Pharmacogenomics and adverse drug reactions in diagnostic and clinical practice 1)

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1 Article in press - uncorrected proof Clin Chem Lab Med 2007;45(7): by Walter de Gruyter Berlin New York. DOI /CCLM /180 Review Pharmacogenomics and adverse drug reactions in diagnostic and clinical practice 1) Vangelis G. Manolopoulos* Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece Abstract Pharmacogenetics and pharmacogenomics deal with genetically determined variations in how individuals respond to drugs. They hold the potential to revolutionize drug therapy. The clinical need for novel approaches to improve pharmacotherapy stems from the high rate of adverse reactions to drugs and their lack of effectiveness in many individuals. Despite the accumulation of research findings showing the potential for clinical benefit for several drug-metabolizing enzymes and some receptors that constitute drug targets, the translation of these findings into tangible clinical applications occurs very slowly. The main steps for clinical implementation of pharmacogenomics include: a) education of clinicians and all other parties involved in the use and benefits of pharmacogenomics; b) execution of large prospective clinical and pharmacoeconomic studies showing the benefit of pharmacogenomic genotyping; c) provision of incentives to develop tests; d) development of specific clinical guidelines; and e) creation of a solid regulatory and ethical framework. Furthermore, the potential should be explored to use existing therapeutic drug monitoring laboratories to introduce pharmacogenomic testing into hospitals. Overall, our thesis is that pharmacogenomics is already a reality in clinical practice and is bound to continue gaining acceptance by clinicians in the coming years. Clin Chem Lab Med 2007;45: Keywords: adverse drug reactions; CYP2D6; CYP2C9; CYP2C19; genetic polymorphism; pharmacogenetics; pharmacogenomics; therapeutic drug monitoring; thiopurine S-methyltransferase (TPMT); uridine diphosphate glucuronosyltransferase (UGT1A1). 1) This article is based on a contribution at the 3rd Santorini Biologie Prospective Conference, Sep 29 Oct 2, *Corresponding author: Dr. Vangelis G. Manolopoulos, Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus, Alexandroupolis, Greece Phone/Fax: q , manolopoulos@med.duth.gr Introduction Pharmacogenetics has been around since the 1950s, representing the study of individual pharmacological response based on genotype. Its objective is to personalize medicine in a continuous quest to offer safer, more efficient, and individual pharmacotherapy to patients, providing clinicians with effective scientific tools that will guide them to choose the correct drug at an adequate dose for every patient. It is an area in which clinical pharmacology incorporates genetics in efforts to provide better drug treatment for patients. The genetic basis of the differential response to drugs has been established by understanding the biological and pharmacological pathways that led to the identification of candidate genes, including single nucleotide polymorphisms that affect drug response. In the last 15 years, renewed interest in the topic has emerged, largely due to the great technical developments in molecular biology methods for rapid and efficient analysis of the human genome. These developments led to the introduction of the term pharmacogenomics (PGx) in 1998 to reflect the evolution of pharmacogenetics into the study of the entire spectrum of genes that determine drug responses, including assessment of the diversity of the human genome sequence and its clinical consequences (1). While it is clear that the terms pharmacogenetics and PGx are not identical (pharmacogenetics focuses on one gene while PGx has a wider brief and covers all the genes), they have increasingly been used interchangeably, perhaps reflecting the increasing use of genomic approaches and techniques to study pharmacogenetic questions. To avoid the confusing use of both terms, we use the term PGx here to represent any influence that genetics may have on drug therapy. Recent years have seen an explosion of research and publication activity in pharmacogenetics and PGx. A recent (March 2007) search in Medline for publications containing the terms pharmacogenetics or pharmacogenetic* revealed 6612 hits, while the terms pharmacogenomics or pharmacogenomic* yielded 4985 hits. A search for the presence of either of the two terms provided 7425 hits, demonstrating that the two terms are often used interchangeably within the same publication. Several scientists have expressed optimistic views for the potential of PGx to revolutionize drug therapy (2 5). However, this increased interest and research activity has yet to be translated into substantial progress in the clinical application of research findings for the benefit of patients. This lag has led to the expression of more skeptical views regarding the potential and future of

2 802 Manolopoulos: Pharmacogenomics in clinical practice Article in press - uncorrected proof PGx (6, 7). A report on personalized medicine by the University of Cambridge concluded that there is widespread recognition that pharmacogenetics may have been oversold, the basic science behind it is still substantially uncertain, and media excitement about genetic applications may be exaggerating investment and research activity in pharmacogenetics (8). In the present review we first present the facts regarding adverse drug reactions (ADRs) that clearly demonstrate the need for therapy individualization. Then we present findings for some molecules for which there is enough clinical evidence to support the translation of basic science into specific clinical applications. Thereafter, we address the actual situation regarding clinical uptake of PGx in various countries, followed by analysis of some key reasons for the hindrance of more widespread clinical application of even the well-founded cases of PGx. Furthermore, we discuss the relation between PGx and therapeutic drug monitoring (TDM), as well as some international initiatives to promote and solidify clinical PGx. We focus only on drugs already in clinical use. The huge impact of PGx on drug development and clinical trials is beyond the scope of this article and the reader is referred to recent excellent reviews on the subject (9, 10). Adverse drug reactions and pharmacogenomics From its initiation, pharmacology has taken a more or less experimental and empirical approach to disease therapy. Large variability in the response to any given drug has always been taken as an inevitable part of pharmacotherapy. The response to medication varies greatly between individuals, according to genetic constitution, age, sex, co-morbidities, environmental factors including diet and lifestyle (e.g., smoking and alcohol intake), and drug-related factors such as pharmacokinetic and pharmacodynamic drug-drug interactions. When a drug is given to an individual to treat a condition on the basis of a specific diagnosis, the following possibilities exist: a) normal drug effect; b) pronounced drug effect; c) none or a suboptimal effect; and d) unwanted effects, usually called ADRs, adverse drug events or simply side effects. For centuries, this spectrum of reactions has been taken for granted by both parties involved (doctors and patients). However, they may be avoidable in some cases if pharmacogenetic factors affecting both the pharmacokinetic and pharmacodynamic aspects of drug action on the patient are taken into consideration. ADRs represent one of the major causes of hospitalization, in some cases leading to death. The large negative impact of ADRs has been shown in several studies, as recently reviewed (11). Ingelman-Sundberg et al. estimated that ADRs cost US society nearly US$100 billion and are the cause of up to 100,000 deaths annually in the US, and that up to 7% of all hospital admissions in the UK and Sweden are due to ADRs (12). A recent report by the US Centers for Disease Control and Prevention (CDC), based on comprehensive data analysis from the first 2 years ( ) of the Cooperative Adverse Drug Event Surveillance CDC project, stated that ADRs accounted for 6.7% of all US emergency department visits leading to hospitalization (13). Very similar findings have been reported in a large UK survey showing that 6.5% of 18,820 admissions to two large general hospitals in England over a 6-month period were a direct result of ADRs (14). Very similar rates have also been reported from Germany, France and Spain (15 17). Most ADRs are type A reactions, i.e., plasma-leveldependent. Both pharmacokinetic and pharmacodynamic mechanisms are involved in the relation between initial drug dose and final biological effect(s). Genetic polymorphisms in drug-metabolizing enzymes, transporters or pharmacological targets of drugs constitute the genetic cause of individual diversity of responses to drugs observed in clinical practice. In addition, it should be remembered that variations in the responses of individuals to drugs may be related not only to pharmacogenetic polymorphisms, but also to non-genetic factors, including co-medications and concurrent diseases. The contribution of pharmacogenomic variation to ADRs is assumed to be significant (2, 18, 19), but remains largely unsubstantiated. Some indirect evidence was provided for PGx as a factor in ADRs by Phillips and co-workers in 2001 (20). They analyzed data for the 27 drugs most frequently cited in ADR studies and showed that 59% (16/27) of them were metabolized in the body by at least one enzyme with a variant allele causing poor metabolism, of which 69% (11/16) were metabolized by CYP2D6 (mainly antidepressants and b-blockers). On the other hand, only 7% 22% of randomly selected drugs are known to be metabolized by enzymes with genetic variability. These data are compelling; however, prospective studies are still missing. Which pharmacogenomic biomarkers are ripe for widespread application to clinics? Intensive research is constantly generating new pharmacogenomic information with potential clinical relevance in almost all fields of medicine. An increasing number of reports describing differences in drug response as a result of genetic polymorphisms in numerous molecules that affect either the pharmacokinetic or pharmacodynamic fate of each drug have been published. These molecules could be considered as novel genetic biomarkers (see below). However, one major drawback of most of these reports is the lack of explicit statements on how to translate this information for use in clinical drug therapy, and often the study designs are inadequate to draw clinically applicable conclusions (21). It is therefore difficult for the clinician to evaluate the available information on each reported pharmacogenomic association and decide if and how it can be translated to specific prac-

3 Article in press - uncorrected proof Manolopoulos: Pharmacogenomics in clinical practice 803 tical guidelines useful and applicable in daily clinical practice. A consortium of scientists met in a workshop organized by the Food and Drug Administration (FDA), Johns Hopkins University, and the Pharmaceutical Research and Manufacturers Association of the US in September 2004 to assess evidence of the clinical utility of pharmacogenomic tests. They first agreed that a molecule should be considered as a known valid biomarker if it is measured in an analytical test system with well-established performance characteristics and for which there is widespread agreement in the medical or scientific community about the physiological, toxicological, pharmacological or clinical significance of the results (22). Accordingly, a pharmacogenomic biomarker would be a molecule that expresses a measurable genetic polymorphism with proven association to variable response to a drug. Such a biomarker could be measured in patients either prospectively (before drug therapy is initiated) or during the course of therapy when the therapeutic course does not meet expectations (lack of effect or ADRs). Following this definition, they suggested that the following enzymes should be included in the category of known valid pharmacogenomic biomarkers: CYP2D6, CYP2C19, CYP2C9, thiopurine S-methyltransferase (TPMT), and uridine diphosphate glucuronosyltransferase (UGT1A1) (22). The evidence available for several other molecules including additional drug-metabolizing enzymes and transporters, as well as receptors and proteins related to the pharmacodynamic aspect of drug action although substantial was not enough to classify them as valid pharmacogenomic biomarkers; these molecules were placed in the category of exploratory pharmacogenomic biomarkers. In this category CYP3A4/5, CYP1A2, ABCB1 (MDR1), CYP2B6 and several others molecules were included. Another attempt to assess the available data and evaluate the clinical maturity of pharmacogenomic tests for their use as diagnostic tools was published in 2005 (21). These investigators introduced the concept of pharmacogenomics-based reasonable diagnostics. They proposed that specific metabolizing enzymes already constitute reasonable diagnostic tools for monogenic influences with strong and evident drug-induced phenotype. In these cases, the genetic variant has a significant influence on pharmacokinetics, drug efficacy, or ADRs. Therefore, these enzymes constitute conventional standards, and pharmacogenetic analysis of all polymorphisms with relevant functional impact should be performed in terms of preclinical data, theoretical assumptions and studies confirming the clinical consequences. The authors proposed the following genes to include in the category of PGx-based reasonable diagnostics: CYP2D6 and CYP2C19 for antidepressants, CYP2C9 for S-warfarin, TPMT for azathioprine and 6-mercaptopurine, CCR5 in human immunodeficiency virus therapy, and factor V (Leiden variant) in anticoagulant therapies and in the evaluation of thrombosis as an adverse drug event. Furthermore, they also suggested that another set of genes including ABCB1, DRD2, ADRB1, ADRB2, SERT, and GNB3 should be considered as pharmacogenomics-based extended exploration genes (21). This category includes all genes that exhibit polymorphic variants with known functional effect, but apparently inconsistent clinical data. In the following paragraphs we introduce some of the enzymes and drug target molecules that exhibit polymorphic genetic nature and briefly present the evidence supporting their use as biomarkers for defined clinical situations. CYP2D6 There is widespread recognition that CYP2D6 (debrisoquine 4-hydroxylase) constitutes the indisputable king in the categories of both known valid metabolizing enzyme biomarkers and reasonable PGxbased diagnostics. CYP2D6 comprises approximately 1.5% of the total hepatic P450 content and is involved in the elimination of approximately 25% of all prescribed drugs, including tricyclic antidepressants, several antipsychotics, selective serotonin re-uptake inhibitors, b-blockers, antiarrythmics, tamoxifen, and opiates (23, 24). Polymorphisms in the CYP2D6 gene influence enzyme activity, leading to the phenotypic appearance of distinct types of responders to the drug, ranging from: a) loss of catalytic enzyme activity in individuals possessing two defective alleles (termed poor metabolizers, PMs); b) decreased enzyme activity in individuals possessing one defective and one functional allele (termed intermediate metabolizers, IMs); c) normal function of the enzyme in individuals possessing two functional alleles (termed extensive metabolizers, EMs); and d) increased enzyme activity in individuals expressing multiple copies of the functional allele (termed ultrarapid metabolizers, UMs). As a consequence of these altered enzyme activities, when the standard prescribed dose of a drug primarily metabolized by CYP2D6 is used, UMs will show low plasma concentrations of the parent drug and thus an inadequate response to therapy, while PMs could develop harmful side effects (25). CYP2D6 appears to be highly polymorphic, with more than 70 different allelic variants; however, analysis of the most common variants, i.e., CYP2D6*3/*4/*5/*6, allows for prediction of more than 95% of the PMs and IMs in Caucasian populations (26, 27). The most obvious discipline to benefit from CYP2D6 genotyping is psychiatry. Variability among individuals in their therapeutic response to psychotropic drugs and susceptibility to adverse effects is considerable. CYP2D6 is the principal enzyme metabolizing most antidepressants, as well as several antipsychotic agents. Tricyclic antidepressants, venlafaxine, typical antipsychotics, and risperidone are some of the prescribed medications for which CYP2D6 genotype information could be important in minimizing potential ADRs. An extensive review has recently been published of the evidence indicating which antidepressant and antipsychotic drugs are affected to a clinically important extent by CYP2D6

4 804 Manolopoulos: Pharmacogenomics in clinical practice Article in press - uncorrected proof genotypes (28). Haloperidol, which is used in the treatment of schizophrenia and other psychoses and in the management of hyperactivity, agitation and mania, has also been suggested as a possible candidate for pharmacogenetic testing (29). Evidence suggests that reduced levels of CYP2D6 activity correlate with some of the side effects of haloperidol and therefore pre-treatment CYP2D6 genotyping might prevent such effects in approximately 5% of all patients being treated with haloperidol (30). A strong case for CYP2D6 genotyping can also be made for risperidone. Several small studies showed that CYP2D6 genotype might influence risperidone ADRs and lack of efficacy (31 33). The strongest evidence so far was provided by a study with a much larger patient sample (38 PMs) showing strong correlation between the CYP2D6 PM phenotype and (i) moderate-to-marked risperidone ADRs and (ii) drug discontinuation due to ADRs (34). Another recent study found a positive correlation between CYP2D6 PM status and length of stay in hospital for patients treated with antipsychotic or antidepressant medications (35). An aspect that has been overlooked is the potential significance for increased ADRs in CYP2D6 IMs. Steimer and co-workers conducted a prospective study seeking correlations between CYP2C19 and CYP2D6 genotypes, amitriptyline concentrations, ADRs, and therapy response (36). They found that carriers of two functional CYP2D6 alleles had a significantly lower risk of ADRs than carriers of only one functional allele (12.1% vs. 76.5%). The lowest risk was observed for carriers of two functional CYP2D6 alleles combined with only one functional CYP2C19 allele w0 of 13 (0%) vs. 9 of 11 (81.8%) for the high-risk groupx. No correlation between drug concentrations or genotypes and therapeutic response was found. The authors concluded that identification of genotypes associated with intermediate metabolism may be more important than currently considered. Cardiovascular disorders are another field in which the identification of CYP2D6 genotype may ameliorate drug therapy. It has been reported that CYP2D6 PMs have a four- to five-fold higher incidence of ADRs when treated with metoprolol (37). Furthermore, monohydroxylation of the anti-angina agent perhexiline is almost exclusively catalyzed by CYP2D6, and CYP2D6 PMs have approximately 100-fold lower activity of CYP2D6 than EMs (38). A recent study found that, in tamoxifen-treated breast cancer patients, women CYP2D6 PMs with the genotype CYP2D6 *4/*4 tend to have a higher incidence of disease relapse and a lower incidence of hot flashes, indicating a possible role of CYP2D6 in the metabolic activation of tamoxifen to endoxifen (24). In a more recent paper, the same investigators confirmed these findings and concluded that CYP2D6 metabolism status is an independent predictor of outcome in women receiving tamoxifen for treatment of early breast cancer (39). These data suggest that CYP2D6 genotyping may provide a means by which hormonal therapy of breast cancer can be individualized. Genotyping for CYP2D6 might also be useful for prodrugs such as codeine. CYP2D6 metabolizes codeine to morphine. As a result, standard doses of codeine are unlikely to offer pain relief in CYP2D6 PMs while, on the contrary, CYP2D6 UMs have very high blood levels of morphine and are at higher risk of developing ADRs associated with morphine overdose (28). Similar results have been reported for tramadol treatment (40) and ADRs have been observed in CYP2D6 UMs treated with ethylomorphine (41), oxycodone and hydrocodone (42). CYP2C9 (and VKORC1) CYP2C9 is another notable phase I metabolic enzyme involved in the metabolism of S-warfarin, phenytoin, tolbutamide, losartan, torasemide, sulfonylureas, and many non-steroidal anti-inflammatory drugs such as diclofenac, ibuprofen, and flurbiprofen (43). CYP2C9 is quite polymorphic and many allelic variants have been associated with reduced enzyme activity wa complete list can be found in ref. (44)x. In Caucasians, the main defective alleles are CYP2C9*2 and CYP2C9*3, with allelic frequencies of 11% and 7% respectively, while in African-Americans and Asians they are rare (45, 46). The principal area of clinical application of CYP2C9 genotyping is treatment with oral anticoagulants. It is known that CYP2C9*2 and CYP2C9*3 variants reduce the metabolism of warfarin by 30% 50% and 90%, respectively. Warfarin acts by interfering with the recycling of vitamin K in the liver, which leads to reduced activation of several clotting factors. This drug is now the most widely used anticoagulant in the world (47). It is used in the prevention of arterial and venous thromboembolism and there is overwhelming evidence of its effectiveness in preventing embolic strokes in patients with atrial fibrillation (47). Unfortunately, warfarin has a very narrow therapeutic index. The main ADR associated with warfarin is bleeding. Major and fatal bleeding events occur at rates of 7.2 and 1.3 per 100 patient-years, respectively, according to a meta-analysis of 33 studies (48). Warfarin is number three on the list of drugs implicated in causing hospital admission due to ADRs in the UK and also tops the list of drugs implicated in ADRs in US emergency departments (13, 14). Warfarin s narrow therapeutic index makes it difficult to maintain patients within a defined anticoagulation range. A recent analysis of 6454 patients with atrial fibrillation taking warfarin showed that for almost 50% of the time, the international normalized ratio (INR) was outside the target range of 2 3 (49). An INR )3 increases the risk of bleeding, while a value -2 increases the risk of thrombotic events (50). Higashi and co-workers have reported that CYP2C9 PMs and IMs need a longer time to reach stable warfarin dosing, have a five- to six-fold increased risk of elevated INR, and are four-fold more likely to develop major bleeding complications during the initiation phase of warfarin therapy (51). Since then, several clinical studies have reported a higher risk of adverse effects,

5 Article in press - uncorrected proof Manolopoulos: Pharmacogenomics in clinical practice 805 such as bleeding complications, at the start of treatment with oral anticoagulants (52). Another gene that affects warfarin anticoagulant activity is VKORC1 (vitamin K epoxide reductase complex subunit 1). Being a carrier of a combination of defective polymorphisms of both CYP2C9 and VKORC1 (C1173T), rather than just one of these polymorphisms, is associated with severe overanticoagulation. The time to achieve stability, another important clinical parameter in relation to anticoagulant drugs, does not appear to be affected by VKORC1 polymorphism and is mainly associated with the CYP2C9 genotype (53). These two genes, together with environmental factors, partly explain the interindividual variation in warfarin dose requirements (54). For example, two studies have shown that combining age and body surface area with genetic polymorphisms in CYP2C9 and VKORC1 accounts for approximately 50% 55% of the variance in dosage requirements (55, 56). Large ongoing studies of genes involved in the actions of warfarin, together with prospective assessment of environmental factors, will undoubtedly increase the capacity to accurately predict warfarin dose. Implementation of CYP2C9 genotyping prior to initiation of drug therapy represents an opportunity to individualize warfarin therapy and minimize the risk of hemorrhage without compromising effectiveness. A substantial case can also be made for CYP2C9 genotyping of patients receiving phenytoin. This drug is primarily eliminated via CYP2C9 hydroxylation, while CYP2C19 also plays a role at higher concentrations (28). Several studies have shown substantial elevations in phenytoin in patients possessing defective CYP2C9 alleles. In one study, the authors went as far as to provide dosage recommendations based on CYP2C9 and CYP2C19 genotypes. For example, wildtype individuals may require mg/kg/day, whereas those with the CYP2C9*1/*3 genotype require 2 4 mg/kg/day, depending also on the presence of defective CYP2C19 alleles (57). Given that central nervous system toxicity (e.g., ataxia and nystagmous) is closely related to concentration, it is likely that individuals who carry defective CYP2C9 alleles will be predisposed to such adverse effects. CYP2C19 Another polymorphic isoform of CYP2C is CYP2C19, which metabolizes several important drugs, including most tricyclic antidepressants, some antipsychotics, S-mephenytoin, diazepam, the proton pump inhibitors omeprazole and lansoprazole, and proguanil (58). Interindividual differences in CYP2C19 activity divide the population into EMs, IMs, and PMs. The CYP2C19*2 and *3 defective alleles are found in 87% of PMs in Caucasians and 98% of PMs in Orientals, while the PM phenotype occurs in 2% 5% of Caucasians and Africans and 10% 23% of Orientals (46, 59). CYP2C19 PMs may suffer harmful side effects under standard prescribed doses of drugs inactivated by CYP2C19, or may not gain therapeutic response from prodrugs activated by CYP2C19, such as the antimalarial drug proguanil (59). CYP2C19 PMs may have poor tolerance of several tricyclic antidepressants that are demethylated by CYP2C19 and also appear to have poor tolerance to citalopram, escitalopram, and sertraline (60). Treatment of CYP2C19 PMs with sertraline was found to result in ADRs such as nausea and dizziness, adverse effects that may be due to toxic concentrations of the accumulated drug (61). Finally, as mentioned in the previous section, carriers of defective CYP2C19 alleles might be at increased risk of experiencing ADRs during phenytoin treatment. Genotyping each patient for both CYP2C19 and CYP2C9 might be helpful in phenytoin patient-tailored therapy, and it should also be complemented with TDM (see below). A different situation exists in relation to CYP2C19 allelic status and therapy with proton pump inhibitors. CYP2C19 is responsible for more than 80% of the metabolism and inactivation of omeprazole, lansoprazole, and pantoprazole (62). CYP2C19 PMs and possibly IMs may experience more effective acid suppression and Helicobacter pylori eradication, resulting in better healing of duodenal and gastric ulcers during treatment with proton pump inhibitors (58, 63). In a study using a relatively low dose of omeprazole (20 mg) to treat ulcers, cure rates were very low in EMs (25%), higher in IMs (50%) and complete in PMs (100%), suggesting that higher plasma levels of the drug are necessary for effective treatment (23). In another study, a 7-day triple therapy regimen resulted in cure rates of 60% in EMs, compared with 84% and 100% in IMs and PMs, respectively (64), while a third study revealed eradication rates of 73%, 92%, and 98% in EMs, IMs, and PMs, respectively (65). CYP3A5 CYP3A5 is the major extrahepatic isoform of the CYP3A gene family and in association with CYP3A4 is responsible for the metabolism of over 50% of all clinically used drugs, including steroids, antidepressants, immunosuppressive agents (calcineurin inhibitors), some antibiotics, and protease inhibitors (66). The primary causal mutation for its polymorphic expression (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mrna splicing and reduced translation of a functional protein (67). The CYP3A5*3 polymorphism is widely detectable in Caucasian populations and homozygosity of the allelic variants is strongly correlated with decreased enzyme activity (66, 68). CYP3A5 was not classified as a valid biomarker in the study discussed earlier (21). This was because it has not been possible to establish the clinical significance of CYP3A5*3 polymorphism, although it has been the subject of intensive study, especially in relation to the immunosuppressant cyclosporin (69). However, the situation is different for tacrolimus. Several studies have shown that individuals with the CYP3A5*3/*3 genotype require greater dose-adjusted tacrolimus concentrations than carriers of the CYP3A5*1/*3 genotype, and that predicted plasma clearance of tacrolimus was higher in individuals with

6 806 Manolopoulos: Pharmacogenomics in clinical practice Article in press - uncorrected proof the CYP3A5*1/*3 genotype than in those with the CYP3A5*3/*3 genotype (70). It appears that CYP3A5 genotyping could serve as a useful tool for the individualization of tacrolimus therapy, together with TDM. Another potential application of CYP3A5 genotyping has emerged recently in the cardiovascular field. Kivisto and co-workers reported that in subjects possessing the CYP3A5*1 allele (CYP3A5 expressors), the mean reduction in serum total cholesterol and lowdensity lipoprotein (LDL)-cholesterol in response to some statins (simvastatin, atorvastatin, lovastatin) was significantly lower compared to subjects possessing the CYP3A5*3 allele (CYP3A5 non-expressors) (71). This finding suggests that CYP3A5 polymorphism may be a genetic determinant of interindividual differences in response to statins. If confirmed by larger studies, this finding has obvious clinical significance and application, since statins are currently among the most widely prescribed drugs globally. TPMT TPMT is a cytoplasmic enzyme that inactivates drugs belonging to the thiopurine family such as 6-mercaptopurine, 6-thioguanine, and azathioprine, used in the treatment of leukemia, autoimmune disorders, and for immune suppression in organ transplant recipients (72). In Caucasians, the activity of TPMT exhibits a trimodal distribution, with 0.3% 0.6% having low or undetectable activity, 10% having intermediate activity, and the remaining 90% having high (normal) activity (73). Many TPMT allelic variants have been identified, with TPMT*2, *3A, *3B, and *3C being responsible for most cases of TPMT deficiency (74). The most prevalent low-activity TPMT alleles are TPMT*3A in Caucasians and TPMT*3C in Chinese, Egyptians, and African-Americans (28). Subjects homozygous for a deficient allele (e.g., TPMT*3A/*3A) have negligible TPMT activity, whereas heterozygotes (e.g., TPMT*1/*3A) have intermediate activity that is approximately half of that in subjects homozygous for the wild-type allele (TPMT*1/*1). Individuals with reduced activity of TPMT are at risk of life-threatening ADRs such as liver toxicity and leukopenia (28, 75). A recent analysis of the cost-effectiveness of TPMT genotyping in acute lymphoblastic leukemia in Europe concluded that it should be considered as an integral part of healthcare prior to the initiation of therapy with thiopurine drugs (76). In addition, Gardiner and Begg, after reviewing all the evidence on TPMT genotyping in relation to thiopurine therapy, suggested that all individuals commencing thiopurine therapy should undergo prospective genotyping (and/ or phenotyping) for TPMT status (28). The main clinical value lies in the ability to identify those with extremely low TPMT activity who will almost certainly develop profound myelosuppression with standard thiopurine doses. Despite the accumulated evidence, however, there is no commercial test available for TPMT genotyping and testing for TPMT is not widespread, or is performed only with biochemical tools, measuring erythrocytes activity (29, 77, 78). UGT1A1 Another important application of PGx in oncology deals with association between the genetic variability of the uridine UGT1A1 gene and irinotecan toxicity. UGT1A1 is responsible for the inactivation of 7-ethyl- 10-hydroxycamptothecin (SN-38), the active metabolite of the anticancer drug irinotecan, to form SN-38 glucuronide (SN-38G) (79). Irinotecan is a camptothecin analog with strong antitumor activity mediated by the inhibition of topoisomerase I. The drug is used widely, especially for colorectal and lung cancers, but exhibits dose-limiting toxicity with severe ADRs such as leukopenia and diarrhea, as well as occasionally fatal events (80, 81). The activity of UGT1A1 varies widely, with an in vitro study demonstrating a 17-fold variation in SN-38 glucuronidation (82). UGT1A1*28 is the variant most frequently implicated in defective SN-38 glucuronidation and involves an extra thymineadenine (TA) repeat in the TATA section of the UGT1A1 promoter (7 TA repeats instead of 6 in the wild-type) resulting in lower expression and activity of the enzyme. This variant occurs commonly, with the homozygous genotype found in 5% 15% of Caucasians and 10% 25% of Africans and South Asians (83). Patients with the UGT1A1*28 genotype are at higher risk of toxicity when treated with irinotecan and evidence has accumulated showing that prospective screening of all patients before irinotecan chemotherapy may reduce the incidence of ADRs. Andersson and co-workers reviewed all the adequately sized studies of UGT1A1*28 and irinotecan/ SN-38 pharmacokinetics and metabolism or toxicity and concluded that UGT1A1*28 is a valid biomarker for decreased UGT1A1 activity and for increased irinotecan toxicity and should be measured along with other clinical markers (e.g., bilirubin levels) when treating patients taking irinotecan (22). On the basis of these conclusions, the irinotecan label was updated in 2005 in the US to provide pharmacogenetic information (84) following the resolution of an FDA advisory committee meeting in November It now indicates that a dose reduction of irinotecan should be considered for patients known to be homozygous for the UGT1A1*28 allele when administered in combination with other agents or as monotherapy. Another significant development was the approval by FDA in August 2005 of the Invader UGT1A1 pharmacogenetic test (85) to identify patients who may be at increased risk of ADRs to irinotecan. The availability of this test is expected to facilitate the translation of the existing pharmacogenetic information on irinotecan/ugt1a1 into clinical practice (86). b 2 -Adrenergic receptor and asthma The last two molecules discussed in terms of their potential as pharmacogenomic biomarkers are receptors and are thus related to the pharmacodynamic aspect of drug action. The first is the b 2 -adrenergic

7 Article in press - uncorrected proof Manolopoulos: Pharmacogenomics in clinical practice 807 receptor (b 2 -AR). b 2 -Adrenergic agonists are the most commonly prescribed asthma drugs and there is controversy among clinicians as to the toxicity and appropriate use of these drugs (87). Several polymorphisms within the coding region of the b 2 -AR gene significantly alter receptor downregulation. The most common polymorphisms are in the amino-terminus of the receptor at amino acid position 16 (B16), where Arg or Gly can be found, and position 27 (B27), where Gln or Glu is common. These functional polymorphisms appear to influence both susceptibility and treatment response in asthma. An early study examining the effects of b 2 -AR genotype on responsiveness to b 2 -AR agonists in children found that 60% of asthma patients homozygous for arginine at B16 (B16 Arg/ Arg) had a positive response to albuterol, compared to only 13% for individuals homozygous for glycine at position 16 (88). Another study in more than 250 patients with mild asthma randomized to regular vs. as-needed albuterol use showed no association at B27, but an association of B16 Arg/Arg with a significant decline in peak expiratory flow with regular use of albuterol compared with B16 Gly/Gly subjects (89). In another study, patients with B16 Arg/Arg genotype had more asthma exacerbations during regular treatment with albuterol than during treatment with placebo (90). Finally, a recent prospective study of patients randomized to regular vs. minimal albuterol use confirmed genotype-specific altered responses in B16 Arg/Arg patients using albuterol regularly (91). These studies suggest that a true association between the B16 Arg/Arg polymorphism and response to b 2 -AR agonists may exist. Wechsler and Israel recently reviewed all the available data regarding the relation of b 2 -AR gene polymorphisms and asthma therapy with b 2 -AR agonists and concluded that the B16 polymorphism of b 2 -AR is associated with a major, clinically significant pharmacogenomic effect (92). They went on to suggest that clinicians should take into consideration potential genotyperelated effects in patients using high doses of b 2 -AR agonists and possibly other medications who continue to have poorly controlled asthma, or who experience ADRs. Furthermore, they suggested if the apparent effects associated with B16 Arg/Arg are replicated in populations using long-acting b 2 -AR agonists, current recommendations for asthma pharmacotherapy may be significantly altered. Given all the evidence accumulated, it is surprising that genotyping for b 2 -AR B16 polymorphism has not yet entered routine clinical practice. 5-Hydroxytryptamine receptor 2C Both pharmacological and genetic studies have revealed that 5-hydroxytryptamine receptor 2C (5- HT 2C ) contributes substantially to the serotonergic regulation of a wide variety of behavioral and physiological processes such as feeding and glucose homeostasis. Weight gain is a common ADR of treatment with antipsychotic agents, and polymorphisms in the 5-HT 2C receptor gene have been associated with it. In particular, the 759C/T polymorphism of the promoter region has been associated with antipsychotic agentinduced weight gain in several studies (93 96). The allelic T variant seems to be a protective factor against weight gain in schizophrenic patients treated with clozapine, olanzapine, risperidone, and chlorpromazine. Prospective studies are necessary; however, the available evidence appears sufficient to support the argument for genotyping for the 5-HT 2C 759C/T polymorphism in all patients starting treatment with antipsychotics or who are already in therapy and experience serious ADRs related to their weight regulation. Clinical application of PGx: reality check Pharmacogenomic genotyping is performed mostly by two types of laboratories: a) laboratories in academic hospitals offering pharmacogenomic testing to clinics in the hospital, usually disease-specific applications in dedicated hospitals/clinics (chemotherapy, psychiatry, cardiovascular, pulmonary etc.) and often within the framework of clinical research protocols; and b) private diagnostics laboratories, set up either to develop PGx services, or as novel business units/ products of established diagnostics companies. A partial list of private laboratories offering pharmacogenomic testing services to patients, physicians and/ or drug developers is shown in Table 1. At present, pharmacogenomic testing is rarely used in clinical practice. The Scandinavian countries perhaps represent an exception, with some clinical genotyping performed, mainly for drug-metabolizing cytochrome P450 enzymes in patients receiving psychotropic drugs. A survey of the four laboratories conducting such tests for Paris hospitals showed that only approximately 750 tests were performed during a 12-month period between 2004 and 2005; most of those tests dealt with genotyping for allelic variants of drug-metabolizing enzymes (97). Some data on the limited application of pharmacogenetic tests in Germany have also been reported (98). A non-representative, explorative survey conducted amongst Table 1 Partial list of commercial laboratories offering pharmacogenomic testing services to patients, physicians and/or drug developers (accessed March 31, 2007). PGXL Laboratories DECODE Genetics Genomas Inc Laboratory Corporation Molecular Diagnostics Laboratories Genelex Speciality Laboratories Gentris Arup Laboratories Quest Diagnostics DNAVISION (Belgium) JURILAB (Finland) EUROFINS Medigenomix (Germany) DNALEX (Greece)

8 808 Manolopoulos: Pharmacogenomics in clinical practice Article in press - uncorrected proof members of the German Society of Laboratory Medicine revealed that the demand for pharmacogenomic testing is limited and has not increased much in recent years, although a certain increase is expected in the future (29). In another recent study, Gardiner and Begg investigated the extent of pharmacogenomic testing for drug-metabolizing enzymes in clinical practice in Australia and New Zealand (78). They sent a questionnaire to 629 laboratories in Australia and New Zealand, 507 of which responded. Ten of these 507 laboratories reported that they were performing clinical genotyping tests, and 18 out of 507 labs had in place clinical phenotyping tests. The most frequent tests (2003 data) were for TPMT (400 tests; ratio of genotyping to phenotyping of 1:5), pseudocholinesterase (250 tests; genotyping to phenotyping 1:8) and CYP2D6 (4200 tests). It is noteworthy that all the CYP2D6 tests involved phenotyping for perhexilline. No other clinical genotyping was performed. In academic settings in Greece, to the best of our knowledge, only the author s laboratory performs clinical pharmacogenomic genotyping of the drug-metabolizing enzymes CYP2D6, CYP2C9, CY2C19, and CYP3A5, and the 5-HT 2C 759C/T polymorphism. Despite our continuous efforts, however, little real interest has been expressed so far by clinicians in implementing PGx into their practice. Several reasons may account for the poor clinical application of PGx. The authors of the New Zealand study explained their findings by concluding that the low clinical utilization most likely reflects a poor evidence base, unestablished clinical relevance and, in the few cases with the strongest rationale, a slow translation to the clinical setting (78). Indeed, indications for genotyping in patients have not yet been evaluated or generally accepted. An important step forward would be the generation of specific recommendations for dose adjustment based on the results of pharmacogenetic genotyping. These recommendations should be simple and easy to understand and use by clinicians. Several noteworthy efforts in this direction have been published. In 2001, a consortium of scientists from several European countries published an article with CYP2D6 and CYP2C19 genotypebased dose recommendations for antidepressants (99). More recently, de Leon and colleagues published specific clinical guidelines for psychiatrists for identifying and treating CYP2D6 PMs and UMs, as well as CYP2C19 PMs based on the literature and their own vast clinical experience (60). Despite the lack of universal consensus on the advice contained in these studies, the contribution of both sets of recommendations to the advancement of clinical implementation of genotyping is indisputable. The lack of approved diagnostic tools for identifying individuals with defective alleles has also hindered clinical uptake of PGx. However, the situation appears to be changing, and several pharmacogenetic tests of clinical relevance are currently been developed by different companies (100). The recent FDA approval of two pharmacogenetic tests for use in the clinical setting is expected to improve the clinical uptake of PGx. These tests are the Roche AmpliChip P450 (101) and the Invader UGT1A1. The Invader UGT1A1, as already discussed in the relevant section, is used to identify patients who may be at increased risk of experiencing ADRs to irinotecan. The Roche AmpliChip CYP450 test, based on microarray technology, is an important step toward introducing personalized medicine into the clinical environment. It classifies subjects into four CYP2D6 phenotypes (PMs, IMs, EMs and UMs) by testing 27 alleles, including seven duplications, and two CYP2C19 phenotypes (PMs and EMs) by testing three alleles. FDA market approval for the AmpliChip test was granted in December 2004, making it the first such test to receive FDA approval. Consequently, CYP2D6 and CYP2C19 genotyping became the first pharmacogenetic tests to officially enter the clinical laboratory (100). Implementation of pharmacogenetic testing in clinical practice: keys to success Although the clinical utility of pharmacogenetic tests in several cases has been substantiated, there are some key barriers hindering the implementation of PGx in clinical practice. One of these is a lack of genetic tools that need to be developed by industry. As mentioned already, this appears to be changing with the introduction of FDA-approved, clinically validated commercial diagnostic tests. Furthermore, the utility of pharmacogenetic tests in reducing ADRs and improving drug efficacy has to be proved by large prospective clinical studies and related pharmacoeconomic studies. One of the most important barriers seems to be education in PGx, not only for health professionals, but also for the general public. Some of these issues are further discussed below. The need for education Clinicians tend to ignore the large amount of new pharmacogenetic information and view it as an additional burden and complication of the complex process of therapeutic decision-making. This appears to be largely due to the lack of education on the science and potential of genomics by all parties involved in the medical application of this technology (77, 102). This is a major obstacle that has hampered the widespread clinical application of PGx. Education in genetics at the undergraduate, postgraduate, and continuing medical education levels has trailed behind the enormous scientific and technical advances in the field (103). In addition to clinicians, this lack of education involves all stakeholders, including: a) other healthcare professionals (including researchers); b) patients and concerned individuals; c) media journalists, who often transmit incorrect information due to their lack of knowledge; d) government-employed regulators and politicians; e) hospital administrators; and f) health insurance executives and decision-makers. The latter are a very important group, since they are the ones who will decide to include pharmacogenetic tests in their coverage.

9 Article in press - uncorrected proof Manolopoulos: Pharmacogenomics in clinical practice 809 A report issued in 2002 by the Consortium on Pharmacogenetics in the UK (104) stated that: Perhaps the greatest single factor affecting the penetration of pharmacogenomics into clinical practice and the pace at which it will occur will be the knowledge and acceptance of physicians. Studies indicate that many physicians lack basic knowledge of genetics and also frequently fail to take into account available information about drugs. This urgent need was pinpointed by the participants of a recent Pharmacogenomics Education Forum of the International Society of Pharmacogenomics (ISP), who issued a set of recommendations and a Call for Action addressed to Medical, Pharmaceutical and Health Schools Deans of Education (105). This document urges Deans of Education to incorporate PGx in the core teaching curricula of pharmacology without further delay. This step is vital for ensuring rapid and successful implementation of personalized medicine into medical practice, in pace with the emergence of the latest genomic diagnostics tools, and for the benefit of society at large. The need for prospective clinical studies Numerous investigators and other specialists in the field have called for prospective clinical studies (21, 22, 28, 77, 106). This is connected to the need for incentives for the pharmaceutical and the diagnostics industry to develop genotyping tools and validate them in the clinical setting via clinical studies. However, it has been pointed out that unless co-developing a diagnostic to accompany a PGx-based drug pharmaceutical companies have few incentives to sponsor randomized controlled clinical trials of PGxbased diagnostics (22). On the other hand, the diagnostics industry has great interest in developing new PGx-based diagnostic tests, but often has insufficient resources to sponsor major clinical trials and is not accustomed to testing the value of its products using randomized clinical research. Overall, it appears that governments should act soon. A thorough analysis of the types of studies required and related problems has recently been published, to which the interested reader is referred (22). As stated by Gardiner and Begg: In addition to clinical studies, formal pharmacoeconomic studies need to be performed whenever a strong evidence-based case is made for pharmacogenetic testing. This is valuable from a population perspective when there are limited funds available for health care expenditure. However, it is also recognized that the business model may intervene, with aggressive marketing, e.g., of genetic tests encouraging clinical uptake before the evidence supports use of the test. From a best evidence perspective, it would be useful for pharmacoeconomists to define standards for conducting such studies that are both feasible and readily comprehensible (28). Furthermore, the advancement of clinical PGx creates an urgent need for the establishment of a solid and clearly defined regulatory framework. However, since regulatory issues are outside the scope of this article, the reader is referred to two recent excellent reviews on the subject (107, 108). Pharmacogenomics initiatives Several efforts to streamline research and clinical activities in PGx have been initiated. The earliest and widest in scope is the National Institutes of Healthsupported Pharmacogenetics Research Network (PGRN). PGRN is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation (109, 110). It consists of several research groups, each of which concentrates on drugs used to treat specific disorders (asthma, depression, cardiovascular disease, nicotine addiction, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug-metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). PharmGKB is an interactive tool for researchers investigating how genetic variation affects drug response. The PharmGKB web site ( displays genotype, molecular, and clinical primary data integrated with literature, pathway representations, protocol information, and links to additional external resources. Users can search and browse the knowledge base by gene, drug, disease, and pathway. Registration is free to the entire research community, but subject to an agreement to respect the rights and privacy of the individuals whose information is contained within the database. As mentioned earlier, a major obstacle to the clinical implementation of PGx is the lack of prospective clinical studies and of economic data showing benefit for the use of these approaches. The EU has identified this gap and has stepped up efforts to assist with validating the PGx concept in the clinic, with an emphasis on economic value. A three-part report from the Institute for Prospective Technological Studies (IPTS) was recently released, titled Pharmacogenetics and Pharmacogenomics: State-of-the-art and potential socioeconomic impact in the EU (111). The first part of this report presents a global picture of the status of PGx by mapping key actors, trends and outputs from current academic and industrial research and development activity in the field, presenting, for example, PGx research groups worldwide, leading countries in PGx research, and biomedical questions addressed by PGx research in the public sector. The second part assesses the clinical impact of PGx (in socio-economic terms) in four EU member states (Germany, Ireland, the Netherlands and the UK) using two case studies (HER-2 and TPMT testing). This part includes information on market size and the role of industry, levels of use, reimbursement, patient support groups, education, and an analysis of cost-effectiveness. Finally, the third part contains a comparison of regulatory and