CYP2B6, and CYP2C19. Clopidogrel and 2-oxo-clopidogrel are extensively hy-drolyzed to inactive metabolites, potentially magnifying the effects of CYP2C19 inhibitors and


polymorphisms.Prasugrel is also a pro-drug that requires biotransformation to active metabolites by cy-

tochrome P-450 enzymes, including CYP3A isoforms, CYP2B6, CYP2C9, and CYP2C19.Prasugrel is hydrolyzed to a thiolactone derivative in the intestine and then oxidized to its ac-tive metabolite in both the intestine and the liver. Reduced-function CYP2C19 alleles are notbelieved to have a clinically meaningful effect in prasugrel-treated patients.

Ticagrelor (AZD6140) is an orally active cyclopentyltriazolopyrimidine adenosine triphos-phate analog that reversibly inhibits P2Y12 platelet receptors. Ticagrelor, which is not yetapproved in the United States, is an active compound and is metabolized by CYP3A4 to anactive metabolite. Ticagrelor and its active metabolite are both metabolized and glucuronidatedin the liver before elimination in the urine. Genetic variations in CYP isoenzymes do not appearto affect metabolism of ticagrelor.

3.6 References1. Desta Z, Zhao X, Shin JG, Flockhart DA. Clinical significance of the cytochrome P450

2C19 genetic polymorphism. Clin Pharmacokinet. 2002;41(12):913-958.

2. Campo G, Fileti L, Valgimigli M, et al. Poor response to clopidogrel: current and futureoptions for its management. J Thromb Thrombolysis. 2010;30(3):319-31.

3. Gurbel PA, Tantry US. Selecting optimal antiplatelet therapy based on platelet functionmonitoring in patients with coronary artery disease. Curr Treat Options Cardiovasc Med.2009;11(1):22-32.

4. Kuliczkowski W, Witkowski A, Polonski L, et al. Interindividual variability in the re-sponse to oral antiplatelet drugs: a position paper of the Working Group on antiplateletdrugs resistance appointed by the Section of Cardiovascular Interventions of the PolishCardiac Society, endorsed by the Working Group on Thrombosis of the European Societyof Cardiology. Eur Heart J. 2009;30(4):426-435.

5. Terpening C. Clopidogrel: A pharmacogenomic perspective on its use in coronary arterydisease. Clin Med Insights Cardiol. 2010;4:117-128.

6. Sharma RK, Reddy HK, Singh VN, et al. Aspirin and clopidogrel hyporesponsivenessand nonresponsiveness in patients with coronary artery stenting. Vasc Health Risk Manag.2009;5:965-972.

7. Simon T, Verstuyft C, Mary-Krause M, et al. Genetic determinants of response to clopi-dogrel and cardiovascular events. New Engl J Med. 2009;360(4):363-375.

8. Sofi F, Marcucci R, Gori AM, Abbate R, Gensini GF. Clopidogrel non-responsivenessand risk of cardiovascular morbidity. An updated meta-analysis. Thromb Haemost.2010;103(4):841-848.

9. Hulot J, Bura A, Villard E, Azizi M, et al. P450 2C19 loss-of-function polymorphism.Cytochrome is a major determinant of clopidogrel responsiveness in healthy subjects.Blood. 2006;108(7):2244-2247.


10. Geisler T, Schaeffeler E, Dipporr J, et al. CYP2C19 and nongenetic factors predict poorresponsiveness to clopidogrel loading dose after coronary stent implantation. Pharma-cogenomics. 2008;9(9):1251-1259.

11. Mega JL, Close SL, Wiviott SD, et al. Cytochrome P-450 polymorphisms and responseto clopidogrel. N Engl J Med. 2009;360(4):354-362.

12. http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationfor Patientsand-Providers/ucm203888.htm. (Accessed Jun 8, 2011).

13. Product information for Plavix (Sanofi/Aventis US). Label information, approved Feb2011. (htt p : //www.accessdata. f da.gov/drugsat f dadocs/label/2011/020839s052lbl.)

14. Kazui M, Nishiya Y, Ishizuka T, et al. Identification of the human cytochrome P450enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to itspharmacologically active metabolite. Drug Metab Dispos. 2010;38(1):92-99.

15. Dahl M, Gunes A. Implications of inter-individual differences in clopidogrel metabolism,with Focus on Pharmacogenetics. Pharmaceuticals. 2010;3(4):782-794.

16. Pare G, Mehta SR, Yusuf S, et al. Effects of CYP2C19 genotype on outcomes of clopi-dogrel treatment. N Engl J Med. 2010;363(18):1704-1714.

17. Sim SC, Risinger C, Dahl ML, et al. A common novel CYP2C19 gene variant causesultrarapid drug metabolism relevant for the drug response to proton pump inhibitors andantidepressants. Clin Pharmacol Ther. 2006;79(1):103-113.

18. Li-wan-po A, Girard T, Farndon P, Cooley C, Lithgow J. Pharmacogenetics of CYP2C19:functional and clinical implications of a new variant CYP2C19*17. Br J Clin Pharmacol.2010;69(3):222-230.

19. Shuldiner AR, OConnell JR, Bliden KP, Ghandhi A, et al. Association of cytochromeP450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel ther-apy. JAMA. 2009;302(8):849-858.

20. Human cytochrome P450 allele nomenclature (htt p : //www.cypalleles.ki.se/cyp2c19.htm)

21. Mega, J.L. et al. Reduced-function CYP2C19 genotype and risk of adverse clinical out-comes among patients treated with clopidogrel predominantly for PCI: a meta-analysis.JAMA. 2010;304(16):1821-1830.

22. Mega JL, Hochholzer W, Frelinger AL, Kluk MJ, et al. Dosing clopidogrel based onCYP2C19 genotype and the effect on platelet reactivity in patients with stable cardiovas-cular disease. JAMA. 2011;306(20):2221-2228.

23. Trenk D, Hochholzer W, Fromm MF, Chialda L, et al. Cytochrome P450 2C19 681G¿Apolymorphism and high on-clopidogrel platelet reactivity associated with adverse 1-Yearclinical outcome of elective percutaneous coronary intervention with drug-eluting or bare-metal stents. J Am Coll Cardiol. 2008;51(20):1925-1934.

24. Giusti B, Goria AM, Marcuccia R, et al. Cytochrome P450 2C19 loss-of-function poly-morphism, but not CYP3A4 IVS10 + 12G/A and P2Y12 T744C polymorphisms, is as-sociated with response variability to dual antiplatelet treatment in high-risk vascular pa-tients. Pharmacogenet Genomics. 2007;17:1057-1064.

Chapter 4

Pharmacogenetics of NAT2

The arylamine N-acetyltransferases (NATs) are found in nearly all species from bacteria tohumans. They catalyse the acetyltransfer from acetylcoenzyme A to an aromatic amine, hete-rocyclic amine or hydrazine compound. In humans, acetylation is a major route of biotransfor-mation for many arylamine and hydrazine drugs, as well as for a number of known carcinogenspresent in the diet, cigarette smoke and the environment. The reaction pathway is catalysed bytwo cytoplasmic acetyltransferases (NAT; EC 2.3.1.5), N-acetyltransferase Type I (NAT1) andN-acetyltransferase Type II (NAT2).

The arylamine N-acetyltransferases (NATs) are involved in the metabolism of a variety ofdifferent compounds that we are exposed to on a daily basis. Many drugs and chemicals foundin the environment, such as those in cigarette smoke, car exhaust fumes and in foodstuffs, canbe either detoxified by NATs and eliminated from the body or bioactivated to metabolites thathave the potential to cause toxicity and/or cancer. NATs have been implicated in some adversedrug reactions and as risk factors for several different types of cancers. As a result, the levels ofNATs in the body have important consequences with regard to an individual’s susceptibility tocertain drug-induced toxicities and cancers.

4.1 NAT localizationTwo NAT isoenzymes have been identified in humans, namely NAT1 and NAT2, which are theproducts of distinct genetic loci, designated NAT1 and NAT2, respectively. A related pseu-dogene, NATP1, has also been identified, which contains multiple frameshift and nonsensemutations. The two functional NAT genes share an 87% nucleotide identity, which translatesto an 81% homology at the amino acid level. While the entire transcript of NAT1 is derivedfrom a single exon, that of NAT2 is derived from the protein encoding exon together with a sec-ond noncoding exon of 100 bp located about 8kb upstream of the translation start site. HumanNAT1 and NAT2, as well as NATP1, have been localised to the short arm of chromosome 8,more specifically in region 8p22. The NAT loci are separated by only 170-360kb and are in theorientation NAT1→NATP1→NAT2.

4.2 Human NAT2 AllelesSince the human NAT2 locus was established as the site of the classical acetylation polymor-phism, the study of NAT2 allelic variation has been an area of intense investigation. To date, 29different NAT2 alleles have been detected in human populations. Each of the variant alleles is

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CHAPTER 4. PHARMACOGENETICS OF NAT2 31

comprised of between one and four nucleotide substitutions, of which 13 have been identified,located in the protein encoding region of the gene.

Nine of these lead to a change in the encoded amino acid (C190T, G191A, T341C, A434C,G499A, G590A, A803G, A845C, and G857A), while the remaining four are silent (T111C,C282T, C481T, and C759T).

Several studies have been performed that show clear correlations between NAT2 genotypeand phenotype. Early genotyping studies screened for the presence of the C481T (M1), theG590A (M2), the G857A (M3) and sometimes the G191A (M4) nucleotide changes, all ofwhich were shown to cause a slow acetylation phenotype. Moreover, there was a gene-dosageeffect. Individuals who were homozygous for NAT2 polymorphisms had a slow acetylatorphenotype, individuals heterozygous for NAT2 polymorphisms had an intermediate acetylatorphenotype, and individuals who lacked NAT2 polymorphisms had a rapid acetylator phenotype.It should be noted that the method of detection of the above polymorphisms only identifies asubset of the variant alleles found in human populations, and there is potential for the misclas-sification of genotype and deduced phenotypes.

4.3 NAT and DiseaseThe association between acetylator status and the risk of various diseases has been extensivelyreported, and reviewed in detail. Altered risk with either the slow or rapid phenotype has beenobserved for bladder, colon and breast cancer, systemic lupus erythematosis, diabetes, Gilbert’sdisease, Parkinson’s disease and Alzheimer’s disease. These associations imply a role for envi-ronmental factors that are metabolised by the NATs, in particular NAT2, in each disorder. How-ever, identifying those factors has remained elusive. Humans are exposed to many toxic NATsubstrates including the food-derived heterocyclics present in the diet as well as arylamines suchas 4-aminobiphenyl and -naphthylamine present in tobacco smoke. Moreover, occupational ex-posure to arylamine carcinogens such as benzidine has also been reported.

Because of the role of acetylation in the metabolic activation and detoxification of ary-lamine and heterocyclic carcinogens, acetylator status and cancer risk has been widely investi-gated. Unlike the relatively rare but highly penetrant genes involved in familial cancers, thosegenes responsible for metabolic polymorphisms have low penetrance and cause only a moder-ate increase in cancer risk. Nevertheless, their widespread occurrence in the general populationsuggests they are a significant contributor to individual risk.

When acetylator phenotype has been linked to carcinogen exposure, more consistent resultshave been reported. For example, the rapid phenotype has emerged as a strong risk factor forcolorectal cancer in those individuals who have a higher exposure to the food-derived hete-rocyclic amines. Recently, the NAT2 acetylator phenotype has been linked to increased riskassociated with neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s dis-ease.

4.4 NAT2 and Drug Response

4.4.1 NAT2 and IsoniazidThe genetic polymorphism in N-acetyltransferase activity was first discovered in patients treatedwith isoniazid for tuberculosis. This drug is primarily excreted following acetylation catalysedby NAT2.


Isoniazid (INH), a key drug of antituberculosis therapy, is metabolized by arylamine N-acetyltransferase2 (NAT2), cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GST).The metabolism of INH into acetyl isoniazid (AcINH) by N-acetyltransferase 2 (NAT2), whichis estimated to account for 50-90% of INH metabolism. A portion of AcINH is further con-verted to acetyl hydrazine (AcHz) by amidase-catalyzed hydrolysis. There is another amidase-catalyzed route where INH is directly hydrolyzed to yield hydrazine (Hz). NAT2 then catalyzesthe acetylation of Hz to yield AcHz and subsequently to diacetyl hydrazine before excretion.AcHz can also enter the cytochrome P450 2E1 (CYP2E1)-mediated metabolic pathway in theliver, which is linked to the glutathione-S-transferase (GST)-mediated metabolic pathway, tobecome an excretable metabolite.

Figure 4.1: Metabolism of Isoniazid

NAT2 genetic polymorphisms and the resulting variation in acetylation capacity of NAT2are implicated in the risk of INH-induced hepatotoxicity. Slow acetylator genotype of NAT2will activate the CYP2E1 pathway resulting in reactive metabolite, hence will cause hepatotox-icity (Hepatic Necrosis). But there are some contradictory result also, like RA (rapid or fastacetylator) genotype also have a significant role in INH induced hepatotoxicity. Hydrazine andN-actyl Hydrazine are supposed to be hepatotoxic so are RA groups. So it is necessary to con-sider the role of CYP2E1 and GST in INH metabolism to draw a conclusion here without anyconflict.

4.4.2 NAT2 and Environmental carcinogenBiological Amines are not reactive and converted into a highly electrophilic reactive metabolite,acts as a carcinogen and later detoxified by the action of NAT2 enzymes into an excretable non-


reactive form. Slow-acetylator phenotype should have a higher risk of developing cancer bythis biological amine than in fast-acetylator phenotype.

4.4.3 NAT2 and ProcainamideProcainamide is a class Ia antiarrhythmic used for the treatment of several different arrhyth-mias. It is metabolized by n-acetyltransferases (NAT) to n-acetylprocainamide (NAPA), an ac-tive metabolite, which also possesses antiarrhythmic properties. NAT1 is consistently expressedin post patients; however, NAT2 is variably expressed and plays a major role in the productionof NAPA. The majority of patients reveiving procainamide therapy will develop autoantibodiesover time and possibly drug induced lupus. An early study showed that subjects who had aslow-acteylator phenotype developed antinuclear antibodies earlier than rapid acetylators. Thiswas later confirmed in a study of subjects who received long term procainamide therapy. The-oretically patients with NAT2 genotype associated with rapid-acetylation may have increasedNAPA concentrations and thus increased anti-arrhythmic effects and possible excessive QTprolongation.

4.5 References1. D W Hein. N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplo-

type on urinary bladder cancer risk. Oncogene (2006) 25, 16491658. doi:10.1038/sj.onc.1209374

2. Alexandra King. NAT2 and GSTM1 polymorphisms affect the risk of bladder cancer.Nature Reviews Clinical Oncology 2, 540 (November 2005) — doi:10.1038/ncponc0307

3. T SOTSUKA, Y SASAKI, S HIRAI, F YAMAGISHI and K UENO. Association ofIsoniazid-metabolizing Enzyme Genotypes and Isoniazid-induced Hepatotoxicity in Tu-berculosis Patients. In Vivo. 2011. 25(5): 803-812.

4. Evans DA (1989). ”N-acetyltransferase”. Pharmacology & Therapeutics 42 (2): 157234.doi:10.1016/0163-7258(89)90036-3. PMID 2664821.


Table 4.1: Common NAT2 alleles and their phenotype

Chapter 5

Pharmacogenetics of CYP2D6

5.1 CYP2D6 allelic variantsMore than 50 human CYP isozymes have been identified to date. Of these, more than 20 areencoded by genes that are functionally polymorphic, including CYP2A6, CYP2C9, CYP2C19,and CYP2D6. Consequently, approximately 40% of CYP-dependent drug metabolism is carriedout by polymorphic enzymes.

Table 5.1: CYP2D6 Substrate

CYP2D6 is the best characterized of these polymorphic CYP isozymes, with more than 75allelic variants currently identified. These variants result from point mutations, deletions oradditions, gene rearrangements, and deletion or duplication of the entire gene, and result in anincrease, reduction, or complete loss of activity. While it accounts for only 2%5% of all hepaticCYP isozymes, CYP2D6 metabolizes approximately 25% of all clinically used medications,including some cytotoxics, tamoxifen, and many agents used to treat associated complicationssuch as antiarrythmics, antiemetics, antidepressants, antipsychotics, and analgesics. Since theidentification of the CYP2D6 polymorphism in the 1970s, several studies have shown that thefrequencies of the alternative CYP2D6 phenotypes vary significantly among different ethnic

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CHAPTER 5. PHARMACOGENETICS OF CYP2D6 36

groups and that such polymorphisms may play a role in the induction of adverse effects fromadministration of some therapeutic agents. Variation in CYP2D6 expression is also thoughtto increase the potential for drugdrug interactions, an important implication because of theincreasing number of drugs being prescribed. For example, in the oncology setting, patientswith advanced cancer receiving palliative therapy receive an average of five or more medicationsfor symptom relief.

Table 5.2: CYP2D6 Allelic variants with enzyme function in different ethnic groups

A number of specific polymorphisms have been found in the CYP2D6 gene that results inenzymatic deficiencies. The frequency of these polymorphisms varies within the major ethnicgroups. CYP2D6 polymorphisms that produce poor metabolizers are found with frequencies of7% to 10% in Caucasians, 2% in Africans and African Americans, and 1% in Asians. Identifica-tion of patient CYP2D6 genotypes can help clinicians individualize drug treatment by selectionof appropriate therapies. These measures may improve patient outcome by ensuring maximumdrug efficacy with minimal adverse drug reactions (ADR).


Table 5.3: Genotype and Phenotype of CYP2D6 Alleles

CYP2D6 polymorphisms can be classified according to one of four levels of activity: poormetabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ul-trarapid metabolizers (UMs) [Figure 5.4]. The EM phenotype is expressed by the majority ofthe population and is therefore considered the norm. PMs inherit two deficient CYP2D6 allelesand, as a result, metabolize drugs at a notably slower rate. This leads to an accumulation of highlevels of unmetabolized drugs that are CYP2D6 substrates, a concomitant greater potential foradverse events and drugdrug interactions, and lower efficacy for drugs requiring CYP2D6 acti-vation. The UM phenotype is caused by the duplication, multiduplication, or amplification ofactive CYP2D6 genes, including primarily the CYP2D6*2 allele, but also involving CYP2D6*1and others. Individuals with the UM phenotype metabolize drugs at an ultrarapid rate, whichmay lead to a loss of therapeutic efficacy at standard doses. Individuals who are heterozygousfor a defective CYP2D6 allele often demonstrate an IM phenotype. This phenotype has a widespectrum of metabolic activity that can range from marginally better than the PM phenotype toactivity that is close to that of the EM phenotype.

5.2 CYP2D6 and Antipsychotics PharmacologyCYP2D6 is described as the most relevant enzyme in the metabolism of many antipsychoticdrugs. Its contribution to the interindividual differences in drug response is reviewed here high-lighting its role in the kinetics of antipsychotic drugs and the occurrence of drug interactions.The activity of CYP2D6 is inherited as a monogenetic trait and the CYP2D6 gene appearshighly polymorphic in humans. The polymorphic alleles may lead to altered activity of the


Table 5.4: Metabolizer classification of CYP2D6

CYP enzymes causing absent, decreased (poor), or increased (ultrarapid) metabolism that inturn influence the disposition of the antipsychotic drugs. Antipsychotic drug biotransformationis mainly determined by genetic factors mediating CYP2D6 gene polymorphism, however theimportance of environmental factors (dietary, smoking, diseases, etc.) is also recognized. Ad-ditionally, the potential interaction between CYP2D6 and the endogenous metabolism must betaken into consideration.

5.2.1 Metabolism of AmytriptylineAmytriptyline (AT) is metabolized mainly by demethylation forming nortriptyline (NT), and byhydroxylation, leading to the formation of E-10-hydroxy (EHAT) and Z-10-hydroxyamitriptyline(ZHAT). NT is further demethylated to desmethylnortriptyline (NNT) and hydroxylated to E-10-hydroxy (EHNT) and Z-10-hydroxynortriptyline (ZHNT). The demethylation of AT and NTis mainly catalyzed by CYP2C19, with the participation of other CYP enzyme forms in higherdrug concentrations. The formation of the E-10-hydroxy metabolites is dependent on the activ-ity of CYP2D6, with stereospecificity to the (-)-EHAT and (-)-EHNT metabolites.

5.2.2 Dosing recommendations for amitriptyline/nortriptyline based onCYP2D6 phenotype

1. Likely phenotype: Ultrarapid metabolizer ( 1-2% of patients)


Figure 5.1: Metabolism of Amytriptyline

(a) Genotypes: An individual carrying more than two copies of functional alleles.

(b) Examples of diplotypes: *1/*1xN, *1/*2xN

(c) Implications for TCA metabolism: Increased metabolism of tricyclics to less ac-tive compounds when compared to extensive metabolizers. Lower plasma concen-trations will increase probability of pharmacotherapy failure.

(d) Therapeutic Recommendations: Avoid tricyclic use due to potential lack of effi-cacy. Consider alternative drug not metabolized by CYP2D6. If a tricyclic is war-ranted, consider increasing the starting dose.a Utilize therapeutic drug monitoringto guide dose adjustments.

(e) Classification of recommendation for amitriptyline/nortriptyline therapy: Strong

2. Likely phenotype: Extensive metabolizer ( 77-92% of patients)

(a) Genotypes: An individual carrying two alleles encoding full or reduced functionor one full function allele together with either one nonfunctional or one reduced-function allele.

(b) Examples of diplotypes: *1/*1, *1/*2, *2/*2, *1/*41, *1/*4, *2/*5, *10/*10

(c) Implications for TCA metabolism: Normal metabolism of tricyclics.


(d) Therapeutic Recommendations: Initiate therapy with recommended starting dose.


3. Likely phenotype: Intermediate metabolizer ( 2-11% of patients)

(a) Genotypes: An individual carrying one reduced and one nonfunctional allele,

(b) Examples of diplotypes: *4/*10, *5/*41

(c) Implications for TCA metabolism: Reduced metabolism of tricyclics to less ac-tive compounds when compared to extensive metabolizers. Higher plasma concen-trations will increase the probability of side effects.

(d) Therapeutic Recommendations: Consider 25% reduction of recommended start-ing dose. Utilize therapeutic drug monitoring to guide dose adjustments.

(e) Classification of recommendation for amitriptyline/nortriptyline therapy: Mod-erate

4. Likely phenotype: Poor metabolizer ( 5-10% of patients)

(a) Genotypes: An individual carrying no functional alleles.

(b) Examples of diplotypes: *4/*4, *4/*5, *5/*5, *4/*6

(c) Implications for TCA metabolism: Greatly reduced metabolism of tricyclics toless active compounds when compared to extensive metabolizers. Higher plasmaconcentrations will increase the probability of side effects.

(d) Therapeutic Recommendations: Avoid tricyclic use due to potential for side ef-fects. Consider alternative drug not metabolized by CYP2D6. If a tricyclic is war-ranted, consider 50% reduction of recommended starting dose. Utilize therapeuticdrug monitoring to guide dose adjustments.


5.2.3 Recommended dosing of codeine by CYP2D6 Phenotype1. Likely phenotype: Ultrarapid metabolizer ( 1-2% of patients)

(a) Genotypes: An individual carrying more than two copies of functional alleles.

(b) Examples of diplotypes: *1/*1xN, *1/*2xN

(c) Implications for codeine metabolism: Increased formation of morphine followingcodeine administration, leading to higher risk of toxicity.

(d) Therapeutic Recommendations: Avoid codeine use due to potential for toxicity.

(e) Classification of recommendation for codeine therapy: Strong

(f) Considerations for alternative opioids: Alternatives that are not affected by thisCYP2D6 phenotype include morphine and non-opioid analgesics. Tramadol, and toa lesser extent hydrocodone and oxycodone, are not good alternatives because theirmetabolism is affected by CYP2D6 activity.

2. Likely phenotype: Extensive metabolizer ( 77-92% of patients)


(a) Genotypes: An individual carrying two alleles encoding full or reduced functionor one full function allele together with either one nonfunctional or one reduced-function allele.

(b) Examples of diplotypes: *1/*1, *1/*2, *2/*2, *1/*41, *1/*4, *2/*5, *10/*10

(c) Implications for codeine metabolism: Normal morphine formation

(d) Therapeutic Recommendations: Use label recommended age- or weight-specificdosing.

(e) Classification of recommendation for codeine therapy: Strong

(f) Considerations for alternative opioids: Not required.

3. Likely phenotype: Intermediate metabolizer ( 2-11% of patients)

(a) Genotypes: An individual carrying one reduced and one nonfunctional allele.

(b) Examples of diplotypes: *4/*10, *5/*41

(c) Implications for codeine metabolism: Reduced morphine formation.

(d) Therapeutic Recommendations: Use label recommended age- or weight-specificdosing. If no response, consider alternative analgesics such as morphine or a non-opioid.

(e) Classification of recommendation for codeine therapy: Moderate

(f) Considerations for alternative opioids: Monitor tramadol use for response.

4. Likely phenotype: Poor metabolizer ( 5-10% of patients)

(a) Genotypes: An individual carrying no functional alleles.

(b) Examples of diplotypes: *4/*4, *4/*5, *5/*5, *4/*6

(c) Implications for codeine metabolism: Greatly reduced morphine formation fol-lowing codeine administration, leading to insufficient pain relief.

(d) Therapeutic Recommendations: Avoid codeine use due to lack of efficacy.

(e) Classification of recommendation for codeine therapy: Strong.

(f) Considerations for alternative opioids: Alternatives that are not affected by thisCYP2D6 phenotype include morphine and non-opioid analgesics. Tramadol, and toa lesser extent hydrocodone and oxycodone, are not good alternatives because theirmetabolism is affected by CYP2D6 activity; these agents should be avoided.

5.3 CYP2D6 and Tamoxifen: Why endoxifen is a better op-tion than Tamoxifen

The selective estrogen receptor modulator (SERM) tamoxifen has been used for many years asendocrine treatment for hormone receptorpositive breast cancer, with indications in the metastatic,adjuvant, and preventative settings. The minimal acute toxicity of endocrine treatments such astamoxifen compared with chemotherapy helps maintain patients quality of life, and increasingemphasis is being placed on these treatments to delay the use of cytotoxic therapies for as longas possible.


Tamoxifens adverse-event profile includes venous thrombosis, endometrial cancer, and,most commonly, hot flashes, which are often treated with an SSRI antidepressant. Tamoxifenis extensively hepatically metabolized to several primary and secondary metabolites by multi-ple CYP enzymes, including CYP2D6. With some SSRIs (e.g., paroxetine) known to inhibitCYP2D6, a study was recently conducted to investigate the effects of SSRI coadministration onplasma concentrations of tamoxifen and its metabolites in addition to the effects of individualpatient genotypes. A strong association was shown between CYP2D6 activity and plasma levelsof the active tamoxifen metabolite, endoxifen. Concomitant use of paroxetine was associatedwith lower endoxifen plasma concentrations, with the magnitude of this difference being de-pendent on CYP2D6 genotype. It is possible, therefore, that patients taking some SSRIs maybe less responsive to tamoxifen because of a lower rate of formation of its active metabolite,which may be exacerbated in individuals with slowed CYP2D6 metabolism. The possibilitythat tamoxifens antitumoral activity could be affected in this way needs further testing in clini-cal trials designed to record clinical outcome in conjunction with genotype and coadministeredmedication with the potential to affect CYP2D6.

5.4 Why Endoxifen?Endoxifen is an active metabolite of Tamoxifen, a widely used breast cancer drug. Treat-ment of breast cancer directly with Endoxifen may eliminate Tamoxifen metabolism associ-ated variability and avoid a potential serious drug-drug interaction. Upon oral administration,tamoxifen (TAM) is extensively metabolized by cytochrome P450(CYP) enzymes into activemetabolites including 4-hydroxy tamoxifen and endoxifen (4-hydroxy N-desmethyl Tamox-ifen) Endoxifen is one hundred times more potent than TAM and is generated via CYP3A4/5-mediated N-demethylation and CYP2D6-mediated hydroxylation. Due to genetic polymor-phism of CYP2D6, there are large variations among patients in both the therapeutic efficacyand side effects. Furthermore, CYP2D6 interacts with specific selective serotonin reuptake in-hibitors (SSRIs), frequently used to prevent the hot flushes experienced by up to 45% of patientstaking TAM. As a consequence, significant numbers of women might not receive optimal bene-fit from TAM treatment. Our pre-clinical studies have validated the concept of using Endoxifenfor the treatment of breast cancer (Ahmad et al. 2010). Treatment of breast cancer directly withEndoxifen may eliminate TAM metabolism associated variability and avoid a potential seriousdrug-drug interaction.

5.4.1 Why Endoxifen Treatment may have better therapeutic outcome• Endoxifen may overcome the unpredictable therapeutic outcome due to pharmacoge-

nomic variation of Tamoxifen and Aromatase inhibitors

• Endoxifen would help women taking anti-depressants who have problems metabolizingTamoxifen

• Endoxifen acts directly at the target

• Tamoxifen have been shown to be more effective than Aromatase inhibitors in patientswith wild type CYP2D6

• Showed enhanced therapeutic efficacy against established human tumor model in mice


Figure 5.2: Tamoxifen metabolism

• Only potential drug for ER positive premenopausal women with CYP2D6 deficiency

5.5 References1. Wang B, Yang LP, Zhang XZ, Huang SQ, Bartlam M, Zhou SF (2009). ”New insights into

the structural characteristics and functional relevance of the human cytochrome P450 2D6enzyme”. Drug Metab. Rev. 41 (4): 573643. doi:10.1080/03602530903118729. PMID19645588.

2. Teh LK, Bertilsson L (2012). ”Pharmacogenomics of CYP2D6: molecular genetics, in-terethnic differences and clinical importance”. Drug Metab. Pharmacokinet. 27 (1):


Figure 5.3: Tamoxifen: Interaction with other drugs

5567. doi:10.2133/dmpk.DMPK-11-RV-121. PMID 22185816.

3. Walko CM, McLeod H (April 2012). ”Use of CYP2D6 genotyping in practice: tamox-ifen dose adjustment”. Pharmacogenomics 13 (6): 6917. doi:10.2217/pgs.12.27. PMID22515611.

4. ”Entrez Gene: CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6”.

5. Ostille DO., Warren AM., Kulkarni, J. The role of pharmacogenomic testing in psychia-try. Aust New Zealand J Psychiatry 2014 Jan 10. PMID 24413808

6. Bertilsson L, Dahl ML, Daln P, Al-Shurbaji A (February 2002). ”Molecular genetics ofCYP2D6: clinical relevance with focus on psychotropic drugs”. Br J Clin Pharmacol 53(2): 11122. doi:10.1046/j.0306-5251.2001.01548.x. PMC 1874287. PMID 11851634.


7. Llerena A, Dorado P, Peas-Lled EM (January 2009). ”Pharmacogenetics of debrisoquineand its use as a marker for CYP2D6 hydroxylation capacity”. Pharmacogenomics 10 (1):1728. doi:10.2217/14622416.10.1.17. PMID 19102711.

8. Lynch T, Price A (August 2007). ”The effect of cytochrome P450 metabolism on drugresponse, interactions, and adverse effects”. Am Fam Physician 76 (3): 3916. PMID17708140.

9. Droll K, Bruce-Mensah K, Otton SV, Gaedigk A, Sellers EM, Tyndale RF (1998). ”Com-parison of three CYP2D6 probe substrates and genotype in Ghanaians, Chinese and Cau-casians”. Pharmacogenetics 8 (4): 32533. doi:10.1097/00008571-199808000-00006.PMID 9731719.

10. Gaedigk A, Bradford LD, Marcucci KA, Leeder JS (2002). ”Unique CYP2D6 activitydistribution and genotype-phenotype discordance in black Americans”. Clin. Pharmacol.Ther. 72 (1): 7689. doi:10.1067/mcp.2002.125783. PMID 12152006.

11. McLellan RA, Oscarson M, Seidegrd J, Evans DA, Ingelman-Sundberg M (June 1997).”Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians”. Pharmacogenet-ics 7 (3): 18791. doi:10.1097/00008571-199706000-00003. PMID 9241658.

12. PHARMACOGENETICS AND PHARMACOGENOMICS. J. Steven Leeder PharmD,PhD Pediatric Clinics of North America - Volume 48, Issue 3 (June 2001). doi:10.1016/S0031-3955%2805%2970338-2.

13. Hoskins JM, Carey LA, McLeod HL (August 2009). ”CYP2D6 and tamoxifen: DNAmatters in breast cancer”. Nat. Rev. Cancer 9 (8): 57686. doi:10.1038/nrc2683. PMID19629072.

14. Foster BC, Sockovie ER, Vandenhoek S, Bellefeuille N, Drouin CE, Krantis A, BudzinskiJW, Livesey J, and Arnason JT (2004). ”In Vitro Activity of St. John’s Wort Against Cy-tochrome P450 Isozymes and P-Glycoprotein”. Pharmaceutical Biology 42 (2): 159169.doi:10.1080/13880200490512034.

Chapter 6

Pharmacogenetics of cancer

Advances in molecular biology and genetics over the past 60 years have facilitated develop-ment of multiple chemotherapeutic agents that are active against most common malignancies.However, significant heterogeneity in the efficacy and toxicity of these agents is consistently ob-served across human populations. Ad-ministration of the same dose of a given anticancer drugto a population of patients can result in any-where from minimal toxicity to severe, potentiallylife-threatening events. Many clinical factors are associated with drug toxicity, but genetic dif-ferences, or polymorphisms, in the proteins affecting drug disposition also have a great impact.In addition, the enzymes that metabolize the majority of chemotherapy agents contain geneticpolymorphisms, but prospective identification of patients likely to be harmed by chemotherapycurrently is not possible for most therapeutic regimens.

6.1 Interindividual variation in Chemotherapeutic drug re-sponse

Interindividual differences in tumor response and normal tissue toxicities are consistently ob-served with most chemotherapeutic agents or regimens. While many clinical variables havebeen associated with drug responses (e.g., age, gender, diet, drug-drug interactions), inheritedvariations in drug disposition (metabolism and transport) genes and drug target genes also likelycon-tribute to the observed variability in cancer treatment outcome.

Pharmacogenomic studies aim to elucidate the genetic bases for interindividual differencesand to use such genetic information to predict the safety, toxicity, and/or efficacy of drugs. Thereexist several clinically relevant examples of the utility of pharmacogenomics that associate spe-cific genetic polymorphisms in drug metabolizing enzymes (e.g., TPMT, UGT1A1, DPD), drugtransporters (MDR1), and drug target enzymes (TS) with clinical outcomes in patients treatedwith commonly prescribed chemotherapy drugs, such as 5-fluorouracil and irinotecan. Tech-niques to discover and evaluate the functional significance of these polymorphisms have evolvedin recent years and may soon be applied to clinical practice and clinical trials of currently pre-scribed anticancer drugs as well as new therapeutic agents.

Clearly, a better understanding of the genetic determinants of chemotherapeutic responsewill enable prospective identification of patients at risk for severe toxicity or those most likelyto benefit from a particular treatment regimen. Such studies can be translated to clinical practicevia molecular diagnostics (genotyping) in order to guide selection of the optimal drug combina-tion and dosage for the individual patient. A number of detailed reviews on cancer pharmacoge-nomics have been published recently. This article focuses on the current and future applications

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CHAPTER 6. PHARMACOGENETICS OF CANCER 47

of pharmacogenomics in clinical cancer therapy and cancer drug development.

6.2 Genetic variations affecting drug response and toxicitywith cancer chemotherapy

Table 6.1: Examples of genetic variations in major Phase-I and Phase-II enzymes asso-ciated with changes in patient responses to chemotherapy agents. Abbreviations: CYP =cytochrome P450; DPD = dihydropyrimidine dehydrogenase; GST = glutathione S-transferase;MDR1 = multidrug resistance 1; MRP = multidrug-resistancerelated protein; MTHFR = 5,10-methylenetetrahydrofolate reductase; NAT = N-acetyl transferase; TPMT = thiopurine methyl-transferase; TS = thymidylate synthase; UGT1A1 = UDP-glucuronosyltransferase 1A1; 5-FU= 5-fluorouracil; 6-MP = 6-mercaptopurine.

Pharmacogenomic approaches have been applied to many existing chemotherapy agents inan effort to identify relevant inherited variations that may better predict patient response tochemotherapy. Genetic variations include nucleotide repeats, insertions, deletions, and singlenucleotide polymorphisms (SNPs), which can alter the amino acid sequence of the encoded pro-teins, RNA splicing, and gene transcription. Such genetic polymorphisms in drug-metabolizing


enzymes, transporters, and molecular targets have been actively explored with regard to func-tional changes in phenotype (altered expression levels and/or activity of the encoded proteins)and their contribution to variable drug response. Recent studies also indicate that genetic varia-tions vary substantially among different ethnic groups and that the evaluation of the haplotypes(combination of polymorphisms that are inherited together) can often result in better correla-tion with phenotypes than with individual polymorphisms. The following sections describesome clinically relevant exam-ples of genetic polymorphisms to illustrate the relevance of can-cer pharmacogenomics in optimizing chemotherapy as a way to enhance efficacy and safety[Figure 6.1].

6.3 Polymorphism in Drug-Metabolizing Enzymes

6.3.1 Thiopurine methyltransferase and 6-Mercaptopurine6-Mercaptopurine (6-MP) is a purine antimetabolite used in the treatment of leukemia. Theantitumor activity of 6-MP is via the inhibition of the formation of nucleotides necessary forDNA and RNA synthesis. Thiopurine methyltransferase (TPMT) catalyzes the S-methylationof 6-MP to form inactive metabolites. Genetic variations in the TPMT gene have profoundeffects on the bioavailability and toxicity of 6-MP. It has been demonstrated that about 1 in 300individuals inherit TPMT deficiency as an autosomal recessive trait. Patients who carry TPMTpolymorphisms are at risk for severe hematologic toxicities when treated with 6-MP becausethese polymorphisms lead to a decrease in the rate of 6-MP metabolism.

Figure 6.1: Metabolism of 6-Mercaptopurine

The molecular basis for polymorphic TPMT activity has been well defined. Three particularTPMTalleles, designated as TPMT*2, TMPT*3A, and TPMT*3C, have been shown to accountfor nearly 95% of the observed cases of TPMT defi-ciency. Each of these mutant alleles encodesTPMT pro-teins that undergo rapid degradation, leading to enzyme deficiency. The types andfrequencies of TPMTalleles have been reported to differ among ethnic groups. A recent trialestimated that 71% of patients with bone marrow intolerance to 6-MP were phenotypicallyTPMT deficient and that these patients were more likely to be hospitalized, receive platelettransfusions, and miss scheduled doses of chemotherapy.


Figure 6.2: 6-Mercaptopurine dosing according to TPMT genotype

Appropriate 6-MP dose reductions for TPMT-deficient patients have allowed for similar tox-icity and survival outcomes as patients with normal TPMT levels. Genotyping methods havebeen established for the molecular diagnosis of TPMT deficiency and can assist with determin-ing a safe starting dose for 6-MP therapy.

6.3.2 UDP-glucuronosyltransferase 1A1 and Irinotecan[Irinotecan metabolism and Genotype based toxicities are discussed in Chapter 1: Figure1.3 and Figure 1.4]

Variations in UGT1A1 activity most commonly arise from polymorphisms in the UGT1A1promoter region that contains several repeating TA elements. The presence of seven TA re-peats (referred to as UGT1A1*28), instead of the wild-type number of six, results in reducedUGT1A1 expression and activity. Accordingly, UGT1A1*28has been shown to be associatedwith reduced glucuronidation of SN-38, increased exposure to SN-38, and increased clini-cal toxicity for patients treated with irinotecan. The frequencies of UGT1A1*28alleles varysignificantly among different ethnic groups: UGT1A1*28alleles are present in approximately35% of Caucasians and African Americans while their frequency is much lower in Asians..A recent prospective study demonstrated, with sufficient statistical power, that patients with aUGT1A1*28 allele are at higher risk of grade 4 neutropenia. While that study clearly illustratesthe importance of UGT1A1 pharmacogenetics as a molecular predictor of irinotecan toxicity,clinical guidelines have yet to be developed with regard to dosage adjustment or selection of


alternative non irinotecan-containing regimens.

6.3.3 Dihydropyrimidine dehydrogenase and 5-FUBased on its activity against a variety of tumors and synergistic interactions with other chemother-apy agents, 5-fluorouracil (5-FU) and its derivatives remain some of the most commonly pre-scribed chemotherapy agents. Approximately 5% of administered 5-FU undergoes anabolisminto cytotoxic nucleotides responsible for its antitumor activity, whereas the other 80%-95%under-goes catabolism into biologically inactive metabolites that are excreted in the urine andbile. Dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step in 5-FU catabolism;therefore, variability in this enzyme activity is one of the major factors that influences systemicexposure to fluorodeoxyuridine monophosphate (FdUMP) and the incidence of adverse effectsto 5-FU. DPD activity is completely or partially deficient in 0.1% and 3%5% of individualsin the general population, respectively, and DPD deficiency has been associated with severetoxicity and fatal outcomes after 5-FU treatment.

Figure 6.3: 5-FU metabolism and Rate limiting role of DPYD enzyme

DPD deficiency appears to be a genetic disorder arising from multiple polymorphisms in theDPYDgene resulting in decreased enzyme activity. Analyses of the preva-lence of the variousmutations in the DPYD allele have shown that a guanidine to adenine point mutation in theinvariant splice donor site (DPYD*2A) is by far the most common .

6.3.4 MTHFR and MethotrexateMethotrexate inhibits dihydrofolate reductase (DHFR), which in turn causes accumulation ofdihydrofolate and depletion of cellular folates needed for DNA synthesis. Methotrexate is asso-ciated with significant side effects and carries multiple black box warnings regarding potentialtoxicities, including acute renal failure and bone marrow suppression. Side effects can range


from mild and self limiting to more severe and sometimes fatal. There is significant interindi-vidual variation in response to methotrexate therapy. As many as one third of patients fail torespond to treatment either due to lack of efficacy or adverse events.

The cytotoxic effect of methotrexate is dependent on inhibition of folate metabolism, so thefolate pathway has been examined for enzyme variants that could be associated with efficacyand/or toxicity. One of these enzymes, 5,10-methylenetetrahydrofolate reductase (MTHFR),plays a key role, modulating DNA synthesis and intracellular folate pools. Two tightly linked,common variants in the MTHFR gene, C677T and A1298C, have been shown to cause reducedlevels of MTHFR enzyme activity and corresponding reductions in available folate. Therefore,these variants may act as a useful marker for predicting individual differences in efficacy ortoxicity of methotrexate.

Table 6.2: Frequency of MTHFR allelic variation among different ethnic groups

To date, numerous studies have examined the effect of the C677T and A1298C variants ontoxicity and efficacy of methotrexate across disease states, methods of dosing, and ethnicities-and have shown conflicting results.

1. Two recent meta-analyses of rheumatoid arthritis patients (RA) concluded that there isno significant relationship between MTHFR variant alleles and toxicity or efficacy ofmethotrexate. The authors acknowledge that the differences in their analyses comparedto the previous meta-analysis cited could be due to the data sets used, heterogeneity ofpatients, and differences in methods of analysis.

2. In recent studies of psoriatic arthritis and juvenile ALL, homozygosity of the C677 varianthas been associated with toxicity.

3. A small study of juvenile ALL patients showed that patients with normal MTHFR allelestolerated a higher dosage of methotrexate with lower risk of toxicity than patient withvariants treated with a standard dosage, suggesting that the MTHFR genotype can helpguide dosage in this population.


4. A small study of osteosarcoma patients found that the 1958G>A variant was associatedwith higher risk for hematological toxicity and nephrotoxicity. Notably, these patientswere on methotrexate + doxorubicin + cisplatin (MAP therapy).

5. A small study of women treated for gestational trophoblastic disease did not show a re-lationship between MTHFR variants (for either the mother or the molar pregnancy) andoutcomes.

6. A small study of juvenile idiopathic arthritis (JIA) patients found that MTHFR variantsshowed no significant association with liver dysfunction.

The association with the most supportive evidence so far is between the C677T variant and apotential increased risk of methotrexate toxicity.

Intracellular metabolism of MTX

MTX enters the cell via active transport across the reduced folate carrier (RFC). It is effluxedfrom the cell by several of the ATP-binding cassette (ABC) transporters, especially ABCC1-5and ABCG2.

Figure 6.4: Illustrates some of the key enzymes involved in metabolism of MTX: RFC,reduced folate carrier; MTX-PGs, polyglutamated MTX; FPGS, folylpolyglutamate syn-thase; GGH, gamma-glutamyl hydrolase; DHF, dihydrofolate; THF, tetrahydrofolate; TYMS,thymidylate synthase; DHFR, dihydrofolate reductase; MTHFR, methylenetetrahydrofolate re-ductase; ATIC, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.


Once inside the cell, MTX undergoes polyglutamation by the addition of two to seven glu-tamic acid groups. This process is catalyzed by the enzyme folylpolyglutamate synthetase(FPGS). The polyglutamated form is not as readily transported across the cell membrane, andthus the intracellular half-life of MTX is increased. This polyglutamation process is reversed viathe enzyme gamma-glutamyl hydrolase (GGH), which removes the glutamic acid groups. TheMTX polyglutamates (MTXPGs) act on several key enzymes including dihydrofolate reductase(DHFR) and thymidylate synthase (TYMS-which is involved in pyrimidine synthesis).

Table 6.3: Clinical Annotation for Methotrexate in different MTHFR alleles

Studies suggest that MTX is a pro-drug, and that it is the polyglutamated form, rather thanthe parent drug, which is responsible for its efficacy in RA. It is, therefore, suggested that defec-tive polyglutamation represents one potential mechanism of non-response to MTX. It has beenshown in both adult leukaemias and RA that MTX-polyglutamate levels correlate well withtherapeutic efficacy. In addition, MTX also influences the activity of the enzyme methylenete-trahydrofolate reductase (MTHFR), via its effects on intracellular folate metabolism. However,the enzyme reaction most potently inhibited by methotrexate polyglutamates is the conversionof 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to formyl-AICAR, which is me-diated by the enzyme AICAR transformylase (ATIC). The accumulation of AICAR and itsmetabolites has a direct inhibitory effect on several other enzymes within the adenosine path-way, including adenosine deaminase and adenosine monophosphate deaminase (AMPD1). Thenet result of this is the release of adenosine, a potent anti-inflammatory mediator. There aretherefore, a number of key enzymes that mediate MTX metabolism and may be potential phar-macogenetic targets.

6.4 Polymorphisms in Drug TransportersMDR1 (P-glycoprotein, ABCB1)

P-glycoprotein (PGP), encoded by the MDR1gene (ABCB1), is the best-characterized ATP-binding cassette (ABC) transporter. PGP was initially found to be over-expressed in multidrug


resistant cancers. PGP is involved in the transport of a large range of hydrophobic drugs, in-cluding cytotoxic chemotherapeutic agents (e.g., doxorubicin, paclitaxel, hormones, and car-cinogens as well as an array of structurally divergent drugs. This membrane efflux transporteris also found in normal tissues, such as the canalicular domain of hepatocytes, proximal tubulesof the kidney, brush border of the small intestine, colon, adrenal glands, and capillary endothe-lium of the brain and testes. In addition to its wide tissue distribution, PGP expression variesmarkedly among individuals. Genetic variations in the MDR 1gene have been correlated withdrug exposure of some commonly prescribed drugs such as digoxin and fexofenadine.

Multiple MDR1 polymorphisms have been described to occur in various allelic combi-nations. Grouping these polymorphisms into haplotypes may serve as a useful predictor ofthe functional consequences of MDR1 polymorphisms. The frequencies of variant MDR1alleles differ, depending on racial background, raising the possibility of racial differences inchemotherapy pharmacokinetics and response. For example, the C3435T polymorphism (exon26) has a frequency of 73%-84% in individuals of African origin and frequencies of 34%-59%in individuals of European and Asian origin. A recent report also indicated that MDR1 poly-morphisms may play a role in determining the pharmacokinetic and clinical toxicity profile ofirinotecan. Other studies evaluating the relationship between allelic variations in MDR1 andchemotherapy disposition and response are in progress.

6.4.1 Clinical Annotation for ABCB1 (rs1045642) and 5-fluorouracil

Table 6.4: Clinical Annotation of ABCB1 rs1045642 and 5-FU


6.4.2 Clinical Annotation for rs1045642 and paclitaxel

Table 6.5: Clinical Annotation of ABCB1 rs1045642 and Paclitaxel

6.5 Polymorphisms in Drug TargetsThymidylate Synthase and 5-FU

One of the primary mechanisms of action of 5-FU is the inhibition of thymidylate synthase(TS) by FdUMP. TS is the critical enzyme in the de novo synthesis of thymidylate, an essen-tial precursor of thymidine triphosphate, which is required for DNA synthesis and repair. TSinhibition is, therefore, an important target for 5-FU as well as other folate-based antimetabo-lites, and clinical resistance to these TS-targeted agents has been linked to overexpression ofTS in tumor. TS expression levels in vivo appear to be regulated by the number of polymor-phic tandem repeats in the TS enhancer region (TSER), where increases in TS expression andenzyme activity have been observed with increasing copies of the tandem repeats. Clinicalstudies have demonstrated that individuals who were homozygous for the TS promoter allelesTSER*3 (three tandem repeats) had significantly higher TS mRNA expression levels in tumortissue than those with TSER*2 (two tandem repeats) and that these findings correlated with alower response rate to 5-FU. Another study in rectal cancer patients reported a very significantcorrelation between TSER genotypes and tumor downstaging after pre-operative chemoradia-tion. In addition, a recent study reported that a G¿C variation within the tandem repeats of theTSER*3genotype affected the transcriptional activa-tion of the TSgene. Together, these studiessuggest that TSER genotyping may be useful in selecting patients who are likely to respond totreatment with 5-FU or its analogues.


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