95

7 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

Anthony Y.H. Lu and Qiang Ma

7.1 INTRODUCTION

It is well established that in a large patient population, individuals may respond to the same medication differ-ently in drug effi cacy, drug safety, or both. One measur-able parameter of variability in drug response is the plasma drug level that sometimes varies as much as several hundredfold among patients. Therefore, even with the best drug available, the standard daily dose that is proven to be effi cacious and safe for the majority of patients may be ineffective or even harmful for a small number of patients.

Although numerous factors can infl uence the outcome of a drug treatment in individual patients, it is generally accepted that genetic variations in humans play a major role in determining the variability of disease phenotypes, drug effi cacy, and drug side effect (Lu, 1998 ; Meyer, 2000 ; Evans and McLeod, 2003 ; Wein-shilboum, 2003a ; Evans and Relling, 2004 ; Eichelbaum et al., 2006 ; Lin, 2007 ). The human genome sequence provides a special record of human evolution. This sequence varies among populations and individuals. As the complete sequence of the human genome became available, the impact of the variability of the human genome on the pathogenesis of important diseases and the responses to drug therapy in humans can be readily analyzed. Parallel to the rapid accumulation of the knowledge on the genome – disease and genome – drug interactions, there arises a high hope that individual-ized medicine will soon become a reality. In this chapter, we will examine the origins of individual variability in

drug treatment; the role of drug targets, drug metabo-lizing enzymes, and drug transporters in determining the individual variability in drug therapy; and the many challenges we face in reaching the goal of individual-ized medicine.

7.2 INDIVIDUAL VARIABILITY IN DRUG THERAPY

It has been known for years that the optimum doses required for many therapeutic agents can vary to a sig-nifi cant extent from patient to patient. For example, the required daily dose of warfarin for inhibition of throm-bosis and embolism in many disease conditions can vary up to 20 – 30 - fold among individual patients, thus neces-sitating frequent blood coagulation testing to ensure effective and safe anticoagulation in patients.

The study by Davidson et al. (1997) on simvastatin, a member of the statin class of 3 - hydroxy - 3 - methyl - glutaryl - coenzyme A ( HMG - CoA ) reductase inhibitors and cholesterol - lowering agents, provides a clear example of large individual variability in drug response and drug safety. In a population of 156 healthy men and women, a 40 - mg daily dose of simvastatin reduced the low - density lipoprotein ( LDL ) cholesterol levels by 41% on average in 6 weeks, whereas the 80 - and 160 - mg doses resulted in median reductions of 47% and 53%, respectively, demonstrating that simvastatin is highly effective in reducing LDL cholesterol levels for the majority of the population. However, a small number of

ADME-Enabling Technologies in Drug Design and Development, First Edition. Edited by Donglu Zhang and Sekhar Surapaneni.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

96 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

genetic loci that exist in linkage disequilibrium, thereby identifying new disease susceptibility genes and path-ways. In this respect, a haplotype defi nes a set of genetic variants that are inherited together in linkage disequi-librium, and thus, is particularly useful in genome - phenotype analyses.

Variations in genome sequences among individuals may well underlie the differences in the human suscep-tibility to diseases, the onset and severity of illnesses, and the way humans respond to a drug treatment. Unraveling the role of genetic variations in the patho-genesis of a disease could lead to the identifi cation of disease genes. Understanding the role of genetic varia-tions in responses to therapeutic agents could stream-line the clinical development of new drugs by customizing drug interventions to specifi c drug target genotypes of patients; it could also improve the drug effi cacy and safety profi le by optimizing a drug dose according to the genotypes of drug metabolism and pharmacokinetics of individuals.

7.4 ORIGINS OF INDIVIDUAL VARIABILITY IN DRUG THERAPY

Multiple factors, either genetic or environmental, con-tribute to individual variability in drug therapy (Table 7.1 ). Genetic polymorphisms of the proteins involved in drug targeting and drug disposition are likely the most important source of individual variations in drug res-ponse and drug safety. Genetic variations can change the protein structure via mutations in the coding region or the amount of the protein expressed by modulating gene regulation, both of which alter the function of the protein, or the rate and kinetic constants in the case of an enzyme. Structural changes of receptors or enzymes can profoundly impact on the interaction between drugs and intended targets, and consequently, the drug res-ponse. Genetic polymorphisms of drug metabolizing enzymes and transporters are known to affect the absorption, distribution, metabolism, and elimination of many drugs. Alterations of DNA repair enzymes may weaken the ability of cells to defend against mutations and other toxic effects induced by many alkylating anti-cancer agents. Glutathione (GSH) plays an important role in protecting cells from oxidative stress and reac-tive intermediates generated from drugs. Structural alterations of enzymes involved in the biosynthesis of GSH may reduce the cellular GSH content to a low level at which cells are susceptible to attacks by reactive species, resulting in cell damage and death.

Whereas genetic factors commonly cause permanent changes in protein structures and individual responses to drug therapy, the effect of environmental factors on

individuals (approximately 5%) showed little or no reduction in their LDL cholesterol levels even at the high dose of 160 mg per day. In addition, a few individu-als ( < 2%) had slight elevations of plasma hepatic trans-aminase activities and signs of myopathy. The reason for such variability is currently unknown but genetic varia-tions are potential contributing factors. Chasman et al. (2004) reported that individuals carrying an HMG - CoA reductase genetic variant allele may exhibit signifi -cantly smaller reductions in the cholesterol levels when treated with pravastatin, another member of the statin cholesterol - lowering drugs. In a separate study, The SEARCH Collaborative Group (2008) identifi ed several common variants of the SLCO1B1 gene, which encodes the solute carrier organic anion transporter family member 1B1 protein; moreover, these variants are strongly associated with an increased risk of statin - induced myopathy.

7.3 WE ARE ALL HUMAN VARIANTS

Despite the overwhelming similarity in the nucleotide sequences of the human genome, there exist millions of points of DNA variations between any two randomly selected individuals. Genetic variations can result from a single - nucleotide polymorphism ( SNP ), nucleotide repeats, insertions, or deletions in DNA nucleotide sequences. Such changes can alter the amino acid sequence of a coded protein or the transcriptional expression of the gene. SNP is likely the most common gene variation. More than 1.42 million SNPs have been identifi ed in the human genome, among which more than 60,000 SNPs are found in the coding regions of the genes (Sachidanandam et al., 2001 ). Most human genes (> 90%) contain at least one SNP and nearly every human gene is marked by a sequence variation. There-fore, at the best approximation, we are all mutants or variants and we are all “fl awed ” to some extents at the genomic level.

The majority of SNPs appear to have no apparent effect on gene function. However, certain SNPs do have profound impact on the function of associated genes, whether the SNPs occur in the coding regions or at a signifi cant distance from the transcription starting sites of the genes. Some SNPs are known to be associated with signifi cant changes in drug effi cacy and drug dispo-sition (Evans and Relling, 1999 ; McLeod and Evans, 2001 ; Eichelbaum et al., 2006 ; Roden et al., 2006 ). It is becoming increasingly clear that an identifi cation of a single SNP may not be suffi cient to relate the variation of a target protein to a disease or a drug response. Therefore, new techniques are being developed to inte-grate sets of SNPs across the entire genome to identify

GENETIC POLYMORPHISM OF DRUG TARGETS 97

warfarin is 4 – 6 mg for individuals carrying the wild - type alleles (Table 7.2 ). A number of VKORC1 muta-tions (Ala41Ser, Arg58Gly, and Leu128Arg) have been identifi ed (Rost et al., 2004 ; Bodin et al., 2005 ; Rieder et al., 2005 ; Rettie and Tai, 2006 ). Although the fre-quencies of these mutations in human populations are rather low, all three variants exhibit a warfarin resis-tance phenotype. In particular, individuals carrying the Leu128Arg variant require very high doses of warfarin (> 45 mg per day) to achieve effective treatment of a thrombotic event.

Mutations of the β2 - adrenoreceptor, which is encoded by ADRB2 , may alter the airway response to β - agonist, such as albuterol. Lima et al. (1999) showed that alb-uterol evokes a larger and more rapid bronchodilation response in Arg16/Arg16 homozygotes (wild type) than in carriers of the Gly16 variant (Arg16/Gly16 and Gly16/Gly16). The maximal percent increase in albuterol - evoked forced expiratory volume in 1 s (FEV 1 ) is 18% for individuals carrying the Arg16/Arg16, but only 5% for those carrying the Gly16 variants after an oral dose of 8 mg of albuterol.

Chemokine receptor 2 (CCR 2) and chemokine receptor 5 (CCR 5) are two cofactors that are essential for the infection of human immunodefi ciency virus (HIV) in humans. The CCR 2 Val64Ile polymorphism is common in Caucasians and African - Americans with an allele frequency reaching 10% of the populations. Individuals carrying the Ile allele progress to acquired immune defi ciency syndrome (AIDS) 2 – 4 years later than those carrying the wild - type receptor (Smith et al., 1997 ). One important variant of CCR 5 carries a 32 base pair deletion ( Δ 32) (Samson et al., 1996 ). About 9% of Caucasians carry this allele, but the polymorphism is generally not found in Africans. Individuals carrying the Δ 32 deletion are protected from the transmission of HIV. These examples reveal that genetic polymorphisms of CCR 2 and CCR 5 can have signifi cant impact on HIV infection and AIDS onset.

drug response may be more transient in nature. Dietary constituents, environmental chemicals, and multiple drug use are known to induce or inhibit drug metaboliz-ing enzymes, particularly cytochrome P450s, resulting in drug levels that are either too low or too high for a proper drug response. In these scenarios, the drug response may return to a normal level after the envi-ronmental factors are removed from the cells. A large individual variability in the induction and inhibition of human cytochrome P450 enzymes has been well estab-lished (Lin and Lu, 2001 ). Physiological factors, such as age and disease state, also contribute signifi cantly to pharmacokinetic variability and drug response in patients.

7.5 GENETIC POLYMORPHISM OF DRUG TARGETS

Genetic variations in drug targets can have a profound effect on drug effi cacy. For instance, vitamin K epoxide reductase complex 1 ( VKORC1 ) is the target of war-farin in the treatment and prevention of thromboem-bolic diseases. Mutations in the VKORC1 coding region lead to warfarin resistance. The effective daily dose of

TABLE 7.1. Origins of Individual Variability in Drug Therapy

Factors Consequences a

Genetic factors Drug targets Alter drug effi cacy Drug metabolizing enzymes Alter drug metabolism Drug transporters Alter drug absorption,

distribution, and elimination

DNA repair enzymes, GSH level

Affect drug safety

Environmental factors P450 induction Decrease drug effi cacy P450 inhibition Cause potential drug – drug

interactions Physiological factors Age, gender, disease,

infl ammatory mediators, and so on

Affect drug absorption, distribution, metabolism, and elimination

a Stated consequences regarding cytochrome P450 induction and inhibition do not apply to prodrugs that require P450 - catalyzed acti-vation steps to form an active metabolite, or drugs that are converted by P450 to biologically active metabolites. In the former case, that is, prodrugs, only the metabolites are active, whereas in the latter case, both the parent drug and its metabolites can be active. One type of prodrugs takes the form of esters to improve drug availability; in this scenario, activation of the prodrugs generally involves hydrolysis by esterases.

TABLE 7.2. Mutations in VKORC1 Coding Region Leading to Warfarin Resistance

Amino Acid Change

Daily Warfarin Dose (mg)

Resistance Phenotype Reference

Wild type 4 – 6 – Ala41Ser 16 Moderate Rieder et

al. (2005) Arg58Gly 34 Major Rost et al.

(2004) Leu128Arg > 45 Severe Rost et al.

(2004) ; Bodin et al. (2005)

98 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

studies of drug metabolizing enzymes is clinically signifi -cant and meaningful.

Since the completion of the human genome project, numerous variants of drug metabolizing enzymes, par-ticularly those of cytochrome P450s, have been identi-fi ed and categorized. At the present, most of the variants were only evaluated for enzyme activities. With a few exceptions, the signifi cance of genetic variations of these variants in pharmacokinetics and clinical outcome of drug therapy has not been established. Thus, even though the DNA sequence - based analysis of variants is much faster than the classical approach used in past research for identifying new variants, the impact of many of the newly identifi ed genetic variants on drug therapy remains unclear.

The CYP2D6 polymorphism is one of the best studied among cytochrome P450s (Eichelbaum et al., 2006 ; Zhou et al., 2008 ). The CYP2D6 variant alleles are clas-sifi ed on the basis of enzymatic activities (Table 7.3 ). The frequency and genetic basis of major CYP2D6 vari-ants are well documented. In addition, methods for rapid and effective clinical testing of these variants are now available. The CYP2D6 phenotypes are particu-larly important in antidepressant therapy, as many of the available antidepressants are metabolized predomi-nantly by this enzyme. If CYP2D6 is mainly responsible for the blood level of a drug in humans, and the genetic polymorphism of the drug target is not an issue, knowing the CYP2D6 phenotype of an individual patient would allow physicians to prescribe a safe and effective dose of the drug to the patient.

CYP2C19 catalyzes the metabolism of many commonly used drugs, including S - mephenytoin (anti-convulsant), omeprazole (antiulcer), and diazepam (antianxiety). To date, more than 20 variants of CYP2C19have been identifi ed (Zhou et al., 2008 ). CYP2C19 * 2and * 3 are null alleles that result in a total loss of the enzyme activity. The majority of PMs of CYP2C19 are due to these two variant alleles. About 15 – 25% of the Chinese, Japanese, and Korean populations are PMs of S - mephenytoin. On the other hand, the frequency of PMs in Caucasians is much lower ( < 5%).

CYP2C19 plays a very important role in the proton pump inhibitor therapy for peptic ulcer and gastro-esophageal refl ux diseases. Furuta et al. (1999) showed that the effect of omeprazole on the intragastric pH value largely depends on the individual CYP2C19 geno-type. At a single dose (20 mg) of omeprazole, the plasma drug area under the curve ( AUC ) is the highest among PM subjects, the lowest among extensive metabolizer (EM) subjects, and medium in heterozygous EMs. These CYP2C19 genotype - based differences in AUC translate into differences in the extent and duration of inhibition of gastric acid secretion by omeprazole. The pharmaco-

Individualized therapy is particularly important for cancer patients, owing in part to the complexity of the disease, the severe toxicity of many anticancer agents, and the existence of many different genotypes of the same disease in patients. Understanding the crucial molecular abnormalities in cancer is an essential step in the design of an effective anticancer drug. Once this is established, the drug can be targeted to patients who have a particular molecular abnormality. The key for the success in this targeted therapy is to separate the nonresponders from the responders in clinical prac-tice. Success has been achieved with this approach for a number of malignancies. For example, imatinib has been used to specifi cally inhibit the tyrosine kinase activity of bcr - abl in tumor cells of chronic myeloid leukemia (Druker and Lydon, 2000 ); gefi tinib selec-tively targets the overexpressed EGFR mutant proteins in malignant cells for the treatment of nonsmall - cell lung cancer (Sordella et al., 2004 ); and trastuzumab is used for the treatment of breast cancer patients who have overexpressed HER2 receptor in cancer cells (Hudis, 2007 ).

7.6 GENETIC POLYMORPHISM OF CYTOCHROME P450 S

Since the 1960s — long before the concept of individual-ized medicine was established — individual variability of drug levels and genetic polymorphisms of drug metabo-lizing enzymes have been studied vigorously by pioneers including A. Conney, W. Evans, M. Eichelbaum, W. Kalow, R. Smith, E. Vesell, W. Weber, and R. Weinshilboum. Exemplary studies that address drug dosing, effi cacy, and toxicity issues include the phenotyping of thiopurine S - methyltransferase ( TPMT ) to identify cancer patients with a low activity in methylating toxic cancer drugs (an inactivation pathway) (Evans and McLeod, 2003 ; Wein-shilboum, 2003a ; Evans and Relling, 2004 ); the identifi ca-tion of “ slow acetylator ” ( N - acetyltransferase 2; NAT2) for isoniazid acetylation in the treatment of tuberculosis (Weber, 1987 ); and the phenotyping of “ poor metabo-lizer” (PM) of CYP2D6 - mediated debrisoquine hydrox-ylation (Eichelbaum et al., 2006 ). All these studies followed a classical approach: identifying individual phe-notypes by measuring drug levels in the urine or plasma before the genetic mechanism was known; establishing the pharmacokinetics of drugs in “ normal or extensive metabolizers” versus “ poor or slow metabolizers ” and their impact on drug effi cacy and safety; and fi nally, establishing the molecular mechanism of the genetic defect to explain the low or lack of the enzyme activity years later. Although this research process appears slow and tedious, the outcome of the genetic polymorphism

GENETIC POLYMORPHISM OF CYTOCHROME P450S 99

CYP2C9 is involved in the metabolism of many clini-cally important drugs including tobutamide (hypoglyce-mic agent), glipizide (hypoglycemic agent), phenytoin (anticonvulsant), S - warfarin (anticoagulant), and fl ubi-profen (anti - infl ammatory agent). At the present, more than 30 variants of CYP2C9 have been identifi ed. Two of the most common allelic variants are CYP2C9 * 2 and CYP2C9 * 3 . The variants exhibit largely reduced enzy-matic activities; the extent of such reduction is substrate dependent. There is a signifi cant difference in the fre-quency of CYP2C9 variants among different ethnic groups (Lee et al., 2002 ). Among Caucasians, about 1% are the CYP2C9 * 2 homozygous carriers and 0.4% the CYP2C9 * 3 homozygous carriers. In Chinese and Japa-nese populations, homozygous * 2, homozygous * 3, and heterozygous * 1/ * 2 carriers are very rare, whereas the heterozygous * 1/ * 3 accounts for 4%. The impaired metabolism of a low therapeutic index drug, such as warfarin, by CYP2C9 variants has very important clini-cal implications, which will be discussed in more detail in Section 7.11 .

CYP3A4 is the most abundant P450 enzyme in the human liver and is responsible for the metabolism of more than 50% of the drugs used clinically. More than twenty CYP3A4 variants have been identifi ed. Many of these variants have altered enzyme activities varying from a modest to signifi cant loss in catalytic effi ciency; the extent of the activity loss is often dependent on the substrate used (Miyazaki et al., 2008 ; Zhou et al., 2008 ). There is also a large difference across ethnic groups in the frequency of CYP3A4 variants. For example, there is a high frequency of CYP3A4 * 2 and * 7 in Caucasian and a high frequency of CYP3A4 * 16 and * 18 in Asian populations (Sata et al., 2000 ; Lamba et al., 2002 ).

Although large individual variations in the CYP3A4 activity have been identifi ed in human populations, the clinical signifi cance of the CYP3A4 variant alleles for many drugs that are metabolized by CYP3A4 remains uncertain. Based on current data, it appears that the clinical impact of CYP3A4 alleles is only minimal to moderate. These coding variants are unlikely to account for the more - than - 10 - fold differences in the CYP3A4 activity observed in vivo because the alleles produce only small changes in the enzyme activity and many of the alleles exist in low frequencies (Lamba et al., 2002 ). One factor that may contribute to the complexity of the CYP3A4 puzzle is CYP3A5, which is another member of the CYP3A family. Virtually all CYP3A4 substrates (with a few exceptions) are also metabolized by CYP3A5. Although CYP3A5 metabolizes these drugs at slower rates in most cases, some drugs can be metabo-lized by CYP3A5 at equal or greater rates than by the CYP3A4 enzyme. Therefore, the metabolic rates of CYP3A4 drugs measured in vivo may not be just a

kinetic data are in a good agreement with the observa-tions that the intragastric pH is 4.5 for the PM subjects, 3.3 for the heterozygous EM subjects, and 2.1 for the EM subjects. Schwab et al. (2004) have shown that the rate of eradication of Helicobacter pylori by a combina-tion therapy with lansoprazole and antibiotics is highly dependent on CYP2C19 in white patients who take a standard dose of lansoprazole (30 mg twice a day). Besides resistance to antibiotics, CYP2C19 polymor-phisms were identifi ed as the most important factor that affects the success of H. pylori eradication. Since individuals with the EM phenotype have lower serum concentrations of lansoprazole and lower rates of H. pylori eradication, the patients would benefi t from a higher dose of the proton pump inhibitor than the patients with the PM phenotype.

TABLE 7.3. CYP2D6 Genetic Polymorphism: Characteristics and Clinical Consequences

Phenotype Characteristics Consequences

Poor metabolizer(PM)

Frequency: 5– 10% Caucasians; 1– 2% Chinese and Japanese

High plasma drug level

Risk of drug - related side effects

Major variants: 2D6 * 3, * 4, * 5, * 6

Use of reduced drug dose

Enzyme inactive Ultrarapid

metabolizer( UM ) a

Frequency: 1 – 2% Caucasian; 30% Ethiopian

Multiple copy of CYP2D6 gene

Very low plasma drug level

Loss of drug effi cacy

Very high enzyme activity

Use of higher drug dose

Intermediate metabolizer( IM )

Major variants: 2D6 * 9, * 10, * 41

Lower dose for somepatients Chinese: very

high frequency of * 10

Low residual enzyme activity

Extensive metabolizer( EM )

Remaining population

Standard dose for most individuals Normal rate of

metabolism Not a uniform

group of individuals

a Does not apply to prodrugs or drugs with active metabolites; see Table 7.1 for more explanation.

100 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

of patients treated with isoniazid developed devastating nerve toxicity due to high blood levels of the drug. Two major genetically distinct phenotypes were identifi ed and were referred to as “ rapid acetylators ” and “ slow acetylators” ; the phenotypes were later attributed to differences in the enzymatic activities of NAT1 and NAT2 (Weber, 1987 ). To date, more than 15 NAT2 alleles have been identifi ed in humans. Individuals carrying NAT2 * 5A , NAT2 * 6A , and NAT2 * 7A are associated with the slow acetylator phenotype (Zhou et al., 2008 ). NAT2polymorphisms are also associated with cancer suscep-tibility to certain industrial chemicals. A poor acetylator phenotype leads to increased risks of lung, bladder, and gastric cancers if the individuals are exposed to carcino-genic arylamines for a long period of time.

Uridine 5 ’ - diphosphate - glucuronosyltransferase (UGT) 1A1 plays an important role in the glucuronidation of many commonly used drugs as well as certain endoge-nous substrates, such as bilirubin (Tukey and Strassburg, 2000 ). More than 100 UGT1A1 variant alleles have been identifi ed so far. There is a signifi cant difference among ethnic groups in the frequency of UGT1A1 vari-ants (Zhou et al., 2008 ). For instance, the frequency of the UGT1A1 * 6 mutation is high in Japanese and Chinese (16– 23%) but low in Caucasian populations ( < 1%). Because UGT1A1 is mainly responsible for the glucuronidation of bilirubin, the high frequency of UGT1A1 * 6 variant appears to contribute to the high incidence of neonatal hyperbilirubinemia in Asian chil-dren. Three inherited forms of unconjugated hyperbili-rubinemia are known to occur in humans (Kadakol et al., 2000 ; Tukey and Strassburg, 2000 ): the Crigler – Najjar syndrome Type I and Type II are caused by variant alleles in the UGT1A1 coding region and the Gilbert’ s syndrome by polymorphisms in the UGT1A1promoter. In the Crigler – Najjar syndrome Type I, bili-rubin glucuronidation in patients is completely lacking (e.g., UGT1A1 * 6), which leads to very high serum levels of unconjugated bilirubin and early childhood death. Patients with the Crigler – Najjar syndrome Type II show markedly reduced activities of bilirubin glucuronidation (10– 30% of normal). A genetic polymorphism in the promoter region of the UGT1A1 gene (e.g., UGT1A1 * 28) results in a reduced expression of the UGT enzyme, leading to the Gilbert ’ s syndrome. The signifi cance of UGT1A1 * 28 variant in the toxicity of the anticancer drug irinotecan will be discussed in Section 7.9 .

7.8 GENETIC POLYMORPHISM OF TRANSPORTERS

Infl ux and effl ux transporters play important roles in the absorption, distribution, and elimination of therapeutic agents. Genetic polymorphisms of transporters can pro-

measurement of the activity of CYP3A4, but also that of CYP3A5. Since about 25% of whites and 50% of blacks express functional CYP3A5 (Kuehl et al., 2001 ), this dual pathway potentially obscures the clinical effects of CYP3A4 variants in human studies. The pre-diction of CYP3A4 phenotypes is also complicated by the fact that the effect of polymorphisms of the CYP3A4 gene on the activity of a variant enzyme is often sub-strate dependent. Additionally, unlike the phenotyping of CYP2D6 and CYP2C19, clinically meaningful pheno-typing of CYP3A4 by using probe drug substrates in a population has not been successfully established.

7.7 GENETIC POLYMORPHISM OF OTHER DRUG METABOLIZING ENZYMES

In addition to cytochrome P450s, many other drug metabolizing enzymes also play an important role in the metabolism of drugs of diverse chemical structures. Genetic polymorphisms of these enzymes are often associated with various human diseases and drug effi -cacy and toxicity issues.

The cytoplasmic TPMT catalyzes the S - methylation of a number of thiopurine drugs, such as 6 - mercaptopurine, azathioprine, and thioguanine, that are commonly used for the treatment of leukemia and autoimmune diseases. The effi cacy and safety of these agents lie in the balance of two metabolic pathways: (1) activation of the drugs to 6 - thioguanine nucleotides followed by incorporation of the nucleotides into nucleic acids that cause the death of leukemia cells; and (2) inactivation of the drugs to inac-tive metabolites (Evans and Johnson, 2001 ; Eichelbaum et al., 2006 ). Since S - methylation by TPMT is the pre-dominant inactivation pathway of thiopurine drugs, indi-viduals carrying defective TPMT variants accumulate higher levels of cytotoxic thioguanine nucleotides than those with the wild - type alleles after receiving a standard dose of the drugs, thereby leading to severe hematologi-cal toxicity. More than 20 variant alleles of the TPMTgene have been documented (Zhou et al., 2008 ). In Cau-casians, about 90% of the population inherit a high enzyme activity, 10% with an intermediate activity (het-erozygous), and 0.3% with a low or no activity. Individu-als carrying defective TPMT alleles, such as TPMT * 2 , TPMT * 3A , and TPMT * 3C , have poor enzymatic activi-ties. They are at risk of developing hematological toxicity if given the normal dose; therefore, a reduced drug dose should be prescribed to these patients.

N - acetyltransferase s ( NAT s) catalyze the acetylation of aromatic amines and hydrazines. Human variability in drug acetylation was discovered more than 50 years ago during the initial clinical trials of isoniazid as an antituberculosis drug (Evans et al., 1960 ). Although iso-niazid is a remarkably effective drug, a high percentage

PHARMACOGENOMICS AND DRUG SAFETY 101

alleles may alter drug exposure by affecting the biliary secretion of drugs and metabolites. Therefore, ABCG2genotypes clearly infl uence the disposition, effi cacy, and safety of drugs.

7.9 PHARMACOGENOMICS AND DRUG SAFETY

One of the most challenging issues in drug safety is related to drug toxicities that are idiosyncratic in nature. Idiosyncratic adverse drug reactions, which are charac-terized by their rare occurrence and the requirement of multiple exposures, represent the most extreme cases of individual variability in drug safety. Because the number of individuals affected by this type of toxicity is rather small, a major challenge is to identify the gene or genes that are responsible for the toxic events and the indi-viduals that are prone to the injury caused by a specifi c drug. It is by no means an easy task, but the large number of genetic variants known to be present in human popu-lations may aid in addressing this diffi cult safety issue.

Statins, such as simvastatin, atorvastatin, and pravas-tatin, are HMG - CoA reductase inhibitors. Statin thera-pies result in a large decrease in LDL cholesterol levels and a reduction in cardiovascular events. In rare cases, however, statins cause myopathy (muscle pain or weak-ness) that occasionally leads to rhabdomyolysis (muscle breakdown and myoglobin release), which in turn causes renal failure and even death, particularly at high doses of statins. In a large SEARCH (Study of the Effectiveness of Additional Reduction in Cholesterol and Homocysteine) trial involving 12,064 subjects, 96 participants from the 80 - mg - simvastatin - daily - dose group known to have developed myopathy during treat-ment were selected for a genome - wide association study using 316,184 SNPs in comparison with 96 control sub-jects (no documented myopathy) from the 80 - mg - daily - dose group (The SEARCH Collaborative Group, 2008 ). A single strong association of myopathy was established with the rs4363657 SNP marker within the SLCO1B1gene on chromosome 12. No SNPs in any other region of the chromosome were clearly associated with myopa-thy. SLCO1B1 encodes the organic anion transporting polypeptide OATP1B1 that is known to regulate the hepatic uptake of statins. It appears that the SLCO1B1variants diminish the hepatic uptake of statins leading to higher drug concentrations in the circulation to cause myopathy. Therefore, genotyping of SLCO1B1 may help screen out those individuals with abnormal SLCO1B1 activities, and thereby achieve the benefi ts of statin therapy safely and effectively. This study illus-trates the power of genome - wide associations in relat-ing genetic variants to drug response and drug toxicity, particularly if a single gene is involved in such events.

foundly affect drug disposition, drug response, and drug safety. P - glycoprotein (Pgp, MDR1, or ABCB1) encoded by ABCB1 has received much attention because many clinically important drugs are substrates of Pgp. The ABCB1 gene is highly polymorphic with numerous documented variants. Ethnic - dependent frequencies of some allelic variants are well known. Among many of the naturally occurring genetic variants of ABCB1 , SNP C3435T is of particular interest due to its high frequency (20– 60%) in many populations (Zhou et al., 2008 ). The functional signifi cance of C3435T has been studied on the disposition of digoxin and other Pgp substrates. Confl icting results have been reported: One study found lower serum digoxin concentrations in individuals car-rying the variant T alleles than in wild - type subjects, whereas another study showed higher plasma digoxin levels in mutant carriers than in carriers of the wild - type gene (Sakaeda et al., 2001 ; Verstuyft et al., 2003 ). The discrepancy between these studies is in part due to the fact that C3435T may not be the only polymorphism that affects the Pgp expression level. In this respect, the expression level of Pgp may be determined by polygenic traits rather than a monogenic one. Thus, the infl uence of the C3435T polymorphism on the pharmacokinetics and pharmacodynamics of Pgp substrates remains to be defi ned.

The human organic anion transporting polypeptide - C ( OATP - C ) coded by SLC21A6 is a liver - specifi c trans-porter and is important for the hepatic uptake of a variety of endogenous compounds and therapeutic agents. Tirona et al. (2001) characterized 16 OATP - Calleles in vitro and found several variants (e.g., OATP - C * 5 , OATP - C * 9 ) that show reduced uptake of OATP - C substrates, such as estrone sulfate and estra-diol 17 β - D - glucuronide. Since the genotypic frequency of the OATP - C * 5 variant is 14% in European - Americans and the frequency of OATP - C * 9 is 9% in African - Americans, these variants may have signifi cant effects on the disposition of OATP - C drug substrates. Indeed, individuals carrying OATP - C * 5 have been found to have high plasma levels of the cholesterol - lowering drug pravastatin (Nishizato et al., 2003 ; Mwinyi et al., 2004 ) and the antidiabetic drug repaglinide (Niemi et al., 2005 ), both of which are OATP - C substrates.

The breast cancer resistance protein (BCRP/ABCG2) plays an important role in regulating the intestinal absorption and biliary secretion of drugs, drug metabo-lites, and toxic xenobiotics (Gradhand and Kim, 2008 ). The ABCG2 C421A (Q141K) genotype is widely present in ethnic groups (30 – 60% in Asians and 5 – 10% in Caucasians and African - Americans). Sparreboom et al. (2004) found that heterozygous patients for Q141K have 300% higher plasma levels of difl omotecan, an anticancer agent, after an intravenous drug administra-tion of the drug. These fi ndings indicate that the ABCG2

102 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

clot control; on the other hand, patients given too high a dose can experience excessive and uncontrolled bleed-ing. Due to high rates of adverse events associated with warfarin therapy, frequent monitoring is necessary and is commonly used clinically to achieve appropriate anti-coagulation. For years, physicians have used the “ pro-thrombin time, ” a blood test for measuring the time required for blood to clot after medication, to adjust the warfarin dose and obtain a desired balance between the clinical benefi t and bleeding risk. Prothrombin time has been standardized to the “ international normalized ratio” ( INR ). The current practice involves the use of a standard 4 – 6 - mg daily dose of warfarin or an initial dose estimated according to the patient ’ s clinical character-istics; prothrombin time is then measured and adjust-ment is made based on the INR response with a goal to maintain INR in a range between 2 and 3. The time period required to reach this INR range can differ sig-nifi cantly among individuals, ranging from several days to several months. Nevertheless, the test does provide a simple, rapid, and relatively cheap way for clinicians to identify warfarin doses at which individual patients can be treated safely and effectively.

The pharmacological target of warfarin therapy is VKORC1 , a gene encoding a subunit of the vitamin K epoxide reductase complex (Rettie and Tai, 2006 ; Limdi and Veenstra, 2008 ; Kim et al., 2009 ). The vitamin K reductase catalyzes the conversion of vitamin K to reduced vitamin K, a component required for the car-boxylation of hypofunctional blood clotting factors including factors II, VII, IX, X, and other regulatory proteins to activated factors by γ - glutamyl carboxylase ( GGCX ). Blocking the generation of the reduced form of vitamin K by warfarin interferes with the formation of functional clotting factors through GGCX. Oral war-farin is well absorbed, and the S - enantiomer of warfarin is responsible for most of the VKORC1 inhibition. In the human liver, the S - warfarin is metabolized pri-marily to the inactive metabolite 7 - hydroxywarfarin by CYP2C9.

In patients carrying the wild - type CYP2C9 * 1 allele, S - warfarin is cleared from the body normally, and the standard daily dose results in a modest elevation of INR. PMs who carry the CYP2C9 * 2 and/or CYP2C9 * 3alleles have impaired capacity of metabolizing S - warfarin, and thus require a reduced daily dose of warfarin. These patients have a two - to threefold higher risk of an adverse event than those with the wild - type allele when warfarin therapy is initiated. The most common polymorphisms of the VKORC1 gene are within the noncoding region, which alter the expression of the enzyme (Rettie and Tai, 2006 ; Limdi and Veen-stra, 2008 ). For example, individuals carrying the 1173T/T allele require about half of the warfarin daily

Irinotecan is a potent DNA topoisomerase I inhibi-tor used in the treatment of colorectal and lung cancers (Tukey et al., 2002 ). Irinotecan is a prodrug that is con-verted by liver carboxylesterases to SN - 38, the active topoisomerase inhibitor. SN - 38 is glucuronidated by UGT1A1 and then eliminated from the body. High levels of SN - 38 are associated with bone marrow toxic-ity (leucopenia) and gastrointestinal ( GI ) toxicity (severe diarrhea). Thus, the benefi t of irinotecan therapy lies in a balance between the inhibition of topoisomer-ase I in cancer cells and the glucuronidation of SN - 38 by UGT1A1 to minimize toxicity. Individuals carrying UGT1A1 variants could face severe toxicity problems in irinotecan therapy due to the accumulation of toxic SN - 38. The UGT1A1 * 28 polymorphism is such an example. The presence of seven, instead of six, TA repeats in the UGT1A1 promoter region results in a decreased expression of the UGT1A1 protein and low glucuronidation of SN - 38 (Kadakol et al., 2000 ; Zhang et al., 2007 ). Patients that are homozygous or heterozy-gous for the UGT1A1 * 28 polymorphism have elevated levels of SN - 38 due to a decrease in the glucuronidation activity, and therefore are more susceptible to bone marrow and GI toxicities if given an irinotecan treat-ment. The FDA has recommended that patients be genotyped for the UGT1A1 * 28 polymorphism and dose adjustments be made before an irinotecan treatment.

Many drug - related toxicity issues remain unsolved. A key to address the issues is to understand the mecha-nism of toxicity (drug related versus drug target related), determine the gene or genes responsible for the toxic events, and develop reliable biomarkers for screening. The two examples discussed above illustrate the utility of these approaches and bring hope that many of the toxicity problems can eventually be resolved and measures can be taken, for instance, by adjusting drug dose or using alternative drugs, to minimize the injury to patients according to individual genotypes and phenotypes.

7.10 WARFARIN PHARMACOGENOMICS: A MODEL FOR INDIVIDUALIZED MEDICINE

The anticoagulant agent warfarin is widely used for the treatment and prevention of thromboembolic diseases. Due to its narrow therapeutic index and wide individual variability in drug response, warfarin has been used as a model drug for studying individualized medicine (Rettie and Tai, 2006 ). The standard daily dose of war-farin is between 4 and 6 mg; however, in a large patient population, the effective daily dose of warfarin can vary from 0.5 to over 30 mg. Patients who receive an insuf-fi cient dose of warfarin will be at risk of failing blood

WARFARIN PHARMACOGENOMICS: A MODEL FOR INDIVIDUALIZED MEDICINE 103

other, yet - to - be identifi ed factors reach almost 50%. Perhaps this is the major reason that many clinicians do not believe that the time is ripe for incorporating geno-typing testing in warfarin therapy, particularly when the “ prothrombin time ” test, though not perfect, is available in clinics and provides reasonable information for dose adjustment. In an editorial titled “ Genetic testing for warfarin dosing? Not yet ready for prime time, ” Bussey et al. (2008) stated that “ some experienced clinicians question whether genetic testing adds signifi cantly to the information one may discern by carefully monitor-ing the INR and by taking into consideration the numer-ous patient - specifi c factors that infl uence warfarin dosing requirements, such as age, underlying disease states, and concomitant drugs. ”

The International Warfarin Pharmacogenetics Con-sortium (2009) has used clinical data (age, height, weight, and race) and genetic data ( CYP2C9 * 1 , * 2 , and * 3 , and VKORC1 variants ) of 4043 patients from nine countries to estimate the warfarin doses to achieve targeted INR. The pharmacogenetic algorithm accurately identifi ed 50% of the patients who require 3 mg of warfarin or less per day and 25% who require 7 mg or more per day to achieve the target dose. Thus, despite impressive progress made in identifying CYP2C9 and VKORC1variants, the successful prediction of a target clinical warfarin daily dose based on clinical and genetic data is only 25 – 50%, which is consistent with the fact that almost 50% of the factors responsible for variable warfarin responses remain unknown.

The clinical use of pharmacogenetic testing, such as in the case of warfarin therapy, is severely limited by a lack of prospective clinical trials that demonstrate that incorporating genetic testing can indeed benefi t the selection of appropriate therapeutic agent and drug dose for individual patients to improve therapeutic response and reduce adverse drug effects. Research efforts designed to evaluate the effectiveness of genotype - guided warfarin therapy in improving

dose compared with patients with the wild - type 1173C/C allele. In other cases, a higher - than - standard daily dose is required for individuals carrying certain VKORC1noncoding SNPs. Polymorphisms in the coding regions of the VKORC1 gene often lead to varying degrees of warfarin resistance (Table 7.2 ). The number of resistant patients examined to date is relatively small.

Because of the large variation and diffi culties in achieving effective and safe anticoagulation associated with warfarin therapy, it is apparently preferable to have a more accurate prediction of the initial dose based on the pharmacogenetics of each individual to improve anticoagulation control. It is believed that genotyping of patients prior to a warfarin therapy potentially reduces warfarin adverse events and facili-tates achieving stable INR effi ciently in patients with genetic variations in CYP2C9 and VKORC1 (Gage and Lesko, 2008 ; Kim et al., 2009 ). In August 2007, FDA revised the warfarin labeling to include pharmacoge-netic information in the labeling. FDA has also approved four genetic tests for warfarin use, one of which pro-vides fast testing in less than an hour. However, shortly after the new warfarin labeling was initiated, FDA issued a brief press release, apparently due to the nega-tive response from the medical community, indicating that the new label is not a mandatory requirement for clinicians to genotype patients before the initiation of a warfarin therapy. In a special article, Limdi and Veen-stra (2008) stated that, although the infl uence of CYP2C9 and VKORC1 genotypes on warfarin dose requirements has been consistently demonstrated in observational studies and randomized clinical trials, evi-dence to date from prospective, controlled studies has not demonstrated an added benefi t of incorporating genotype - guided therapy in improving anticoagulation control or in preventing or reducing adverse effects. Therefore, they concluded that the routine use of CYP2C9 and VKORC1 genotyping in the general patient population who begin warfarin therapy is not supported by evidence currently available.

Variability in warfarin therapy is apparently a complex issue involving more than just the genotypes of CYP2C9 and VKORC1 and several other known physiological factors (Table 7.4 ). Although the variable metabolism of S - warfarin by CYP2C9 was identifi ed initially as a major contributor to variable responses of the drug, it is now recognized that the contribution of the variable metabolism by CYP2C9 is rather small, estimated to be about 10% of warfarin dose variations. The contribution of VKORC1 genotypes to dose varia-tions is approximately 25%, and clinical factors such as age, gender, diet drugs, and body mass index another 20%. Therefore, the identifi ed factors that contribute to variable warfarin responses are roughly 50%, whereas

TABLE 7.4. Contributions of Genetic and Nongenetic Factors to Population Variance in Warfarin Dose

Factors % Contribution

to Dose Variance

Vitamin K epoxide reductase complex 1 (VKORC1)

25

CYP2C9 10 γ - Glutamyl carboxylase 2 Clinical factors (age, gender,

drugs, diet, body mass index) 20

Unknown 43

Adapted from data in Au and Rettie (2008) .

104 PHARMACOGENOMICS AND INDIVIDUALIZED MEDICINE

genotype. Therefore, it remains unclear whether indi-vidualized drug therapy will ever be achievable by means of DNA testing. It was suggested that, perhaps in combination with proteomics, metabonomics might complement genomics in achieving individualized drug therapy.

7.12 CONCLUSIONS

The ultimate goal of individualized medicine is for phy-sicians to prescribe an appropriate medication for the right target of the disease and at the right dose for indi-vidual patients to achieve maximal effi cacy with minimal adverse effects. Although the concept is very attractive and the goal is noble, good clinical data to support the use of genetic testing for drug treatment for most dis-eases are still not yet available. Take warfarin dosing as an example: Despite excellent research and high hopes, the target dose can be successfully predicted based on genetic and clinical information for only 25 – 50% of the patients. Factors responsible for the remaining 50% of drug variations remain unknown.

The challenge for achieving individualized drug therapy is enormous. It is perceivable that a long and rough road lies ahead. The achievements so far have been limited. Perhaps, instead of asking for a full package to address all questions involved in individual-ized medicine, we may deal with individual issues, one at a time, at different stages in the quest for answers. From this prospective, several important achievements can be highlighted at what is the starting point in this long journey. Target therapies with imatinib, gefi tinib, and trastuzumab for certain genotype cancer patients have pointed to the direction for future cancer therapy. The doses for antidepressant drugs can be adjusted in patients based on their CYP2D6 phenotypes to mini-mize drug toxicity. Genotyping of UGT1A1 * 28 in colon cancer patients before irinotecan treatment is an impor-tant measure to decrease the GI and bone marrow tox-icities of irinotecan. Genotyping of SLCO1B1 in patients undergoing a statin therapy potentially helps achieve the benefi t of cholesterol - lowering drugs more safely and effectively.

Based on these successes, it is believed that basic research will continue to be essential to understanding the mechanism of the pathogenesis of diseases and the roles of genetic variations of disease gene(s), drug targets, and all proteins important for drug disposition in determining drug response variations. The integration of genomics, proteomics, and metabonomics in genome - wide association studies will facilitate the identifi cation of predisposing genetic factors associated with multifac-torial diseases and drug response. Finally, prospective

therapeutic outcomes are under way. Defi nitive clinical outcomes in such studies would certainly be welcome news for the new era of individualized medicine.

7.11 CAN INDIVIDUALIZED DRUG THERAPY BE ACHIEVED?

The answer to this question varies from optimistic to pessimistic, in part depending on to what extent one considers it a success in achieving the goal of individual-ized medicine. When asked about the status of pharma-cogenomics and individualized medicine in drug therapy, Richard Weinshilboum (2003b) replied, “ The future is here! In psychiatry, where many of the drugs are metab-olized by CYP2D6, our psychiatrists at Mayo Clinic have begun to request that the CYP2D6 genotype infor-mation be made available before drug therapy. ” He was, of course, referring to the fact that individuals car-rying CYP2D6 * 3 , * 4 , * 5 , or * 6 variants should be given reduced doses of antidepressants to avoid or reduce drug side effects. In this regard, individualized medicine could be achieved when the metabolism of a drug is the major factor affecting drug response and safety. Of course, this is the simplest case in individualized medi-cine in which the variable factor is simple and well defi ned. However, in most cases, variable drug responses involve multiple factors, such as in the case of warfarin. Therefore, achieving individualized medicine for many diseases requires one to understand the pathogenesis of the disease, identify the gene(s) responsible for the disease, and establish the role of genetic polymorphisms of drug targets, transporters, and drug metabolizing enzymes involved in the drug therapy. It is particularly challenging in achieving these goals if disease genes are unidentifi ed.

On the other hand, Nerbert and coworkers (Nebert and Vesell, 2006 ; Nebert et al., 2008 ) questioned whether personalized drug therapy can ever be achieved by means of DNA testing alone. At the present time, the major goal of clinical pharmacology and pharmacoge-nomics is to establish phenotype – genotype associations through genetic tests that reveal genetic predispositions to a disease and drug toxicity. The practical purpose is to identify patients who are drug responders and patients who are prone to drug toxicity. Although some success has been achieved in recent years in establishing such phenotype– genotype associations for monogenic disor-ders, it was argued that, because of the complexity of the genome, this task is far more challenging than origi-nally anticipated. Based on their analysis of literature data, the authors concluded that, for complex diseases involving multiple genes, it would be very diffi cult to determine unequivocally either an exact phenotype or

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clinical trials that evaluate the utility and effectiveness of genotyping and individualized medicine are critical in guiding the research and clinical practice of individu-alized medicine in the future.

DISCLAIMER

The fi ndings and conclusions in this chapter are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

CONTACT INFORMATION

Q. Ma: Receptor Biology Laboratory, Toxicology and Molecular Biology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Preven-tion, 1095 Willowdale Rd., Morgantown, WV, 26505, USA; Telephone: (304) 285 - 6241; Email: qam1@cdc.gov .

A.Y.H. Lu: Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscat-away, NJ, 08854 USA; Telephone: (732) 445 - 3400; Email: antylu@rci.rutgers.edu

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