Pharmacogenetics and drug response

Pharmacogenetics and Drug Response
Author & Course Teacher
MAIZBHA UDDIN AHMED
Assistant Professor
Department of Clinical Pharmacy and Pharmacology
Faculty of Pharmacy
University of Dhaka

Contents
1 Pharmacogenetics 5
1.1 Pharmacogenetics and Drug Response . . . . . . . . . . . . . . . . . . . . . . 6
1.2 Pharmacogenetics and Drug safety: Pharmacogenetics of Irinotecan . . . . . . 6
1.3 Genotype Phenotype correlation: CYP3A4 . . . . . . . . . . . . . . . . . . . . 8
1.3.1 Genotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.2 Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.3 Phenotypic diversity due to heterogeneity of mutations . . . . . . . . . 9
1.4 Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.5 Classification of Metabolizer groups . . . . . . . . . . . . . . . . . . . . . . . 11
1.6 CYP3A4 genotype and Phenotype . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6.1 CYP3A4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6.2 CYP3A4 genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6.3 CYP3A4 phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2 Pharmacogenetics of Warfarin 17
2.1 Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1.1 Warfarin Pharmacgology . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1.2 Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2 Metabolism of Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3 Pharmacogenetics of Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.1 CYP2C9 and Warfarin dosing . . . . . . . . . . . . . . . . . . . . . . 21
2.3.2 VKORC1 and Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.3 CYP4F2 and Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.4 CPIC dosing guideline for Warfarin . . . . . . . . . . . . . . . . . . . . . . . 21
2.5 References: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3 Pharmacogenetics of Clopidogrel 23
3.1 Clopidogrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1.1 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1.2 Why it is important to study CYP2C19 polymorphism . . . . . . . . . 24
3.2 Bioactivation of Clopidogrel and Prasugrel . . . . . . . . . . . . . . . . . . . . 24
3.3 Clopidogrel and CYP2C19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.4 Genetic variation in CYP2C19 influences individual response to clopidogrel . . 25
3.5 Choosing the best option: Clopidogrel? Prasugrel? or Ticagrelor? . . . . . . . 26
3.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1

CONTENTS 2
4 Pharmacogenetics of NAT2 30
4.1 NAT localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.2 Human NAT2 Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.3 NAT and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.4 NAT2 and Drug Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.4.1 NAT2 and Isoniazid . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.4.2 NAT2 and Environmental carcinogen . . . . . . . . . . . . . . . . . . 32
4.4.3 NAT2 and Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 Pharmacogenetics of CYP2D6 35
5.1 CYP2D6 allelic variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.2 CYP2D6 and Antipsychotics Pharmacology . . . . . . . . . . . . . . . . . . . 37
5.2.1 Metabolism of Amytriptyline . . . . . . . . . . . . . . . . . . . . . . 38
5.2.2 Dosing recommendations for amitriptyline/nortriptyline based on CYP2D6
phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5.2.3 Recommended dosing of codeine by CYP2D6 Phenotype . . . . . . . . 40
5.3 CYP2D6 and Tamoxifen: Why endoxifen is a better option than Tamoxifen . . 41
5.4 Why Endoxifen? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
5.4.1 Why Endoxifen Treatment may have better therapeutic outcome . . . . 42
5.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6 Pharmacogenetics of cancer 46
6.1 Interindividual variation in Chemotherapeutic drug response . . . . . . . . . . 46
6.2 Genetic variations affecting drug response and toxicity with cancer chemotherapy 47
6.3 Polymorphism in Drug-Metabolizing Enzymes . . . . . . . . . . . . . . . . . 48
6.3.1 Thiopurine methyltransferase and 6-Mercaptopurine . . . . . . . . . . 48
6.3.2 UDP-glucuronosyltransferase 1A1 and Irinotecan . . . . . . . . . . . . 49
6.3.3 Dihydropyrimidine dehydrogenase and 5-FU . . . . . . . . . . . . . . 50
6.3.4 MTHFR and Methotrexate . . . . . . . . . . . . . . . . . . . . . . . . 50
6.4 Polymorphisms in Drug Transporters . . . . . . . . . . . . . . . . . . . . . . . 53
6.4.1 Clinical Annotation for ABCB1 (rs1045642) and 5-fluorouracil . . . . 54
6.4.2 Clinical Annotation for rs1045642 and paclitaxel . . . . . . . . . . . . 55
6.5 Polymorphisms in Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . 55
6.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

List of Figures
1.1 Principle of Pharmacogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2 Pharmacogenetics based Dosing/Diagnostics . . . . . . . . . . . . . . . . . . . 7
1.3 Metabolism of Irinotecan. Abbreviations: APC, 7-ethyl-10[4-N-(5-aminopentanoic
acid)-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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4 Irinotecan Metabolizer and Toxicities . . . . . . . . . . . . . . . . . . . . . . 8
1.5 Genetics of Sickle cell anaemia . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6 Overview of Drug Metabolism and Excretion . . . . . . . . . . . . . . . . . . 10
1.7 Genotype-Phenotype Association of Drug Metabolism and Pharmacology . . . 11
1.8 Genotyping protocol of CYP3A4 . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1 Warfarin Metabolism and Mechanism of Action . . . . . . . . . . . . . . . . . 18
2.2 Warfarin Metabolic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.1 Comparative metabolic pattern of Clopidogrel and Prasugrel . . . . . . . . . . 25
3.2 Comparative Pharmacokinetic pathway of Clopidogrel, Prasugrel and Ticagrelor 27
4.1 Metabolism of Isoniazid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
5.1 Metabolism of Amytriptyline . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.2 Tamoxifen metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
5.3 Tamoxifen: Interaction with other drugs . . . . . . . . . . . . . . . . . . . . . 44
6.1 Metabolism of 6-Mercaptopurine . . . . . . . . . . . . . . . . . . . . . . . . . 48
6.2 6-Mercaptopurine dosing according to TPMT genotype . . . . . . . . . . . . . 49
6.3 5-FU metabolism and Rate limiting role of DPYD enzyme . . . . . . . . . . . 50
6.4 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
3

List of Tables
1.1 Metabolizer’s Genotype and Phenotype in different CYP450 isoenzymes . . . . 12
1.2 PCR mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3 PCR condition and PCR size . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.4 Primers for PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1 Enzymatic activity of different CYP2C9 allelic variants . . . . . . . . . . . . . 20
2.2 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.2 CYP2D6 Allelic variants with enzyme function in different ethnic groups . . . 36
5.3 Genotype and Phenotype of CYP2D6 Alleles . . . . . . . . . . . . . . . . . . 37
5.4 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-methylenetetrahydrofolate
reductase; 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 . . . . . 51
6.3 Clinical Annotation for Methotrexate in different MTHFR alleles . . . . . . . . 53
6.4 Clinical Annotation of ABCB1 rs1045642 and 5-FU . . . . . . . . . . . . . . 54
6.5 Clinical Annotation of ABCB1 rs1045642 and Paclitaxel . . . . . . . . . . . . 55
4

Chapter 1
Pharmacogenetics
Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways
which can affect individual responses to drugs, both in terms of therapeutic effect as well as
adverse effects.The term pharmacogenetics is often used interchangeably with the term phar-macogenomics
which also investigates the role of acquired and inherited genetic differences in
relation to drug response and drug behavior through a systematic examination of genes, gene
products, 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 treated
with 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 drugs
will undergo life-threatenting toxicities. In particular, genetic deregulations affecting genes
coding for DPD, UGT1A1, TPMT, CDA and Cyp2D6 are now considered as critical issues
for patients treated with 5-FU/capecitabine, irinotecan, mercaptopurine/azathioprine, gemc-itabine/
capecitabine/AraC and tamoxifen, respectively. The decision to use pharmacogenetic
techniques 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-based
approaches proved their usefulness while being cost-effective.
Pharmacogenetics has several advantages:
The genotype of an individual is essentially invariable and remains unaffected by the
treatment 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 individual
genes 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.
5

CHAPTER 1. PHARMACOGENETICS 6
1.1 Pharmacogenetics and Drug Response
1. 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) from
fields of drug therapy where compensation for differences in individual drug exposure
can 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 can
affect the probability of obtaining efficacy for that drug. It is unlikely that any physician
would restrict a patient from treatment with a drug because the chance of response might
be only 40% in carriers of a certain PGx variant, when the average chance is 70% in
carriers of the respective wild-type genotype. However, such PGx information might be
helpful if alternative medications are available or if a better response might be achieved
by a change in dose or by a specific additive medication. [Figure 1.2]
1.2 Pharmacogenetics and Drug safety: Pharmacogenetics of
Irinotecan
Irinotecan is another example of a drug that is metabolized differently in some patients; these
differences can predispose a subset of patients to increased drug toxicity. The addition of
irinotecan to the combination of fluorouracil and leucovorin in 1996 represented the first major
advance in the treatment of colorectal cancer since the late 1950s, when fluorouracil originally
became the standard of care. Regimens that contained irinotecan resulted in a near-doubling
of response rates, from around 20% to 40%, and prolongation of overall survival by up to 3
months. 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. Death
often resulted from drug-related gastrointestinal and vascular syndromes. Differences between
individuals with respect to the drug’s metabolism were a rational route of exploration because
of 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 UGT1A1
to 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 also
be metabolized by carboxylesterase to SN-38. [Figure 1.3]
Patients with the UGT1A1*28 allele have lower glucuronidating ability than patients with
wild-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 a
lesser extent in the Asian population; only 14% of individuals are homozygous and 1531% are
heterozygous for the UGT1A1*28 allele. [Figure 1.4]

Figure 1.3: Metabolism of Irinotecan. Abbreviations: APC, 7-ethyl-10[4-N-(5-aminopentanoic
acid)-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: CYP3A4
An organisms genotype is the set of genes that it carries. An organisms phenotype is all of its
observable 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 up
a population from generation to generation. However, since an organisms genotype generally
affects its phenotype, the phenotypes that make up the population are also likely to change.

1.3.1 Genotype
This is the ”internally coded, inheritable information” carried by all living organisms. This
stored information is used as a ”blueprint” or set of instructions for building and maintaining a
living creature. These instructions are found within almost all cells (the ”internal” part), they
are written in a coded language (the genetic code), they are copied at the time of cell division
or 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 control
everything from the formation of protein macromolecules, to the regulation of metabolism and
synthesis.
1.3.2 Phenotype
This is the ”outward, physical manifestation” of the organism. These are the physical parts, the
sum of the atoms, molecules, macromolecules, cells, structures, metabolism, energy utilization,
tissues, organs, reflexes and behaviors; anything that is part of the observable structure, function
or behavior of a living organism.
1.3.3 Phenotypic diversity due to heterogeneity of mutations
Mutations that alter the structure of gene products, missense mutations, may give rise to a
remarkable diversity of phenotypes associated with mutations at the same gene locus. One of
the 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 fetal
haemoglobins have a chains combined with b (HbA a2b2) or g chains (HbF a2g2). Over 400
structural 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 the
haemoglobin molecule, result in a disease phenotype. For example, the substitution of glutamic

acid for valine in the sixth position in the b chain causes the haemoglobin molecules to form
linear stacks in the deoxy configuration which, in turn, causes the red cells to assume a sickled
configuration. The resulting disease in homozygotes, sickle cell anaemia, is characterized by
chronic anaemia and tissue damage due to blockage of the micro-circulation with aggregates
of sickled red cells. Other amino acid substitutions result in instability of the haemoglobin
molecule, which precipitates in the red cells during their life in the circulation. This results in
damage to their membranes and increased rigidity, and hence in a variable degree of anaemia
due to their premature destruction.
1.4 Drug Metabolism
Drug 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 into
more 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 accessible
for renal excretion. During drug metabolism, sometimes toxic compounds are formed. In many
cases, metabolism is responsible for the activation of the prodrug. This process can be divided
into 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 the
originally lipophilic compound more hydrophilic. The genes coding the proteins responsible for
these processes are usually highly polymorphic. This means that in some cases, the individual
response after the administration of a specific drug can be attributed to the genetic background
of 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 might
help not only in quickly establishing the correct dose of certain drug, but also in avoiding the
dangerous, sometimes life-threatening side effects.
1.5 Classification of Metabolizer groups
Drug or other xenobiotics metabolizers can be classified according to their metabolic capacities
and their genetic make up.
1. Ultra Rapid Metabolizer/Ultra Metabolizer: Ultra-rapid metabolizers have one or
more 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 allele
and one normally functioning allele, and therefore have decreased enzyme activity.
4. Poor Metabolizer: Poor metabolizers have two non-functional alleles and therefore have
little 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 CYP3A4
Cytochrome P450 3A4 (abbreviated CYP3A4) (EC 1.14.13.97), is an important enzyme in the
body, mainly found in the liver and in the intestine. Its purpose is to oxidize small foreign
organic molecules (xenobiotics), such as toxins or drugs, so that they can be removed from
the body. While many drugs are deactivated by CYP3A4, there are also some drugs which are
activated by the enzyme. Some substances, such as grapefruit juice and some drugs, interfere
with the action of CYP3A4. These substances will therefore either amplify or weaken the action
of those drugs that are modified by CYP3A4.
CYP3A4 is a member of the cytochrome P450 family of oxidizing enzymes. Several other
members of this family are also involved in drug metabolism, but CYP3A4 is the most common
and the most versatile one. Like all members of this family, it is a hemoprotein, i.e. a protein
containing a heme group with an iron atom. In humans, the CYP3A4 protein is encoded by
the CYP3A4 gene. This gene is part of a cluster of cytochrome P450 genes on chromosome
7q21.1.
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, thus
contributing to the presystemic and systemic metabolism of 30% of all drugs.
1.6.2 CYP3A4 genotyping
Genotyping is normally done by two step gel electrophoresis: a) one after PCR and b) Another
after restriction enzyme digestion. A typical PCR mixture contains: forward primer, reverse
primer, dNTP, Taq DNA polymerase, Buffer and a template DNA. PCR condition will be initial
denaturation 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 machine
stops. 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 phenotyping
CYP3A is involved in the metabolism of endogenous cortisol to 6b-hydroxy-cortisol. The
urinary excretion of 6b-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 6b-hydroxy-cortisol/cortisol ra-tio
is a good indication of CYP3A activity. Hsieh et al. reported that, from the 6b-hydroxy-cortisol/
cortisol ratio, those withCYP3A4*4, *5and*6 alleles have shown below average CYP3A4
activity implying reduced catalytic activity for the corresponding protein variants compared to
those with no mutations shown.
Phenotyping is normally done by measuring the ratio of 6b-hydroxycortisol (6b-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 cortisol
metabolite concentration.

1.7 References
1. Klotz, U. (2007). ”The role of pharmacogenetics in the metabolism of antiepileptic
drugs: pharmacokinetic and therapeutic implications.”. Clin Pharmacokinet 46 (4): 2719.
doi:10.2165/00003088-200746040-00001. PMID 17375979
2. ”Center for Pharmacogenomics and Individualized Therapy”. Retrieved 2014-10-28.
3. Roses AD (June 2000). ”Pharmacogenetics and the practice of medicine”. Nature 405
(6788): 85765. doi:10.1038/35015728. PMID 10866212.
4. Lazarou J, Pomeranz BH, Corey PN (April 1998). ”Incidence of adverse drug reactions
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Chapter 2
Pharmacogenetics ofWarfarin
2.1 Warfarin
Warfarin (also known by the brand names Coumadin, Jantoven, Marevan, Uniwarfin) is an
anticoagulant 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 vegetable
foods or ”greens,” since these typically contain large amounts of vitamin K1) and its activity
has to be monitored by blood testing for the international normalized ratio (INR) to ensure an
adequate yet safe dose is taken. A high INR predisposes to a high risk of bleeding, while an
INR below the therapeutic target indicates the dose of warfarin is insufficient to protect against
thromboembolic events.
2.1.1 Warfarin Pharmacgology
Warfarin and related 4-hydroxycoumarin-containing molecules decrease blood coagulation by
inhibiting vitamin K epoxide reductase, an enzyme that recycles oxidized vitamin K1 to its
reduced 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 does
not antagonize the action of vitamin K1, but rather antagonizes vitamin K1 recycling, depleting
active 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 the
risk of embolism (migration of a thrombus to a spot where it blocks blood supply to a vital
organ).
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 in
blood pooled in dysfunctional cardiac atria. Thus, common clinical indications for warfarin
use are atrial fibrillation, the presence of artificial heart valves, deep venous thrombosis, and
pulmonary embolism (where the embolized clots first form in veins). Warfarin is also used
in antiphospholipid syndrome. It has been used occasionally after heart attacks (myocardial
infarctions), 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
17

CHAPTER 2. PHARMACOGENETICS OF WARFARIN 18
mechanism from warfarin (which normally has no effect on platelet function).
2.1.2 Mechanism of Action
Warfarin 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, protein
S, and protein Z. Other proteins not involved in blood clotting, such as osteocalcin, or matrix
Gla 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 inside
blood vessels, on the vascular endothelium. The enzyme that carries out the carboxylation of
glutamic acid is gamma-glutamyl carboxylase. The carboxylation reaction will proceed only
if 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 back
to vitamin K and vitamin K hydroquinone by another enzyme, the vitamin K epoxide reduc-tase
(VKOR). Warfarin inhibits epoxide reductase (specifically the VKORC1 subunit), thereby
diminishing available vitamin K and vitamin K hydroquinone in the tissues, which inhibits the
carboxylation activity of the glutamyl carboxylase. When this occurs, the coagulation factors
are no longer carboxylated at certain glutamic acid residues, and are incapable of binding to
the 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 inactive
factors, the anticoagulation effect becomes apparent. The coagulation factors are produced,
but have decreased functionality due to undercarboxylation; they are collectively referred to as
PIVKAs (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 diminish
blood clotting in the patient.
2.2 Metabolism of Warfarin
Warfarin is a natural product and given as racemic mixture of the R and S stereoisomers of
the drug. S-warfarin is 3-5 times more potent an inhibitor of the vitamin K epoxide reductase
complex, 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 whereas
metabolism of R-warfarin is mainly via CYP3A4 with involvement of CYP1A1, CYP1A2,
CYP2C8, CYP2C9, CYP2C18 and CYP2C19 as depicted in the Warfarin Pharmacokinetics
Pathway [Figure 2.2]. Phase 2 metabolism of warfarin has not been well studied and is not
depicted 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 to
interact with the ABCB1 transporter in liver.
Figure 2.2: Warfarin Metabolic Pathway
2.3 Pharmacogenetics ofWarfarin
The association between warfarin dose and the CYP2C9 and VKORC1genes is now unquestion-able,
as evidenced by numerous studies. Single nucleotide polymorphisms in the cytochrome
P450 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 warfarin
make 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 to
the use of pharmacogenetics-guided dosing in the initiation of warfarin therapy has not shown
improved outcomes in either safety or efficacy of therapy. Better clinical evidence of beneficial
effects 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 pharmacogenomic
approach to oral anticoagulation therapy in clinical practice can be established.
Patients carrying CYP2C9 variant alleles had a higher rate of above-range INR values, took
longer to reach stable dosing, and had a higher risk of serious and life-threatening bleeding
events 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 (INR6.0) anticoagulation and bleeding
com-pared with wild-type patients.
Table 2.1: Enzymatic activity of different CYP2C9 allelic variants

2.3.1 CYP2C9 andWarfarin dosing
CYP2C9*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 andWarfarin
In the VKORC1 1639 (or 3673) SNP, the common G allele is replaced by the A allele. Because
people with an A allele (or the ”A haplotype”) produce less VKORC1 than do those with the G
allele (or the ”non-A haplotype”), lower warfarin doses are needed to inhibit VKORC1 and to
produce an anticoagulant effect in carriers of the A allele. The prevalence of these variants also
varies by race, with 37% of Caucasians and 14% of Africans carrying the A allele.
2.3.3 CYP4F2 andWarfarin
Recent genome wide association studies have not only confirmed these observations but also
identified 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 Warfarin
Approach to pharmacogenetic-based warfarin dosing without access to dosing algorithms: In
2007, the FDA modified the warfarin label, stating that CYP2C9 and VKORC1 genotypes may
be useful in determining the optimal initial dose of warfarin. The label was further updated in
2010 to include a table describing recommendations for initial dosing ranges for patients with
different combinations of CYP2C9 and VKORC1 genotypes. Genetics-based algorithms also
better predict warfarin dose than the FDA-approved warfarin label table. Therefore, the use of
pharmacogenetic algorithm-based dosing is recommended when possible, although if electronic
means for such dosing are not available, the table-based dosing approaches are suggested.
Table 2.2: Genotype based Dosing guideline by Clinical Pharmacogenetics Implementation
Consortium (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 Implementation
Consortium Guidelines for CYP2C9 and VKORC1 genotypes and warfarin dosing. Clin
Pharmacol 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 of
Warfarin Understanding the Dose-Effect Relationship”. Clinical Pharamacokinetics (Springer
International Publishing) 11 (6): 483504. doi:10.2165/00003088-198611060-00005. PMID
3542339
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 vitamin
K antagonists: American College of Chest Physicians evidence-based clinical practice
guidelines (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/American
College 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 Association
for 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 the
cytochrome 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 VKORC1
gene is associated with an interindividual variability in the dose-anticoagulant effect of
warfarin.Blood105:64549
10. Scordo MG, Aklillu E, Yasar U, et al. 2001. Genetic polymorphism of cytochrome P450
2C9 in a Caucasian and a black African population.Br. J. Clin. Pharmacol.52:44750

Chapter 3
Pharmacogenetics of Clopidogrel
3.1 Clopidogrel
Clopidogrel (INN) is an oral, thienopyridine-class antiplatelet agent used to inhibit blood clots
in coronary artery disease, peripheral vascular disease, cerebrovascular disease, and to prevent
myocardial infarction (heart attack). It is marketed by Bristol-Myers Squibb and Sanofi under
the trade name Plavix. The drug works by irreversibly inhibiting a receptor called P2Y12,
an adenosine diphosphate (ADP) chemoreceptor on platelet cell membranes. Adverse effects
include hemorrhage, severe neutropenia, and thrombotic thrombocytopenic purpura.
Clopidogrel is used to prevent myocardial infarction (heart attack) and stroke in people who
are at high risk of these events, including those with a history of myocardial infarction and other
forms of acute coronary syndrome, stroke and those with peripheral artery disease. Treatment
with clopidogrel or a related drug is recommended by the American Heart Association and the
American 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 14
days
2. Present for treatment of a non-ST elevation myocardial infarction (NSTEMI) or unstable
angina.
(a) Including a loading dose and maintenance therapy in those receiving PCI and unable
to tolerate aspirin therapy
(b) Maintenance therapy for up to 12 months in those at medium to high risk for which
a 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 treatment
with clopidogrel in combination with aspirin in certain high risk patients.
3.1.1 Pharmacology
Clopidogrel 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
23

CHAPTER 3. PHARMACOGENETICS OF CLOPIDOGREL 24
of ADP receptor, which is important in activation of platelets and eventual cross-linking by the
protein 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 administered
when a rapid effect is needed.
3.1.2 Why it is important to study CYP2C19 polymorphism
1. 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 form
blood clots.
2. Antiplatelet drug does not have its antiplatelet effects until it is metabolized into its active
form by CYP2C19.
3. Study by De Morais et al, 1994 detects the *17 allele as an ultra fast metabolizer of
antiplatelet drugs. A recent report indicates the increased risk for bleeding in individuals
treated with clopidogrel who carry the *17 allele.
4. People who have reduced functioning of their CYP2C19 liver enzyme due to inactivating
polymorphisms in their CYP2C19 gene cannot effectively convert Plavix to its active
form. 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 may
remain 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 Prasugrel
Clopidogrel is a prodrug, and its activation is complex (Figure 3.1). Some 85% of the dose
administered is hydrolyzed in the liver, where it is converted into an inactive metabolite (SR
26334); the remainder is metabolized by CYP2C19 and to a lesser degree by CYP1A2 and
CYP2B6 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 by
the 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 to
inactive metabolites), the complex activation of the prodrug requiring two oxidation steps, and
its 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 prevalence
of around 25%), are the main pharmacokinetic factors affecting the variability of response to
clopidogrel.
3.3 Clopidogrel and CYP2C19
One of the main causes of this clopidogrel nonresponsiveness is interindividual variability in the
metabolic activation of clopidogrel. Polymorphisms in the CYP2C19 gene affect clopidogrel
pharmacokinetics, causing the CYP2C19 enzyme that converts Plavix to its active form to be
more or less effective. The most common CYP2C19 polymorphisms,*2, *3, and *17, result
in 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 to
identify clopidogrel nonresponders (carrying the *2 or *3 alleles) who may benefit by taking an
alternative antiplatelet agent, such as prasugrel and ticagrelor, or individuals who may be at risk
of bleeding due to clopidogrel overactivity (*17 allele carriers).
A loss-of-function polymorphism in the CYP2C19 gene, known as the CYP2C19*2 allelic
variant, has been associated with higher levels of ADP-induced platelet aggregation values in
clopidogrel-treated patients and consequently a higher risk of major adverse cardio vascular
events, including the occurrence of stent thrombosis (ST).
A novel allelic variant, CYP2C19*17, results in an increased enzyme function of CYP2C19
because of a mutation (806CT) in the 5-flanking region of the gene that causes an increased
transcription of CYP2C19. Such increased transcriptional activity of CYP2C19 may confer
a rapid metabolization of CYP2C19 substrates, which may lead to an enhanced response to
antiplatelet treatment. Although this may improve the prevention of thrombotic events, it also
may 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 highly
variable 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 less
effective 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 active
metabolite).
Table 3.1: Common polymorphisms in the CYP2C19 gene that influence anti-platelet response
to clopidogrel
Approximately 2-4% of Caucasian, 4% of African or African/American, and 14-20% of
Asian populations are poor metabolizers (PM) because these individuals have two loss-of-function
alleles and very low CYP2C19 activity. Intermediate metabolizers have one CYP2C19
loss-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? or
Ticagrelor?
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 around
2.5% 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 the
liver, 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 inhibitors
of 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 not
believed 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 yet
approved in the United States, is an active compound and is metabolized by CYP3A4 to an
active metabolite. Ticagrelor and its active metabolite are both metabolized and glucuronidated
in the liver before elimination in the urine. Genetic variations in CYP isoenzymes do not appear
to affect metabolism of ticagrelor.
3.6 References
1. 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 future
options for its management. J Thromb Thrombolysis. 2010;30(3):319-31.
3. Gurbel PA, Tantry US. Selecting optimal antiplatelet therapy based on platelet function
monitoring 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 antiplatelet
drugs resistance appointed by the Section of Cardiovascular Interventions of the Polish
Cardiac Society, endorsed by theWorking Group on Thrombosis of the European Society
of Cardiology. Eur Heart J. 2009;30(4):426-435.
5. Terpening C. Clopidogrel: A pharmacogenomic perspective on its use in coronary artery
disease. Clin Med Insights Cardiol. 2010;4:117-128.
6. Sharma RK, Reddy HK, Singh VN, et al. Aspirin and clopidogrel hyporesponsiveness
and 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-responsiveness
and 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 poor
responsiveness 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 response
to 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 Feb
2011. (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 P450
enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its
pharmacologically 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 causes
ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and
antidepressants. 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 cytochrome
P450 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 on
CYP2C19 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¿A
polymorphism and high on-clopidogrel platelet reactivity associated with adverse 1-Year
clinical 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 to
humans. 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 carcinogens
present in the diet, cigarette smoke and the environment. The reaction pathway is catalysed by
two cytoplasmic acetyltransferases (NAT; EC 2.3.1.5), N-acetyltransferase Type I (NAT1) and
N-acetyltransferase Type II (NAT2).
The arylamine N-acetyltransferases (NATs) are involved in the metabolism of a variety of
different compounds that we are exposed to on a daily basis. Many drugs and chemicals found
in the environment, such as those in cigarette smoke, car exhaust fumes and in foodstuffs, can
be either detoxified by NATs and eliminated from the body or bioactivated to metabolites that
have the potential to cause toxicity and/or cancer. NATs have been implicated in some adverse
drug reactions and as risk factors for several different types of cancers. As a result, the levels of
NATs in the body have important consequences with regard to an individual’s susceptibility to
certain drug-induced toxicities and cancers.
4.1 NAT localization
Two NAT isoenzymes have been identified in humans, namely NAT1 and NAT2, which are the
products of distinct genetic loci, designated NAT1 and NAT2, respectively. A related pseu-dogene,
NATP1, has also been identified, which contains multiple frameshift and nonsense
mutations. The two functional NAT genes share an 87% nucleotide identity, which translates
to an 81% homology at the amino acid level. While the entire transcript of NAT1 is derived
from 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. Human
NAT1 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 the
orientation NAT1!NATP1!NAT2.
4.2 Human NAT2 Alleles
Since 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, 29
different NAT2 alleles have been detected in human populations. Each of the variant alleles is
30

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 genotype
and phenotype. Early genotyping studies screened for the presence of the C481T (M1), the
G590A (M2), the G857A (M3) and sometimes the G191A (M4) nucleotide changes, all of
which were shown to cause a slow acetylation phenotype. Moreover, there was a gene-dosage
effect. Individuals who were homozygous for NAT2 polymorphisms had a slow acetylator
phenotype, individuals heterozygous for NAT2 polymorphisms had an intermediate acetylator
phenotype, 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 a
subset 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 Disease
The association between acetylator status and the risk of various diseases has been extensively
reported, and reviewed in detail. Altered risk with either the slow or rapid phenotype has been
observed for bladder, colon and breast cancer, systemic lupus erythematosis, diabetes, Gilbert’s
disease, 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 NAT
substrates including the food-derived heterocyclics present in the diet as well as arylamines such
as 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, those
genes responsible for metabolic polymorphisms have low penetrance and cause only a moder-ate
increase in cancer risk. Nevertheless, their widespread occurrence in the general population
suggests they are a significant contributor to individual risk.
When acetylator phenotype has been linked to carcinogen exposure, more consistent results
have been reported. For example, the rapid phenotype has emerged as a strong risk factor for
colorectal 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 risk
associated with neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s dis-ease.
4.4 NAT2 and Drug Response
4.4.1 NAT2 and Isoniazid
The genetic polymorphism in N-acetyltransferase activity was first discovered in patients treated
with isoniazid for tuberculosis. This drug is primarily excreted following acetylation catalysed
by 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), which
is 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 catalyzes
the 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 the
liver, which is linked to the glutathione-S-transferase (GST)-mediated metabolic pathway, to
become an excretable metabolite.
Figure 4.1: Metabolism of Isoniazid
NAT2 genetic polymorphisms and the resulting variation in acetylation capacity of NAT2
are implicated in the risk of INH-induced hepatotoxicity. Slow acetylator genotype of NAT2
will 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 fast
acetylator) genotype also have a significant role in INH induced hepatotoxicity. Hydrazine and
N-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 any
conflict.
4.4.2 NAT2 and Environmental carcinogen
Biological 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 by
this biological amine than in fast-acetylator phenotype.
4.4.3 NAT2 and Procainamide
Procainamide 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 expressed
in post patients; however, NAT2 is variably expressed and plays a major role in the production
of NAPA. The majority of patients reveiving procainamide therapy will develop autoantibodies
over time and possibly drug induced lupus. An early study showed that subjects who had a
slow-acteylator phenotype developed antinuclear antibodies earlier than rapid acetylators. This
was 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 increased
NAPA concentrations and thus increased anti-arrhythmic effects and possible excessive QT
prolongation.
4.5 References
1. DWHein. 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 of
Isoniazid-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 variants
More than 50 human CYP isozymes have been identified to date. Of these, more than 20 are
encoded by genes that are functionally polymorphic, including CYP2A6, CYP2C9, CYP2C19,
and CYP2D6. Consequently, approximately 40% of CYP-dependent drug metabolism is carried
out by polymorphic enzymes.
Table 5.1: CYP2D6 Substrate
CYP2D6 is the best characterized of these polymorphic CYP isozymes, with more than 75
allelic variants currently identified. These variants result from point mutations, deletions or
additions, gene rearrangements, and deletion or duplication of the entire gene, and result in an
increase, reduction, or complete loss of activity. While it accounts for only 2%5% of all hepatic
CYP isozymes, CYP2D6 metabolizes approximately 25% of all clinically used medications,
including some cytotoxics, tamoxifen, and many agents used to treat associated complications
such as antiarrythmics, antiemetics, antidepressants, antipsychotics, and analgesics. Since the
identification of the CYP2D6 polymorphism in the 1970s, several studies have shown that the
frequencies of the alternative CYP2D6 phenotypes vary significantly among different ethnic
35

CHAPTER 5. PHARMACOGENETICS OF CYP2D6 36
groups and that such polymorphisms may play a role in the induction of adverse effects from
administration of some therapeutic agents. Variation in CYP2D6 expression is also thought
to increase the potential for drugdrug interactions, an important implication because of the
increasing number of drugs being prescribed. For example, in the oncology setting, patients
with advanced cancer receiving palliative therapy receive an average of five or more medications
for 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 in
enzymatic deficiencies. The frequency of these polymorphisms varies within the major ethnic
groups. CYP2D6 polymorphisms that produce poor metabolizers are found with frequencies of
7% 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 selection
of appropriate therapies. These measures may improve patient outcome by ensuring maximum
drug 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: poor
metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ul-trarapid
metabolizers (UMs) [Figure 5.4]. The EM phenotype is expressed by the majority of
the population and is therefore considered the norm. PMs inherit two deficient CYP2D6 alleles
and, as a result, metabolize drugs at a notably slower rate. This leads to an accumulation of high
levels of unmetabolized drugs that are CYP2D6 substrates, a concomitant greater potential for
adverse events and drugdrug interactions, and lower efficacy for drugs requiring CYP2D6 acti-vation.
The UM phenotype is caused by the duplication, multiduplication, or amplification of
active CYP2D6 genes, including primarily the CYP2D6*2 allele, but also involving CYP2D6*1
and others. Individuals with the UM phenotype metabolize drugs at an ultrarapid rate, which
may lead to a loss of therapeutic efficacy at standard doses. Individuals who are heterozygous
for a defective CYP2D6 allele often demonstrate an IM phenotype. This phenotype has a wide
spectrum of metabolic activity that can range from marginally better than the PM phenotype to
activity that is close to that of the EM phenotype.
5.2 CYP2D6 and Antipsychotics Pharmacology
CYP2D6 is described as the most relevant enzyme in the metabolism of many antipsychotic
drugs. 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 appears
highly 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 in
turn influence the disposition of the antipsychotic drugs. Antipsychotic drug biotransformation
is mainly determined by genetic factors mediating CYP2D6 gene polymorphism, however the
importance of environmental factors (dietary, smoking, diseases, etc.) is also recognized. Ad-ditionally,
the potential interaction between CYP2D6 and the endogenous metabolism must be
taken into consideration.
5.2.1 Metabolism of Amytriptyline
Amytriptyline (AT) is metabolized mainly by demethylation forming nortriptyline (NT), and by
hydroxylation, 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 NT
is mainly catalyzed by CYP2C19, with the participation of other CYP enzyme forms in higher
drug 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 on
CYP2D6 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 monitoring
to 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 function
or 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 to
less active compounds when compared to extensive metabolizers. Higher plasma
concentrations 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 therapeutic
drug monitoring to guide dose adjustments.
5.2.3 Recommended dosing of codeine by CYP2D6 Phenotype
1. 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 following
codeine 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 this
CYP2D6 phenotype include morphine and non-opioid analgesics. Tramadol, and to
a lesser extent hydrocodone and oxycodone, are not good alternatives because their
metabolism 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 function
or 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-specific
dosing.
(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-specific
dosing. 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 this
CYP2D6 phenotype include morphine and non-opioid analgesics. Tramadol, and to
a lesser extent hydrocodone and oxycodone, are not good alternatives because their
metabolism 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 as
endocrine treatment for hormone receptorpositive breast cancer, with indications in the metastatic,
adjuvant, and preventative settings. The minimal acute toxicity of endocrine treatments such as
tamoxifen compared with chemotherapy helps maintain patients quality of life, and increasing
emphasis is being placed on these treatments to delay the use of cytotoxic therapies for as long
as possible.

Tamoxifens adverse-event profile includes venous thrombosis, endometrial cancer, and,
most commonly, hot flashes, which are often treated with an SSRI antidepressant. Tamoxifen
is extensively hepatically metabolized to several primary and secondary metabolites by multi-ple
CYP enzymes, including CYP2D6. With some SSRIs (e.g., paroxetine) known to inhibit
CYP2D6, a study was recently conducted to investigate the effects of SSRI coadministration on
plasma concentrations of tamoxifen and its metabolites in addition to the effects of individual
patient genotypes. A strong association was shown between CYP2D6 activity and plasma levels
of the active tamoxifen metabolite, endoxifen. Concomitant use of paroxetine was associated
with 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 may
be 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 possibility
that 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 coadministered
medication 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 active
metabolites 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 efficacy
and 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 patients
taking 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 Endoxifen
for the treatment of breast cancer (Ahmad et al. 2010). Treatment of breast cancer directly with
Endoxifen may eliminate TAM metabolism associated variability and avoid a potential serious
drug-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 metabolizing
Tamoxifen
Endoxifen acts directly at the target
Tamoxifen have been shown to be more effective than Aromatase inhibitors in patients
with 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 References
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the structural characteristics and functional relevance of the human cytochrome P450 2D6
enzyme”. Drug Metab. Rev. 41 (4): 573643. doi:10.1080/03602530903118729. PMID
19645588.
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. PMID
22515611.
4. ”Entrez Gene: CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6”.
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Aust New Zealand J Psychiatry 2014 Jan 10. PMID 24413808
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7. Llerena A, Dorado P, Peas-Lled EM (January 2009). ”Pharmacogenetics of debrisoquine
and 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 drug
response, interactions, and adverse effects”. Am Fam Physician 76 (3): 3916. PMID
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of three CYP2D6 probe substrates and genotype in Ghanaians, Chinese and Cau-casians”.
Pharmacogenetics 8 (4): 32533. doi:10.1097/00008571-199808000-00006.
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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 drug
to a population of patients can result in any-where from minimal toxicity to severe, potentially
life-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 genetic
polymorphisms, but prospective identification of patients likely to be harmed by chemotherapy
currently 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 have
been associated with drug responses (e.g., age, gender, diet, drug-drug interactions), inherited
variations in drug disposition (metabolism and transport) genes and drug target genes also likely
con-tribute to the observed variability in cancer treatment outcome.
Pharmacogenomic studies aim to elucidate the genetic bases for interindividual differences
and to use such genetic information to predict the safety, toxicity, and/or efficacy of drugs. There
exist several clinically relevant examples of the utility of pharmacogenomics that associate spe-cific
genetic polymorphisms in drug metabolizing enzymes (e.g., TPMT, UGT1A1, DPD), drug
transporters (MDR1), and drug target enzymes (TS) with clinical outcomes in patients treated
with commonly prescribed chemotherapy drugs, such as 5-fluorouracil and irinotecan. Tech-niques
to discover and evaluate the functional significance of these polymorphisms have evolved
in 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 response
will enable prospective identification of patients at risk for severe toxicity or those most likely
to benefit from a particular treatment regimen. Such studies can be translated to clinical practice
via 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
46

CHAPTER 6. PHARMACOGENETICS OF CANCER 47
of pharmacogenomics in clinical cancer therapy and cancer drug development.
6.2 Genetic variations affecting drug response and toxicity
with 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 in
an effort to identify relevant inherited variations that may better predict patient response to
chemotherapy. Genetic variations include nucleotide repeats, insertions, deletions, and single
nucleotide 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 describe
some 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-Mercaptopurine
6-Mercaptopurine (6-MP) is a purine antimetabolite used in the treatment of leukemia. The
antitumor activity of 6-MP is via the inhibition of the formation of nucleotides necessary for
DNA and RNA synthesis. Thiopurine methyltransferase (TPMT) catalyzes the S-methylation
of 6-MP to form inactive metabolites. Genetic variations in the TPMT gene have profound
effects on the bioavailability and toxicity of 6-MP. It has been demonstrated that about 1 in 300
individuals inherit TPMT deficiency as an autosomal recessive trait. Patients who carry TPMT
polymorphisms are at risk for severe hematologic toxicities when treated with 6-MP because
these 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 particular
TPMTalleles, designated as TPMT*2, TMPT*3A, and TPMT*3C, have been shown to account
for nearly 95% of the observed cases of TPMT defi-ciency. Each of these mutant alleles encodes
TPMT pro-teins that undergo rapid degradation, leading to enzyme deficiency. The types and
frequencies of TPMTalleles have been reported to differ among ethnic groups. A recent trial
estimated that 71% of patients with bone marrow intolerance to 6-MP were phenotypically
TPMT deficient and that these patients were more likely to be hospitalized, receive platelet
transfusions, 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 have
been 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: Figure
1.3 and Figure 1.4]
Variations in UGT1A1 activity most commonly arise from polymorphisms in the UGT1A1
promoter 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 reduced
UGT1A1 expression and activity. Accordingly, UGT1A1*28has been shown to be associated
with 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 vary
significantly among different ethnic groups: UGT1A1*28alleles are present in approximately
35% 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 a
UGT1A1*28 allele are at higher risk of grade 4 neutropenia. While that study clearly illustrates
the 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-FU
Based 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 anabolism
into 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 and
bile. 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 systemic
exposure to fluorodeoxyuridine monophosphate (FdUMP) and the incidence of adverse effects
to 5-FU. DPD activity is completely or partially deficient in 0.1% and 3%5% of individuals
in the general population, respectively, and DPD deficiency has been associated with severe
toxicity 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 the
DPYDgene resulting in decreased enzyme activity. Analyses of the preva-lence of the various
mutations in the DPYD allele have shown that a guanidine to adenine point mutation in the
invariant splice donor site (DPYD*2A) is by far the most common .
6.3.4 MTHFR and Methotrexate
Methotrexate inhibits dihydrofolate reductase (DHFR), which in turn causes accumulation of
dihydrofolate and depletion of cellular folates needed for DNA synthesis. Methotrexate is asso-ciated
with significant side effects and carries multiple black box warnings regarding potential
toxicities, 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 to
respond to treatment either due to lack of efficacy or adverse events.
The cytotoxic effect of methotrexate is dependent on inhibition of folate metabolism, so the
folate pathway has been examined for enzyme variants that could be associated with efficacy
and/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 reduced
levels 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 or
toxicity 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 on
toxicity 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 is
no significant relationship between MTHFR variant alleles and toxicity or efficacy of
methotrexate. The authors acknowledge that the differences in their analyses compared
to the previous meta-analysis cited could be due to the data sets used, heterogeneity of
patients, and differences in methods of analysis.
2. In recent studies of psoriatic arthritis and juvenile ALL, homozygosity of the C677 variant
has been associated with toxicity.
3. A small study of juvenile ALL patients showed that patients with normal MTHFR alleles
tolerated a higher dosage of methotrexate with lower risk of toxicity than patient with
variants treated with a standard dosage, suggesting that the MTHFR genotype can help
guide dosage in this population.

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