Pharmacogenetics Testing Introduction Pharmacogenetics is the study of how genetic differences among individuals correspond to variability in drug response.

Researchers track such differences by identifying single nucleotide polymorphisms (SNPs), single base pair alterations in the normal human genetic sequence found in small percentages of the population. (Variant genetic sequences are labeled polymorphisms if they are found in more than one percent of the population).

Figure 01: Molecular mechanisms of genetic polymorphisms. A polymorphism is a variation in the DNA sequence that is present at an allele frequency of 1% or greater in a population. Two major types of sequence variation have been associated with
3

variation in human phenotype: single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). These variations, especially those affecting pharmacokinetics (drug absorption, distribution, metabolism, and excretion) can significantly alter drug effectiveness or increase side effects in individuals carrying them. Historical Highlights In an address before the British Medical Association in1914. Archibald Garrod proposed that enzymes were somehow implicated in the detoxification of exogenous substances. During the intense flurry of research stimulated by the rediscovery of Mendels laws of heredity around 1900, Cuenot, from studies of mice, and Garrod, from human studies, anticipated the connection of enzymes (diastases) with the genetic material. Observations of physiological chemists, intrigued by the fate of chemicals in human subjects, had shown by this time that most drugs were excreted in forms that differed from those that were ingested. These observations, and Garrods observations pertaining to a case of porphyria brought on by the hypnotic drug sulfonal, led Garrod to conclude that the ability of individuals to transform drugs into nontoxic conjugates, such as hippurates and glycuronates, protected them from the poisonous effects of these agents. Garrod was thus far ahead of his contemporaries in attributing unexpected drug responses of individuals to failure of their enzymes to detoxify these substances. These ideas regarding the detoxification of chemicals surfaced again and again in Garrods writings and teachings until the end of his life in the 1930s. He observed that substances in foods, in certain drugs, and in exhalations of animals and plants might produce effects in some persons wholly out of proportion to any they produce in most persons. During the 1920s and 1930s, others began to scrutinize person-to-person differences in the perception of odor and taste. For example, studies of taste blindness to the bitter tasting substance pethoxyphenylurea was demonstrated to be a Mendelian character transmitted from parents to children as an autosomal trait. These deficits in sensory perception were the first indication of the high order of specificity to be expected in human response to chemicals, and the first to establish the heritable nature of such responses. By the time Garrod presented his ideas before the British Medical Association, chemists had identified virtually every major type of conjugation reaction that we know today. The exact
4

dates of those discoveries is less relevant than the fact that they preceded by about 50 years the discovery of the other major category of detoxifying enzymes (P450s), commonly known as the microsomal enzymes or the drug-metabolizing enzymes. In 1954 uncommon severe drug-induced phenotypes served as the triggers to investigate and document pharmacogenetic phenotypes. Prolonged neuromuscular blockade following normal doses of succinylcholine, neurotoxicity following isoniazid therapy, and in 1956 methemoglobinemia in glucose-6-phosphate dehydrogenase (G6PD) deficiency were discovered to have a genetic basis in the first half of the 20th century. In the 1970s and 1980s, debrisoquine hydroxylationand exaggerated hypotensive effects from that drug were related to an autosomal recessive inherited deficiency in the cytochrome P450 isoenzyme 2D6 (CYP2D6). Since the elucidation of the molecular basis of the phenotypic polymorphism in CYP2D, the molecular bases of many other monogenic pharmacogenetic traits have been identified in 1997. History of pharmacokinetics in pharmacogenetics The term pharmacogenetic describes the study of inheritable genetic variation in drug response. Observations of individual differences in response to food and drugs date to as early as the 6th century BC. Pythagoras first made the observation that some but not all individuals fall sick after ingestion of uncooked fava beans. It was not until the 1950s that the association between glucose-6-phosphate dehydrogenase deficiency, hemolytic anemia, and fava beans was known.

Figure: Historical timeline of Pharmacogenetics Inter-individual Variability in Drug Response: The understanding of Pharmacogenomics may be approached by considering the effect of genetic variation on pharmacokinetics and pharmacodynamics. The target list for potential pharmacogenetic tests includes polymorphic genes that code for any protein that interacts with drugs or drug metabolites. Although most currently employed genes relate to pharmacokinetic processes, some genes also reflect pharmacodynamic parameters, for example VKORC1. Combinations of genes such as CYP2C9 and VKORC1 for Warfarin dosing consider aspects of
6

both pharmacokinetics and pharmacodynamics. Such predictive information could then be used by physicians to optimize drug selection and dosage prior to initiating drug treatment. Pharmacokinetic variability: The term pharmacokinetic variability refers to variability in the amount of drug delivered to a receptor, otherwise known as drug disposition. Drug concentration depends on a number of factors including absorption, distribution, metabolism and elimination. Conventional pharmacogenetics has tended to concentrate on inter-individual or interracial differences in drug metabolism. But due to improvement in genomics there have been exciting insights into genetic variation in processes controlling drug absorption and distribution. Furthermore, drug receptors have received attention following a realization that these could also be affected by genetic factors like SNPs altering drug efficacy. Figure: The plasma drug concentration in a patient homozygous for the normal variant of drug

metabolizing enzyme (DME) is shown.

Figure: Drug disposition is depicted for a patient heterozygous for an abnormal variant.

Figure: An individual homozygous for the abnormal allele, the inability of the patient to

metabolize leads to toxicity. Such patients are designated poor metabolizers (PMs). The source of inter-individual variability There are two main types of inter-individual variation which have been found in the genome. 1. Non coding DNA: Variation in the number of recurring small sequences (microsatellites) which occur among the non-coding junk DNA and which are known as variable number tandem repeats (VNTRs). These are commonly used, particularly in forensic pathology, as a means of DNA fingerprinting and in paternity testing. 2. SNP: The second and most important source of variation in the genome is the single nucleotide polymorphism (SNP or snip). A consortium set up in 1999 to chart the extent of these differences found 1.4 million SNPs among the 3 billion or so base pairs which comprise the genome (only 1% of these, however, are in exonic region). Present condition of pharmacogenetic testing Pharmacogenetic research has been advanced by the recent development of DNA chips silicon chips embedded with large numbers of distinct bits of DNA. DNA chips could allow large scale screening to be performed quickly, either to search for thousands of SNPs in one individuals genes or to study the gene expression of thousands of individuals who share a particular disorder, in order to determine whether they share a particular SNP. There is evidence, for example, that some of the variant alleles linked to adverse drug reactions are associated with certain ethnic groups, and such experiments can illuminate these types of connections. The knowledge about individualized drug response that such new technology will bring promises to lead to a new, potentially significant use of genetic testing. Labeled pharmacogenomics, it involves using genetic information to customize medical treatment to a persons particular genetic makeup.
8

Statistics Todays medicines are marketed as one-size-fits-all for any particular medical problem, despite the fact that individuals can differ dramatically in the nor-responses to a drug treatment. Variability based on dosage responses, allergic reactions, and other often hereditary factors leads to side effects that kill 100,000 Americans a year, make 2 million others seriously ill, and, some estimate, lead to health care costs of up to $100 billion annually. Al-though it is unlikely that tracking SNPs will completely eliminate adverse drug reactions, pharmacogenetics and pharmacogenomics promises an enormous improvement over the current system, under which doctors guess proper dosages or drug choice based on generalized adverse reaction and dosage data and then alter the prescription based on observable patient reaction. A problem that the FDA has begun to respond to only in the last decade that the danger of the current trial-anderror system is particularly great for children, women, and minority groups, since clinical trials that provide data on side effects and dosage are usually performed on a sample made up mostly of adult white males. Within five years, tailored prescriptions may begin to significantly diminish such problems, and it may become commonplace for doctors to rely on genetic testing to aid dosage or drug prescription decisions. Pharmacogenetics Testing Protocol A protocol of pharmacogenetic testing was published by Committee for Medicinal Products for Human Use (CHMP) on 22 April 2010. This guideline applies to Marketing Authorization Applications for new medicines for human use submitted in accordance with Article 8(3) of the Directive 2001/83/EC, as amended. This guideline should be read in conjunction with the Introduction and general principles paragraph (4) and Part I, Module 5 of the Annex I to Directive 2001/83, as amended, and all other relevant information included in current and future EU and ICH guidelines and regulations especially: Pharmacokinetic studies in man (Notice to applicants, Vol 3C, C3a, 1987). Guideline on reporting the results of population pharmacokinetic analyses

(CHMP/EWP/185990/06). The investigation of drug interactions (CPMP/EWP/560/95).

9

Guideline on the investigation of bioequivalence (CPMP/EWP/QWP/1401/98 Rev.1). Guideline on the role of pharmacokinetics in the development of medicinal products in

the paediatric population (EMEA/CHMP/EWP/147013/2004). Guideline on the evaluation of the pharmacokinetics of medicinal products in patients

with impaired hepatic function (CPMP/EWP/2339/02). Note for guidance on the evaluation of the pharmacokinetics of medicinal products in

patients with impaired renal function (CHMP/EWP/225/02). Position paper on terminology in Pharmacogenetics (EMEA/CPMP/3070/01). Rules governing medicinal products in the European Union Volume 2C Notice to

applicants; A guideline on summary of product characteristics (SmPC) September 2009. ICH Topic E15. Note for Guidance on definitions for Genomic biomarkers,

pharmacogenomics, pharmacogenetics, genomic data and sample coding categories (EMEA/CHMP/ICH/437986/2006).

ICH Topic E16. Note for Guidance on genomic biomarkers related to drug response: structure and format of qualification submissions.

context,

(EMEA/CHMP/ICH/380636/2009). [11] Procedure Any individual may have a number of permutations of these factors, which may result in numerous phenotypes. The ever-increasing number of recognized SNPs has led to the realization that each individual may handle any medication differently and that only by a priori genetic testing can treatment be optimized, in other words, personalized therapy. Estimation of Pharmacokinetic Parameters: Mean pharmacokinetic parameters and their interindividual variations were estimated using the NONMEM analysis. The pharmacokinetic parameters in individual subjects were obtained from the population estimates according to Bayes theorem using the NONMEM post-hoc option. The one-compartment model with very rapid absorption was parameterized in terms of the oral clearance (CL/F) and the apparent volume of distribution (V/F), with NONMEM-PREDPP library subroutines, ADVAN1 and

10

TRANS2. The oral clearance in the ith individual (CL/Fi) was modeled using the following equation: CL/Fi = 1 s3 WTi (1 + CL/Fi). (1) Where 1 is the predicted population mean of the oral clearance, and 3 is the S/R ratio for CL/F. The S value is xed to one for S-isomer of the experimental compound, and to zero for Risomer of the experimental compound. WTi is the individual body weight, and CL/Fi is a random variable dis-tributed with a mean of zero and variance of 2 CL/F. The apparent volume of distribution in the ith individual (V/Fi) was modeled using the following equation: V/Fi = 2 s4 WTi (1 + V/Fi). (2) Where 2 is the predicted population mean of the apparent volume of distribution, 4 is the S/R ratio for V/F, and V/Fi is a random variable distributed with a mean of zero and variance of 2 V/F. In the present study, we assumed that CL/F,V/F. Finally, the jth observed blood concentration in the ith subject (Cbij) was assumed to be randomly and normally distributed from the predicted value (Cb*ij): Cbij = Cb*ij + Cbij*1/2 ij . (3) Where ij is a random variable that describes intraindividual variability with a mean of zero and variance of 2. [10]
1. Drug metabolizing enzymes: Metabolism converts foreign substances or xenobiotics,
CL/Fi

is correlated with

V/Fi

and that the covariance is

which include drugs and the majority of anaesthetic agents, into water-soluble metabolites, by the introduction of small polar groups on to the parent drug. These are
11

then more readily excreted. The responsible enzymes commonly referred to as xenobiotic metabolizing enzymes (XMEs) or drug metabolizing enzymes (DMEs). DMEs may convert an inactive substance like Codeine into one which is pharmacologically active in this case morphine. DMEs may also produce a toxic or even carcinogenic metabolite. Variation in the activity of these metabolizing enzymes may therefore carry a biological advantage or disadvantage depending on the substance being metabolized. In the case of a poorly metabolized drug that is in itself active, poor metabolism will lead to accumulation and potential toxicity. For example the phase I oxidative enzymes of the CYP450 family, phase II conjugative enzymes and drug transporters such as P-glycoprotein, the multidrug transporter multidrug resistance (MDR1) gene product etc. [4] To date, the best studied pharmacogenetic targets are genes that code for Drug metabolizing enzymes (DMEs), with the Cytochrome P450 iso-enzymes (CYPs) being the best characterized. The CYP P450 enzymes, a superfamily of microsomal DMEs, are the most important of the enzymes that catalyze phase-I metabolism. The advances in molecular biology which have taken place over recent decades have allowed the characterization and classification of these enzymes based upon their amino acid structure. Enzymes with more than 40% amino acid homology are grouped together in a single superfamily. This is designated by a number, for example CYP2. If amino acid homology is greater than 55% the enzymes are grouped together in a subfamily, designated by a capital letter, for example CYP2E. In the 18 superfamilies which exist in humans, enzymes belonging to superfamilies are those which are involved in the breakdown of drugs, pollutants and chemicals. It has been estimated that 90% of drug and xenobiotic metabolism can be attributed to six main enzymes CYP 1A2, 2C9, 2C19, 2D6, 2E1 and 3A4 CYP enzymes are found primarily in the liver but have also been identified in the lungs, kidneys, gut and brain. Most drugs are metabolized by at least one CYP with the exception of CYP3A4. Specific genetic variants in CYPs affect the metabolic phenotype through changes in protein expression, structure, function, stability, and/or substrate specificity. Four major phenotypes are described: a) The extensive or normal metabolizer (EM),
12

b) The poor metabolizer (PM), c) The intermediate metabolizer (IM), d) The ultrafast or rapid metabolizer (UM).

Figure 01: The figure illustrates the approximate percentage of drugs metabolized by the number of Cytochrome P450 superfamily. Sequence variants responsible for these phenotypes include single point mutations, insertions and deletions of several nucleotides and replications and deletions of an entire gene. [3] Important factors underlying the marked pharmacokinetic variability of drugs include their dependence on intracellular phosphorylation for generation of the active drug moiety (nucleoside reverse transcriptase inhibitors NRTIs) and their role as substrates of genetically polymorphic drug-metabolising enzymes and transporters (protease inhibitors PIs and nonnucleoside reverse transcriptase inhibitors NNRTIs).[4]
13

Individualize Drug Development: Concerning metabolic pharmacogenetics the main question during drug development is which iso-enzymes are involved in drug metabolism and what are their contributions to the wanted and unwanted effects of a drug. Studies have to be performed to serve to: - Determine the overall metabolism of a drug - Identify the (polymorphic) enzymes involved and establish their relative contribution - Determine the contribution of the various pathways to the overall elimination of a drug - Identify different levels of expression of enzymes and predict interindividual variability This information can be obtained in several ways and at several stages during drug development and is vital for the estimation of the clinical impact of a polymorphic enzyme to drug metabolism. These data are especially important if it concerns a drug with a narrow therapeutic index or if a drug is metabolized from a prodrug. In clinical studies information on drug metabolising status of volunteers / patients is often needed for inclusion or to explain pharmacokinetic or pharmacodynamic observations. Rather than selecting subjects at random, genotyping on for example genes that code for drug metabolising enzymes, could be helpful in selecting subjects that are most likely to respond or might be helpful in the elucidation of side effects.[7] Genome-wide approaches hold promise for identification of new drug targets and therefore new drugs. In addition, accounting for genetic/genomic inter-individual variability may lead to genotype-specific development of new drugs, and to genotype-specific dosing regimens. Recently, the U.S. Food and Drug Administration (FDA) altered the labels of several drugs in clinical use to indicate a pharmacogenetic issue. [6] Pharmacogenetics may identify subsets of patients who will have a very high or a very low likelihood of responding to an agent. This will permit testing of the drug in a selected population that is more likely to respond, minimizing the possibility of adverse events in patients who derive no benefit, and more tightly defining the parameters of response in the subset more likely to benefit. [6] Pharmacogenetic Testing Process Approaches 1. Phenotype-to-Genotype Approach:
14

Demonstration of Significant Phenotypic Variability: Traditionally the phenotype-togenotype approach begins with identifying the phenotype of interest that has demonstrated significant interindividual variability. The phenotype of interest may be toxic or therapeutic response to a drug. The identification of variable traits or phenotypes, such as succinylcholine apnea and isoniazid peripheral neuropathy, and the subsequent discovery of butyrylcholinesterase and N-acetyltransferase 2 polymorphisms, respectively, are classic examples of the phenotype-to-genotype approach. In oncology, the observation of severe unexpected treatment toxicity to 5uorouracil (5-FU), 6-mercaptopurine (6-MP), and irinotecan led to the discovery of polymorphisms in genes for dihydropyrimidine dehydrogenase (DPYD), thiopurine methyltransferase (TPMT), and UDP glucuronasyltransferase 1A1 (UGT1A1) respectively.

Clinicians should be suspicious of a potential genetic basis for the observed phenotypic variability if there is evidence suggesting that typical variables (like sex, age, body weight, physiologic state, organ function, disease state, concomitant drugs, and diet) do not account for the bulk of the phenotypic variability. After identifying the phenotype of interest, the next step is to identify the genetic variants responsible for the observed phenotypic variability. Case-control studies are often used in the phenotype-to-genotype approach, allowing the comparison of genotypic association between phenotypically different groups, such as responder versus non-responder or those with severe treatment toxicity versus those without. In addition, selective genotyping or resequencing of phenotypic extremes or outliers, such as subjects who have high/low drug clearance, may also improve the chance of detecting clinically relevant genetic variants.

Candidate Gene Studies: Candidate gene studies are the foundation of pharmacogenetic knowledge. The purpose of a candidate gene study is to test the hypothesis of a phenotype-genotype association. Single nucleotide polymorphisms (SNPs) and, to a lesser extent, nucleotide insertions, deletions, and repeats, as well as copy number alterations, contribute to genetic variability. It is estimated that there is approximately 1 SNP in every 1,0003,000 base pairs in the human genome. Genetic variations within the coding region may result in a change in amino acid sequence of the encoded protein, and such mutations are known as nonsynonymous mutations. Genetic variations that do not cause a change in the amino acid sequence are termed
15

synonymous mutations. Genetic variations within the noncoding portion of a gene, including the promoter region, may still affect RNA splicing, mRNA stability, or transcription efficiency. The selection of candidate genes is based on a priori knowledge of the drug pathway and the biology of the target. Earlier pharmacogenetic research utilizing the candidate gene approach focused predominantly on genes of drug metabolism. Over the years, the research focus has broadened to encompass genes that regulate drug trans-porters and biologic targets. Pharmacokinetic parameters such as drug exposure and clearance are frequently used as phenotypic intermediates for toxicity and response because they are readily quantiable and may provide the proof-of-principle evidence of candidate gene polymorphisms and altered function. This approach has been elegantly illustrated by Flockhart and colleagues, who initially demonstrated a strong association between the Cytochrome P450 (CYP) 2D6 genotype and plasma levels of endoxifen and then showed that CYP2D6 metabolism is an independent predictive factor in women with breast cancer treated with adjuvant tamoxifen.

Drug Pathway Approach: Most candidate gene studies favor the examination of several candidate genes rather than a single gene or polymorphism because drug disposition and response might be influenced by a network of genes, each contributing to a different extent to a patients phenotype. Examination of multiple candidate genes relevant to a drug pathway may improve the chance of identifying the correct candidate gene polymorphisms and permit assessment of the relative predictive value of each polymorphism and gene-gene interactions. Evaluating a large number of candidate genes decreases the overall power of the study and increases the cost of genotyping. In order to limit the number of candidate genes evaluated, it is necessary to prioritize and select genes for testing based on their predicted likelihood to exert a clinical effect on drug disposition or response. Data from previous preclinical functional studies, epidemiologic studies, linkage analyses and pharmacologic data help researchers select and prioritize candidate genes of interest. In addition, several online tools, such as FASTSNP (fastsnp.ibms.sinica.edu.tw/pages/input_CandidateGeneSearch.jsp), MEME/MAST system (meme.sdsc.edu/meme/intro.html), and TRANSFAC 7.0 database (www.gene-regulation.com/index.html) allow researchers to predict the functional significance of genetic variants on gene function or expression by comparing the DNA sequence of interest with known motifs such as transcription factor binding site and exonic splicing enhancers.
16

Haplotype Strategy: SNPs that are physically close to each other tend to be inherited together. This association between neighboring SNPs is known as linkage equilibrium and the regions where the genetic variants are shared are termed haplotypes. The genetic diversity across a region or haplotype block can be uniquely represented by variants known as haplotype tagging SNPs (htSNPs). Thus, the number of tags necessary to represent all common polymorphisms across a candidate gene is estimated to be less than one tenth of the total number of the variants present. When a phenotype of interest is found to be associated with a particular htSNP it is very likely that the genetic variant influencing the phenotype is located within or near the same haplotype block. Limited re-sequencing can then be performed across the haplotype block to identify the functional variants. The International HapMap Project is set up to generate detailed haplotype structures of four different population groups. It has already completed the first phase of its project, which aims to provide at least one htSNP for every 5,000 bases of DNA sequence. This information is an invaluable resource to aid

researchers in designing haplotype-phenotype association studies.

17

Figure 02: Haplotype blocks in UGT1A1 generated by Haploview version 4.1. [6]
2. Genotype-to-Phenotype Approach: Since the completion of the Human Genome

Project, there is a wealth of information on genetic variants readily accessible to researchers in public databases, such as the Single Nucleotide Polymorphisms database (dbSNP; www.ncbi.nlm.nih.gov/projects/SNP/) and the Japanese Single Nucleotide Polymorphisms database (JSNP; snp.ims.u-tokyo.ac.jp/). The effects on gene expression and function of the encoded protein are yet to be determined for most of these polymorphisms. The first step of the genotype-to-phenotype approach involves discovering new polymorphisms by sequencing and generating the haplotype structure, followed by conducting in vitro functional studies to determine the underlying mechanism for differential expression or function. Once the functional significance of these variants is characterized, they can be prioritized and validated clinically. At present, in vitro and ex vivo models such as hepatocytes, patient-derived tumor cells, and transfected cell lines are frequently used to characterize genetic variants. Coding nonsynonymous variants can be transfected via cDNA into a cell line with an expression vector to generate the variant proteins of interest. The effect of variants located in the promoter region can be assessed using cells cotransfected with a reporter assay, allowing for quantitative evaluation of transcriptional activity. The main benefit of an in vitro model is that the environmental variables can be easily controlled. However, extrapolation of in vitro data to the clinical setting is fraught with inherent difficulties. In vivo models such as transgenic models and tumor xenografts enable assessment of drug response and resistance in a relatively intact tumor/host microenvironment and can provide useful data on the Pharmacokineticpharmacodynamic relationship.
3. Genome-wide Approach: Recent advances in genomics enable us to examine tens of

thousands of genes in the whole genome at the same time. Genome-wide approaches are usually used to describe patterns of variation in expression (expression arrays), for gene amplification/deletion analysis, and to describe the pattern of SNP variation in a given individual. Gene expression studies (RNA and protein) demonstrate the upregulation and downregulation of genes or gene signatures that are associated with phenotype such as treatment outcome, response, or resistance. Genome- wide approaches offer a comprehensive and relatively unbiased way to scan the entire genome and identify genes that are not previously known to be
18

involved in the treatment response, providing insight into the mechanism of drug response. Using genome-wide microarray analysis of primary acute lymphoblastic leukemia (ALL) cells with 14,500 probe sets, coupled with ex vivo drug sensitivity assays, researchers were able to identify 124 genes associated with treatment response, of which only three were previously known to be associated with drug resistance to the four drugs tested (prednisolone, vincristine, asparaginase and daunorubicin). Using a similar approach, the same group was able to identify a subset of patients with poorer treatment outcome based on the expression profile of 45 genes associated with cross-resistance to all four drugs. This suggests that genome-wide expression analysis may be used to predict treatment outcome and direct cancer therapy in the future. However, there are several challenges intrinsic to the genome-wide approach. First, a much larger sample size is required compared to the candidate gene approach because of the large number of analyses involved. Hence, the expense is generally greater for the genome-wide approach; the high cost of the SNP chips also contributes. Second, the number of potential marker SNPs or candidate genes generated using a genome-wide approach is often large, with a high risk of false-positive findings from multiple testing. One solution to this problem is to include another independent sample to validate the SNPs or candidate genes generated from the initial screening sample. In order to narrow the search for candidate genes, several strategies have been attempted, including the comparison of expression profiles from different biologic models. This approach has been used to compare the expression profile of lymphoblast derived from glucocorticoid sensitive children with ALL to those from adult ALL, healthy donors, glucocorticoid sensitive and resistant cell line models, and mouse thymocytes. Twenty two candidate genes involved in glucocorticoid mediated apoptosis were identified using this approach. [5] An Example of Application of One of the Above Methods: To evaluate the relationship between the disposition of sertraline and the presence of the CYP2C19 gene and to define the contribution of Cytochrome P450 2C19 (CYP2C19) to sertraline N-demethylation the researchers determine the CYP2C19 genotypes and phenotypes by using Genome-wide approach. Regulatory Aspects At the time of writing, regulatory agencies in both Europe and the United States are beginning to show keen interest in the potential role that pharmacogenetic approaches may play in the
19

development and clinical use of new drugs and in the potential challenges that such approaches may present to the regulatory approval process. A formal guideline has been issued by FDA (Food and Drug Administration) named as Guidance for Industry and FDA Staff: Pharmacogenetic Tests and Genetic Tests for Heritable Markers, issued on: June 19, 2007 and the draft of this guidance was issued on February 9, 2006.[8] It will be of key importance for all concerned to engage in an intensive dialogue at the end of which, it is hoped, will emerge a joint understanding that stratification according to DNA-based markers is fundamentally nothing new, and not different from stratification according to any other clinical or demographic parameter, as has been used all along. Still, based on the perception that DNA-based markers represent a different class of stratification parameters, a number of important questions will need to be addressed and answered, hopefully always in analogy to conventional stratification parameters, including those referring to ethical aspects. Among the most important ones are questions concerning: The need and/or ethical justification (or lack thereof) to include likely non-responders in a trial for the sake of meeting safety criteria, which, given the restricted indication of the drug, may indeed be excessively broad. The need to use active controls if the patient/disease stratum is different from that in which the active control was originally tested. The strategies to develop and gain approval for the applicable first-generation diagnostic, as well as for the regulatory approval of subsequent generations of tests to be used to determine eligibility for prescription of the drug, as well as a number of ethical and legal questions relating to the unique requirements regarding privacy and confidentiality for genetic testing that may raise novel problems with regard to regulatory audits of patient data. A concerted effort to avoid what has been termed genetic exceptionalism- the differential treatment of DNA-based markers as compared with other personal medical data, should be made so as not to further complicate the already very difficult process of obtaining regulatory approval. This seems justified based on the recognized fact that in the field of common complex disease, DNA-based markers are not at all different from conventional medical data in all relevant aspects, namely specificity, sensitivity, and predictive value. [9] Pharmacogenetic Testing for Drug Efficacy versus Safety
20

In principle, pharmacogenetic approaches may be useful both to raise efficacy and to avoid adverse events, by stratifying patient eligibility for a drug according to appropriate markers. In both cases, clinical decisions and recommendations must be supported by data that have undergone thorough biostatistical study. Based on the substantially different prerequisites and opportunities for acquiring such data, and applying them to clinical decision making, we expect the use of pharmacogenetics for enhanced efficacy to be considerably more common than for the avoidance of adverse events. The chances of generating adequate data on efficacy in a subgroup is reasonably high, given the fact that unless the drug is viable in a reasonably significant number of patients, it will probably not be developed for lack of a viable business case or at least only under the protected environment of orphan drug guide lines. Implementation of pharmacogenetic testing to stratify for efficacy, provided that safety in the non-responder group is not an issue, will primarily be a matter of physician preference and sophistication, and potentially of customer directives, but would appear less likely to become a matter of regulatory authorization, unless a drug has been developed selectively in a particular level of the overall indication (in which case the indication label will be restricted to this level). Indeed, an argument can be made against depriving those who carry the likely non-responder genotype regarding eligibility for the drug, but who individually, of course, may respond to the drug with a certain, although lower probability. From a regulatory aspect, the use of pharmacogenetics for efficacy, if adequate safety data exist, appears largely unproblematic; the worst case scenario (a genotypically inappropriate patient receiving the drug) would result in treatment without expected beneficial effect, but with no increased odds to suffer adverse consequences. The usefulness and clinical application of pharmacogenetic strategies for improving safety, particularly with regard to serious adverse events, will meet with considerably greater hurdles and is less likely to become practical. A number of reasons are cited for this. First, in the event of serious adverse events associated with the use of a widely prescribed medicine, withdrawal of the drug from the market is usually based largely on subjective evidence from a rather small number of cases, in accordance with the Hippocratic mandate primum non nocere. If the sample size is insufficient to demonstrate a statistically significant association between drug exposure and event, as is typically the case, it will most certainly be insufficient to allow meaningful testing for genotypephenotype correlations; the biostatistical hurdles become progressively more difficult as many markers are tested and the number of
21

degrees of freedom applicable to the analysis for association continues to rise. Therefore, the fraction of attributable risk shown to be associated with a given at risk (combination of) genotype(s) would have to be very substantial for regulators to accept such data. Indeed, the low prior probability of the adverse event, by definition, can be expected to yield an equally low positive (or negative) predictive value. Second, the very nature of safety issues raises the hurdles substantially because in this state the worst situation, administration of the drug to the wrong patient, will result in a higher probability of harm to the patient. Therefore, it is likely that the practical application of pharmacogenetics for the purpose of limiting adverse events will be restricted to diseases with an awful prognosis, where a high medical need exists, where the drug in question offers unique potential advantages, and where, therefore, the tolerance even for relatively severe side effects is much greater than for other drugs. This applies primarily to areas such as oncology or HIV/ AIDS. In most other indications, the sobering biostatistical and regulatory considerations discussed represent barriers that are unlikely to be overcome easily; and the proposed, conceptually highly attractive, routine deployment of pharmacogenetics as generalized drug surveillance or pharmacovigilance practice following the introduction of a new pharmaceutical agent faces these scientific as well as formidable economic hurdles. [9] Applications of Pharmacogenetic Testing The importance of genetic variations in drug response was recognized about 50 years ago, when in some individuals, live threatening adverse drug reactions following application of the muscle relaxant succinylcholine were observed and in patients treated with the tuberculostatic drug isoniazid, pronounced differences in pharmacokinetic parameters (bimodal distribution) were measured. Later, it was determined that these prime examples of variable drug disposition were caused by inherited differences in genes coding respective drug metabolizing enzymes. Since that time, contribution of genetic polymorphisms in drug metabolizing enzymes, transporters and targets (e.g. receptors) to drug disposition and/or drug effects has been investigated in numerous in vitro and clinical studies. Although more prospective studies with clinical endpoints are required to establish a definite role of molecular genetic diagnostics in individually tailored pharmacotherapy, in many situations pharmacogenetics/pharmacogenomics allows for an improved drug response, yet. Possibilities of individual dose adjustment in some important medical fields are briefly discussed below.

22

Application of Pharmacogenetics in Diabetes:

Type II diabetes is one of the most important public health problems and its complications like angio- and neuropathy are associated with pronounced morbidity and mortality. In addition to lifestyle modification programs, an appropriate therapy with oral antidiabetic drugs plays a key role in blood glucose control. Several classes of antidiabetics such as sulfonylureas, meglitinides, biguanides, a-glucosidase inhibitors, thiazolidinediones or insulins belong to the approved drugs for patients with type II diabetes. The action of oral antidiabetic drugs and their adverse drug reactions such as hypoglycemia are subject to wide inter-individual variability. Most oral antidiabetic drugs are metabolized with participation of cytochrome P450 enzymes of the class 2C, which is genetically polymorphic. Whereas sulfonylureas are mostly CYP2C9 substrates, CYP2C8 is the main enzyme responsible for the biotransformation of thiazolidinediones (rosiglitazone and pioglitazone) and repaglinide. For tolbutamide, an oral sulfonylurea hypoglycemic agent used in the treatment of type II diabetes for many years, the contribution of CYP2C9 genetic polymorphisms to pharmacokinetics and blood glucose lowering effects was very well documented. Consequently, a careful monitoring of the hypoglycemic effects upon tolbutamide administration in patients heterozygous and especially those homozygous for CYP2C9*3, which is an allele with decreased enzymatic activity, was recommended. Moreover, dose adjustments for carriers of CYP2C9*3 polymorphism were suggested i.e. half and 20% of tolbutamide standard dose, respectively, for heterozygous and homozygous carriers of CYP2C9*3. The impact of CYP2C9 polymorphism on pharmacokinetics of the second generation sulfonylurea drugs like glibenclamide (glyburide), glimepiride and glipizide have also been studied. Similarly, it could have been shown that total clearance of these oral antidiabetics in carriers of CYP2C9*3/*3 genotype was only about 20% of that in wild types (CYP2C9*1/*1), whereas in heterozygotes, this parameter was reduced to 50-80%. Interestingly, the resulting magnitude of differences in drug effects (insulin concentrations) seems to be much less pronounced than for the pharmacokinetic parameters. Nevertheless, it has been considered that respective CYP2C9 genotype-based dose adjustments may reduce the incidence of possible adverse reactions. At the same time, the presence of another common CYP2C9 variant allele i.e. CYP2C9*2 seems to be without clinical relevance for the therapy with sulfonylureas since it has been considered to reduce the CYP2C9 enzymatic activity to a minor extent only.

23

Both nateglinide and repaglinide are meglitinides, which, like sulfonylureas, act by stimulating insulin release from beta cells of the pancreas via ATP-sensitive K+ channels and on voltagesensitive Ca 2+ channels. For nateglinide, predominantly metabolized via CYP2C9, it could be shown that CYP2C9*3 polymorphism, but not CYP2C9*2, has a moderate impact on pharmacokinetics and pharmacodynamic effects of the drug in healthy volunteers. Furthermore, following administration of repaglinide, which is metabolized via CYP2C8, reduced plasma concentrations have been determined in carriers of CYP2C8*3 variant allele. The possible role of CYP2C8*3 polymorphism in pharmacokinetics of thiazolidinediones rosiglitazon and pioglitazone should be assessed in further clinical studies. Biguanide metformin belongs to oral antidiabetics widely used in overweight patients with type 2 diabetes. It could be shown that organic cation transporter 1 (OCT1) is mainly responsible for metformin entry into enterocytes and hepatocytes. To date, several genetic polymorphisms in OCT1, some of them leading to reduced transporter activity, have been identified. In one clinical study, carriers of at least one OCT1 variant allele, determining reduced function of the transporter, showed higher glucose levels following administration of metformin. However, before OCT1 genotyping could be established as a reliable method for prediction of clinical response to metformin, prospective clinical studies in large numbers of patients must be performed. It appears that personalized medicine could promise an optimization of treatment choices in patients with type II diabetes, however, due to pronounced complexity of the disease and individual drug response, further research is needed to establish the role of pharmacogenetics in therapy of diabetes.

Application of Pharmacogenetics in Psychiatry:

Pharmacogenetic testing allows physicians to identify patients who are likely to have adverse reactions to certain medications because they metabolize them poorly or too rapidly. Much research has focused on the gene P-450 2D6, which has been shown to affect patients response to various drugs including antidepressants and antipsychotics. Patients with certain genotypes may experience negative side effects from medications or may see no benefit at all. This article describes the first pharmacogenetic test to be widely used, which identifies variation in the P450 2D6 gene, and the patient populations that would benefit from it.

24

Basic pharmacogenetic research over the last 30 years has set the stage for tailoring drug treatment to the patients genetic constitution. Clinical applications of that work are emerging, and it is now possible for practicing physicians to use new principles that are shaping the field of personalized medicine. One of the genes of particular interest to psychiatrists is P-450 2D6. Variability in the structure of this gene is associated with dramatic differences in the availability of the 2D6 enzyme, which metabolizes more than 70 drugs, including atomoxetine (a selective norepinephrine reuptake inhibitor used for treating attention deficit hyperactivity disorder), antidepressants, and antipsychotics as well as commonly prescribed nonpsychiatric drugs such as codeine, dextromethorphan, and tamoxifen. For a decade, we have known that genetic variation in this gene affects metabolism of antidepressant medication.2 Genotyping of the cytochrome P-450 2D6 gene is the first pharmacogenomic test to be widely used. The test became available to practicing clinicians at Mayo Clinic in February of 2003. In April of 2004, it became available to all physicians who use the Mayo Medical Laboratories. Since then, Mayo Medical Laboratories has expanded its capabilities to include pharmacogenomic tests for the cytochrome P-450 2C19 gene, the cytochrome P-450 2C9 gene, the serotonin transporter gene (SLC6A4), and two of the serotonin receptor genes (2A and 2C). All provide information related to the response of patients to a variety of antidepressant medications. In 2005, the Food and Drug Administration (FDA) approved a standard genotyping method developed by Roche Diagnostic Laboratories that can be easily adopted by any hospital clinical laboratory with the microarray platform for determining 2D6 and 2C19 genotypes. A small blood sample is all that is required from the patient. Although the FDA has suggested that people who are poor 2D6 metabolizers are at increased risk for adverse reactions to drugs metabolized by the 2D6 enzyme, the agency has not required that patients be tested to determine their metabolic status prior to being prescribed certain drugs. That may well change in the coming year. Indications for 2D6 Genotyping: There are basically 2 reasons to determine the 2D6 genotype of a patient. The first is to identify individuals who are poor metabolizers and, thus, are more likely to have adverse side effects when prescribed 2D6 substrate medications. The second is to identify ultra-rapid metabolizers of these same medications. If a patient metabolizes a drug too quickly, the drug doesnt have the intended effect. Knowing whether a patient is a poor or
25

ultra-rapid metabolizer can help physicians determine which drugsand which doses of those medicationswill work best in certain individuals. It can also help them assess whether a patients symptoms are caused by certain medications or whether medications are interfering with one another. A growing body of research supports the use of pharmacogenomics testing for psychiatric patients. Ongoing studies are showing that these tests may be especially valuable for certain populations. For example, about 1 in 10 Caucasians in Minnesota is a poor metabolizer of 2D6 substrate medications and only about 1 in 50 is an ultra-rapid metabolizer. However, slightly more than half of patients of Somali origin treated in Minnesota have a normal 2D6 genotype and will do well on normal doses of these medications, while approximately 3 in 10 are ultrarapid metabolizers and will experience no therapeutic benefit of 2D6 substrate medications at standard doses. Physicians can use patients racial appearance or self-declaration of ethnic origin to make prescribing decisions that will increase the odds of their avoiding adverse drug reactions, but these clinical inferences are a poor proxy for genotyping. Both psychiatrists and primary care physicians should be concerned about the potential of patients to react adversely to selective serotonin reuptake inhibitors (SSRIs). Although lethal reactions are quite rare, some tragic, avoidable deaths of patients who were poor metabolizers of 2D6 medications and were treated with high doses of those medications have been reported.4 The FDAs black-box warning associated with prescribing such medications has highlighted the importance of monitoring suicidality during the initiation of treatment. Careful monitoring is particularly important for patients who are poor 2D6 metabolizers, as they are likely to have much higher levels of these medications in their bloodstream during the initiation of treatment. Poor 2D6 metabolizers are also believed to be at increased risk for manic or hypomanic symptoms for the same reason. Aside from these concerns, poor 2D6 metabolizers are more susceptible to sexual side effects and are at increased risk for the development of common side effects such as headaches and diarrhea. Although most of these adverse effects do not provide an immediate threat to patients, they are often associated with decreased compliance with their medication schedule, which can have a major impact on the effectiveness of the intervention. Children are more vulnerable to the side effects of medications than adults because their complaints are often taken less seriously. Few adults will continue taking an SSRI if they experience intense headaches or other side effects, but children have less autonomy. By testing children to determine whether they are poor or ultra-rapid metabolizers of 2D6 medications,
26

physicians can help them avoid adverse reactions and help their parents understand the possible reason behind complaints of side effects. Pharmacogenomics testing is particularly indicated for children whose mother or father has been shown to be either a poor or ultra-rapid 2D6 metabolizer. Older patients also may benefit from pharmacogenetic testing. They may not remember which medications they have taken, whether they suffered side effects from those medications, or whether they responded well to them. Geriatric patients often take many medications for a variety of medical problems and may be at risk for drug interactions. For patients who are poor metabolizers, these interactions can be particularly dangerous. Patients who require pain medication may be candidates for pharmacogenetic testing as well. Ideally, physicians should determine whether the patient has sufficient 2D6 enzyme to metabolize the pro-drug codeine to its active metabolite, morphine. A previous history of nonresponse would strongly indicate that the patient is a poor metabolizer. Alternatively, ultrarapid metabolizers often experience a sudden euphoria, which can be unsettling. They may also experience less efficient pain relief as the gradual onset of increasingly severe pain develops before the patient anticipates a need for the next dose. Fortunately, most patients who experience these reactions quickly learn to avoid taking pain medications that include codeine. Patients who take cold medications and are poor metabolizers of 2D6 can develop a high serum level of dextromethorphan after only a few doses. In extreme cases, this can lead to transient psychotic symptoms. Ultra-rapid metabolizers benefit very little from using this medication because of its rapid clearance. One of the most important stories of 2006 in the area of genomics was the discovery that women who are poor metabolizers of 2D6 do not benefit fully from treatment with tamoxifen. Goetz et al. demonstrated this problem, which has been confirmed by other investigators. Furthermore, women who are receiving strong 2D6 inhibitors such as paroxetine and fluoxetine for depression or hot flashes do not appear to respond as well to tamoxifen as those who do not receive these inhibitors, even if they have a genotype that indicates adequate production of the 2D6 enzyme. Genotyping in the Future: The only significant concern related to pharmacogenomics 2D6 genotyping is the cost of the test. The 2D6 test varies in price from less than $300 in some settings to more than $1,000 in others. However, the price is certain to drop with new
27

technological advances and growing demand. Currently, many insurance companies pay for pharmacogenomics testing; but given that this is a relatively new procedure, there is some variability in how these tests are billed and reimbursed. As the test becomes more widely used, knowing a patients genotype will be much like knowing their blood type. Within the next decade, pediatricians will probably gather this information by ordering a standard panel of pharmacogenomically relevant genes during the initial medical evaluation of a healthy infant. Other physicians will simply need to consult the medical records of their patients to clarify their drug metabolic status. However, for the time being, if a patient does not already know his or her 2D6 genotype, it is wise to test them in order to avoid easily detected adverse drug reactions. Clearly, the ancient admonition of Hippocrates that physicians should do no harm has already shifted the practice of many psychiatrists who are scrupulous about trying to minimize the side effects associated with psychotropic medications. With the increased use of pharmacogenomics testing, both improved response to medications and a decrease in adverse reactions should be expected. These are the goals of personalized psychiatric care and ultimately will be an expectation of medical care provided by all specialties. Application of Pharmacogenetic in Oncology:

Application of pharmacogenetics to individualization of therapy with antineoplastic drugs, most of them characterized by a narrow therapeutic index and life-threatening adverse reactions, seems to promise improvement of drug effects in some cases. Thiopurines, like 6-mercaptopurine and thioguanine, largely used in the treatment of acute leukemia, are one of the earliest examples of importance of pharmacogenetics in individualized drug therapy. Following the activation to thioguanine nucleotides via the purine salvage pathway and incorporation into DNA as false purine bases, they are metabolized by the enzyme thiopurine-S-methyltransferase (TPMT) to inactive compounds. The individual enzymatic capacity is a subject to large inter-individual variability which is determined by genetic polymorphisms, with three variant alleles *2, *3A and *3C explaining about 80-95% of enzymatic deficiency. In the Caucasian population, about 89% of people exhibit a high TPMT activity, whereas in 11 and 0.3% of individuals, respectively, intermediate and low activity, is observed. Following a treatment with conventional doses of thiopurines, patients showing
28

diminished catalytic TPMT activity are at increased risk of bone marrow suppression, which may result in fatal outcomes and require discontinuation of therapy. Hepatic TPMT activity can be reliably determined by genotyping or measurement of the catalytic activity of cytosolic TPMT in erythrocytes using established radiochemical or HPLC methods (i.e. phenotyping). Measurement of TPMT activity should routinely precede onset of therapy with thiopurinederived drugs in order to minimize myelotoxic adverse events. For patients being carriers of two non-functional TPMT, thiopurine dose reduction to 5-10% of standard dose was recommended to allow for an efficacious therapy. In heterozygous patients, the therapy begins with a full dose, but a subsequent dose reduction may be required. Although only a small percentage of patients could be affected by inherited differences in TPMT activity, the clinical consequences may be crucial. For that reason the Food and Drug Administration has already implemented respective pharmacogenetic data into the product label of 6-mercaptopurine, widely used for childhood leukemia. Another antineoplastic drug for which pharmacogenetic diagnostics prior to therapy onset would promise selection of potentially toxic patients is 5-Fluorouracil (5-FU). Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the hepatic metabolism of 5-FU and its derivatives such as capecitabine, so that the enzyme activity affects pharmacokinetics, efficacy, and toxicity of the drugs. Diminished enzymatic activity has been observed in about 3-5% of Caucasians and can potentially result in severe adverse drug reactions like mucositis or granulocytopenia in cancer patients treated with 5-FU. DPD is genetically polymorphic and allelic variants in the gene coding the enzyme have been associated with reduced catalytic activity. One of the best described mutations is the the so-called exon 14-skipping mutation at the 5'-splice donor site of exon 14. Although this polymorphism is present in only about 1% of Caucasians, it has been detected in 24% of patients developing severe toxicity (WHO grade IV) following treatment with 5-FU. Nevertheless, further research is needed to evaluate possible benefits of pharmacogenetic strategies upon therapy with 5-FU. At the same time, pharmacogenetics of irinotecan, a potent antineoplastic agent used in the treatment of colorectal cancer and small-cell lung cancer, seems to be one of few promising examples of the implementation of pharmacogenetics to individualized drug therapy. Following its application, irinotecan is metabolized to the active compound SN-38, which is a topoisomerase I inhibitor. In the next step, SN-38 is glucuronidated to its inactive form by various isoenzymes of uridine diphosphate glucuronosyltransferase (UGT), first of all UGT1A1, which is also responsible for glucuronidation of bilirubin. Reduced glucuronidation
29

activity of the UGT1A1 enzyme has been connected to elevated levels of SN-38 and toxic effects like severe diarrhea and neutropenia in patients treated with irinotecan. To date, several genetic polymorphisms leading to impaired UGT1A1 activity have been determined in the gene coding for the enzyme. In the Caucasian population, the UGT1A1*28 polymorphism (TA repeat in the promoter region) is the most frequent variant contributing to reduced glucuronidation activity. It could be shown that even in heterozygous carriers of the variant allele, pronounced changes in irinotecan disposition and severe toxicity occur. For that reason, genotyping for UGT1A1 polymorphisms before the onset of ironotecan therapy has been recommended. Interestingly, the measurement of total bilirubin level seems to be an easy surrogate parameter, if genotyping is not possible. Patients with diminished glucuronidation capacity should be administered a reduced initial dose of irinotecan to avoid the above mentioned severe toxicities. Possible implications of polymorphisms in genes coding for other drug metabolizing enzymes like CYP2D6 and CYP3A, drug transporters like ATP-binding cassette transporter ABCB1 (Pglycoprotein) and drug targets like thymidylate synthase in patients treated with common prescribed antineoplastic drugs have also been considered in numerous studies, but their potential impact on clinical outcomes is still controversial. In summary, oncology is the clinical area where achievements of modern pharmacogenomic diagnostics have already been used to tailor individual therapy with some antineoplastic drugs, but for a wide implementation of genotyping in cancer patients, more clinical data and a precise cost effectiveness analysis of this approach are required. Application of Pharmacogenetics in Cardiology:

Cardiovascular diseases like coronary heart disease, hypertension or heart failure are still a leading health problem in developed countries and respective pharmacotherapy is an established approach in affected patients. It appears that pharmacogenetics throws some new light on the question of treatment amendment with respect to cardiovascular diseases. For several beta-blockers, which belong to the most often prescribed drugs in patients with cardiovascular diseases, possible effects of genetic polymorphisms in drug metabolizing enzymes like CYP2D6 were assessed. CYP2D6 is the key enzyme in metabolism of metoprolol and pronounced differences between CYP2D6 extensive and rapid metabolizers with respect to the phramacokinetics of the drug have been observed. Moreover, CYP2D6 polymorphism has
30

been shown to contribute to pharmacodynamic response following the administration of metoprolol, since reduction of exercise induced heart rate by the drug in the group of ultrarapid metabolizers (carrying a duplication of the CYP2D6 gene) was only circa half of that observed in extensive metabolizers. Also for carvedilol, the role of the CYP2D6 polymorphism was studied. However, respective pharmacokinetic differences resulted from the genetic polymorphism seem to be without any effects on heart rate and blood pressure so that they will have no clinical significance. Another class of drugs, AT 1 (angiotensin II type 1) receptor antagonists (sartans), used to treat hypertension or heart failure, could be potential candidate for consideration of pharmacogenetic data in therapy optimization. Most sartans are metabolized with participation of genetically polymorphic CYP2C9. Losartan is a pro-drug which is transformed to its active form, i.e. E3174, via CYP2C9 and CYP3A4. Unfortunately the role of the CYP2C9 polymorphism for therapy with losartan is quite controversial. Whereas in one study, presence of CYP2C9*3 was shown to be associated with decreased formation of E-3174, in another study, no differences with respect to the pharmacokinetics of the parent drug and its active metabolite between the wild types and carriers of the best investigated CYP2C9 variant alleles related to impaired intrinsic enzymatic activity CYP2C9*3 and CYP2C9*2 were determined. There is also some clinical data suggesting the role of CYP2C9 polymorphism in the pharmacokinetics and/or -dynamics of other AT 1 receptor antagonists like irbesartan or candesartan. However, if potential dose adjustment of sartans according to the CYP2C9 genotype might be beneficial is furthermore doubtful. Recently, importance of pharmacogenetic implications has also been discussed for statins (HMG-CoA reductase inhibitors), administered to lower cholesterol level in numerous patients with or at risk for cardiovascular problems. Statins are the most prescribed and most effective drugs in lipid lowering therapy but large variability in response is observed and in nearly one of three patients treatment goals could not be met. It has been reported that in patients treated with pravastatin, cholesterol lowering effects are poorer in carriers of two common and tightly linked single nucleotide polymorphisms localized in the gene coding for HMG-CoA reductase, which is the target enzyme for statin therapy. However, no data is available; if possible genotyping approach with a following dose adjustment, in terms of application of a higher dose of pravastatin in patients carrying the variant haplotype, could be advantageous in clinical practice.
31

Last but not least, the meaning of pharmacogenetic approaches for therapy with oral anticoagulants (coumarin anticoagulants) should be briefly discussed. These vitamin K antagonists, used widely in patients at risk of thromboembolic disorders, are characterized by a narrow therapeutic index, so that the therapy with them is often complicated by dangerous bleeding episodes or lack of efficacy, in case of under- or over coagulation, respectively. Two polymorphic genes, CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKORC1), can contribute significantly to the known inter-individual variability in the effectiveness of oral anticoagulants. The role of the enzyme CYP2C9 in metabolism of the warfarin and its analogues acenocoumarol and phenprocoumon is well documented. The variant alleles with decreased enzymatic activity CYP2C9 *2 and CYP2C9 *3 have been demonstrated to impact considerably the pharmacokinetics of S-warfarin (which is 3 to 5 times more potent than the Risomer) and so to influence the antithrombotic activity of the drug. Patients carrying at least one variant allele show a longer induction period to achieve a stable warfarin dosing and tend to have increased values of international normalized ratio (INR). They are also at increased risk of life threatening bleedings. Similarly, there is a good evidence for the role of CYP2C9 polymorphism in the anticoagulation effects of acenocoumarol and phenprocoumon in the literature data. For that reason, CYP2C9 genotyping was suggested as a useful approach to select a population of patients who are potentially at risk of complications associated with oral anticoagulants and who may require a reduced dose of the drugs. VKORC1 is the target molecule of vitamin K antagonists and polymorphisms in VKORC1 gene, in addition to CYP2C9 and demographic factors seem to explain a significant part of the inter-individual variability in pharmacokinetics and dynamics of the drugs and consequently could be essential for determination of the individual dose. For warfarin, an algorithm for individual dosing adjustment on the base of CYP2C9 and VKORC1 genotype, age and height has been proposed, but prior to introduction into clinical practice it should be proved in prospective clinical studies. In summary, in the light of current knowledge, it seems that with respect to cardiovascular diseases, only for vitamin K antagonists, there is a place for pharmacogenetic approaches to optimize the therapy and avoid adverse events. Looking back at more than 50 years of pharmacogenetic experience, we have learnt that an important part of the inter-individual variability in drug response is caused by polymorphisms in drug metabolizing enzymes, transporters or target molecules. For some treatments, it was shown that efficacy and safety profile of pharmacotherapy could be improved if respective allelic variations are taken into
32

account. Although it seems that the first genotype-specific dose recommendations have already reached clinical practice in some medical fields, unquestionably more prospective clinical studies validating pharmacogenetic approaches as well as cost-effectiveness evaluations are needed before pharmacogenetics makes a great jump from bench to bedside. Estimation of the Drug Dose with Clinical and Pharmacogenetic Data:

Genetic variability among patients plays an important role in determining the dose of drugs that should be used when oral dosage form is initiated, but practical methods of using genetic information have not been evaluated in a diverse and large population. Scientists developed and used an algorithm for estimating the appropriate drug dose that is based on both clinical and genetic data from a broad population base. For example, Warfarin is the most widely used oral anticoagulant agent worldwide; more than 30 million prescriptions were written for this drug in the United States in 2004.1 The appropriate dose of warfarin is difficult to establish because it can vary by a factor of 10 among patients, and the consequences of taking an incorrect dose can be catastrophic. Because incorrect doses contribute to a high rate of adverse effects, there is interest in developing improved strategies for determining the appropriate dose. Clinical factors, demographic variables, and variations in two genes cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), and vitamin K epoxide reductase complex, subunit 1 (VKORC1) contribute significantly to the variability among patients in dose requirements for warfarin. In 2007, the Food and Drug Administration added pharmacogenetic information to the warfarin product label but did not propose a specific method for using genetic information to predict the dose required in individual patients. Proposed algorithms for predicting the appropriate dose of warfarin are usually based on relatively small clinical populations, and their general predictive accuracy is uncertain. Small trials have recently been performed and large, prospective trials are ongoing or planned in the United States and Europe to test whether algorithms for warfarin dosage that use genetic information improve the outcomes for patients (e.g., better anticoagulation control and a shorter time to achieving a stable dose). We developed a pharmacogenetic dose algorithm for warfarin with the use of a large and diverse data set that included data from patients at centers around the world and used it to determine retrospectively whether the dosage recommendations that were based on this

33

algorithm were significantly better than those that were based on an algorithm that used only clinical variables or those that were based on a fixed-dose strategy. Application of Pharmacogenetics in Drug Development:

Interindividual variability in uptake and metabolism of many drugs makes dose-response relationships difficult to predict. A dose that produces the desired therapeutic response in one individual may not be efficacious or may even be toxic for another patient. Therefore, it is desirable to understand the metabolism and activity pathways that contribute to these differences to optimize the therapeutic effects of new chemical entities. Consequently, pharmacogenetics affects all phases of the research and development process and eventually influences the practice of medicine. Many pharmaceutical research laboratories screen compounds to determine whether they are metabolized by highly polymorphic metabolizing enzymes before full scale efficacy and safety testing (Kleyn and Vesell, 1998). The following figure illustrates the pharmacokinetic outcomes of a hypothetical drug metabolized by a polymorphic enzyme. Normal metabolizers (wild type) have plasma concentrations within the therapeutic range and benefit from the therapy. Poor metabolizers have plasma drug concentrations exceeding the maximal harmful concentrations and are at risk of experiencing drug side effects and toxicity. Ultrafast metabolizers do not reach therapeutic concentrations and fail to achieve the desired therapeutic effect at regular dose regimens. When a compound has a narrow therapeutic window, slight variations in plasma drug concentration can point to the drug's success or failure for a particular treatment.

34

Figure: Schematic plot of the plasma drug concentrations in different patients after giving same doses of a drug metabolized by a polymorphic enzyme Pharmacogenetic data can aid in the selection of a compound for future clinical development and offer a powerful tool for optimizing therapeutic efficacy. Pharmacogenetics may also help design therapeutic agents targeting specific groups of patients with a set of genotypic characteristics that otherwise would deprive them of a cure. For example, an investigative drug showed no statistically significant effect when given to 400 Alzheimer's patients, and a clinically significant response was elicited when patients were stratified according to ApoE subtype (Richard et al., 1997). Applying Pharmacogenetics in Clinical Research:

Pharmacogenetics holds great potential for facilitating the drug discovery process and subsequent clinical study. With the progress of the Human Genome Project and functional genomics, massive increases in the information available on individual genes and functionally important polymorphisms related to disease will emerge (Evans and Relling, 1999). In 1997, the U.S. Food and Drug Administration issued guidance for industry and supported pharmacogenetic testing throughout the drug development process (United States Food and Drug Administration, 1997). Understanding how to adjust dose to minimize toxicity may allow marketing a drug that otherwise would have an unacceptable rate of adverse effects because its toxicity was unpredictable and unpreventable without pharmacogenetic tools. When genetic
35

polymorphisms affect important metabolic routes of elimination, dosing adjustments may achieve the safe and effective use of a drug. Identifying metabolic differences in patient groups based on genetic polymorphisms would provide improved treatment recommendations and product labeling, thereby promoting the safe and effective use of a drug. An example of this is omeprazole (Prilosec), an inhibitor of the H+/K+ ATPase enzyme system at the secretory surface of the gastric parietal cell, used for treatment of ulcer and gastroesophageal reflux disease (Bustamante and Stollman, 1999). In pharmacokinetic studies of omeprazole (single dose), an increase in area under the curve of approximately 4-fold was noted in Asian subjects compared with Caucasians (Johnson, 1997; AstraZeneca, 1999). The area under the curve difference was due to different metabolic rates of the drug, which is a substrate for CYP2C19 (Cupp and Tracy, 1998). Approximately 20% of Asians are homozygous for variants of the CYP2C19 gene resulting in poor metabolizer phenotype (De Morais et al., 1993). Therefore, the dose administered to Asian patients with poor metabolizer genotypes and patients with impaired hepatic function is reduced (Johnson, 1997). These examples of pharmacogenetic information will help to control or reduce adverse responses to drugs and reduce the costs associated with therapeutic failures. For drugs prescribed on a limited basis due to a high incidence of adverse effects, pharmacogenetics may provide the means to identify those most likely to benefit therapeutically without the development of adverse reactions. Another potential application of pharmacogenetics is in the strategic design of clinical trials to increase the information obtained from each study. Identification of potential responder populations through genetic screening before clinical trial enrollment will allow demonstration of drug efficacy in a smaller set of subjects. This approach was relevant for trastuzumab (Herceptin), a monoclonal antibody for treatment of late-stage breast cancer, and only patients with tumor cells overexpressing HER2 gene would benefit from this drug (Shak, 1999). Therefore, patients were tested for this marker before receiving the drug. With the rapid progress in this area, the advent of validated pharmacogenetic markers could be included in clinical trials to increase the demonstration of therapeutic benefits without exposing nonreceptive subjects. Genotyping information also can be used to understand outliers in plasma concentration or therapeutic profiles related to genetic determinants.

36

With the emerging global development, new pharmaceutical agents are tested or developed in multiple countries. However, due to the differential distribution of genetic polymorphisms in ethnic groups (Weber, 1997), a well-developed and extensively tested drug evaluated in one country might not be suitable for patients with pharmacogenetic traits from other countries or continents. Genotyping data from multiple ethnic groups will allow an early identification of drug efficacy and toxicity, and has the potential to reduce the need for conducting pivotal clinical trials in multiple countries, with significant resource savings, shortening development time and reducing the costs of new drugs.

Germline Genetic Testing to Predict Drug Response and Toxicity:

There is an increasing interest in personalized medicine, perhaps none greater than in the eld of oncology. The idea of making therapeutic decisions based on an individuals genetic makeup, with the ability to predict tumour response, as well as minimize toxicity is extremely appealing to the oncologist. This is especially because many chemotherapeutic agents have a narrow therapeutic index. To date, there are at least 5 United States Food and Drug Administration (US FDA) drug labels with recommendations for germline genetic testing to be considered prior to administration of the drug or drug group for the treatment of cancer. This review aims to provide a brief insight on the genetic basis for inter-individual variations in therapeutic outcome relevant to key classes of anticancer agents and the potential application of genetic testing for treatment. 6-Mecaptopurine (6-MP): 6-MP is one of the first chemotherapeutic agents recommended by the US FDA to consider genetic testing prior to commencement of therapy. It is used predominantly for the treatment of childhood acute lymphocytic leukemia (ALL). Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyses the methylation of aromatic and heterocyclic sulphydryl compounds. The substrates of TPMT include 6-MP, 6-thioguanine and azathioprine. Although there are 2 other pathways competing with TPMT for the metabolism of 6-MP, the activity of the competing pathways in haematopoietic tissues is negligible and TPMT is the major inactivating enzyme for 6-MP in these tissues. TPMT activity has a trimodal distribution and is inherited in an autosomal codominant pattern. Genetic polymorphisms in TPMT have been associated with 6-MP toxicity and therapeutic efcacy. Patients with TPMT deciency require dose reduction to prevent life-threatening toxicity. Even when treated at 10% of the standard dose of 6-MP, patients homozygous for TPMT variants have similar or superior survival compared with patients with at least one wild-type (normal)
37

allele. Patients homozygous for the wild-type allele are less likely to have severe treatment toxicity but may be at higher risk of disease relapse. To date, at least 21 TPMT polymorphisms have been identied, 17 of which were shown to have reduced TPMT activity. Approximately 1 in 300 Caucasians has almost no detectable TPMT activity, and 1 in 10 has intermediate TPMT activity. TPMT*2 (238G>C, P240A), TPMT*3A (460G>A, A154C; 719A>G, C240Y), and TPMT*3C (719A>G, C240Y) account for over 90% of low or intermediate activity phenotypes in Caucasians, with reported allelic frequencies of 0.2%, 4.4%, and 0.4%, respectively. TPMT protein variants are prone to enzymatic degradation, resulting in lower catalytic activity. The mean calculated cost per lifeyear gained by TPMT genotyping in ALL patients was 2100 (~SGD$3650), based on genotyping costs of 150 (~SGD$260) per patient, and this is expected to further improve following wider use and decreasing cost due to availability of lower cost genotyping methods. With measurement of TPMT red blood cell activity being time-consuming and may be unreliable following blood transfusions, pretreatment genetic testing is an attractive alternative to screen for patients with TPMT deciency. In 2003, a US FDA advisory committee made a landmark decision to recommend the addition of pharmacogenetic information on TPMT polymorphisms and treatment toxicity to the prescribing information for 6-MP. However, several questions remain unanswered. There are substantial differences in the frequency of TMPT variants across various population groups. In Asian populations, TPMT*3C is the most common TPMT variant, with estimated allele frequency of TPMT*3C in the region of 2% to 4%. This difference means that the cost effectiveness of testing in specic populations is still unclear. In addition, variable number tandem repeats (VNTR) have been found in the promoter region of TPMT. Although there is in vitro evidence to suggest that VNTR polymorphisms correlate negatively with TPMT activity, the importance of VNTR polymorphisms has not been clearly established in clinical studies. There is currently still no consensus regarding the optimal dose of chemotherapy for TMPT heterozygotes in all populations. Similarly, there has been no study to date to compare if dose individualization for heterozygotes is superior to dose adjustment based on toxicity. Tamoxifen: Tamoxifen is used in the treatment of estrogen receptorpositive breast cancer and also for chemoprevention for patients at high risk for developing breast cancer. It is metabolized via the cytochrome P450 (CYP) pathway to form several metabolites, including endoxifen and 4-OH-tamoxifen which are the most potent. Endoxifen is present at a much higher plasma concentration than 4-OH-tamoxifen and is thought to be largely responsible for
38

the therapeutic effect of tamoxifen. CYP2D6 is the predominant CYP isoform that catalyses the formation of endoxifen. At least 88 allelic variants of CYP2D6 have been described, many of which are nonfunctional or have reduced catalytic activity, resulting in a tetra-modal distribution: poor, intermediate, extensive and ultra-rapid. It is estimated that 5% to 10% of Caucasians have nonfunctional variants. The most common nonfunctional variants, CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*6, constitute approximately 97% of the poor-metabolizer phenotype, resulting in up to a 4-fold difference in endoxifen concentration in homozygotes for these variants, compared to wild-types. Ultra rapid metabolizers carry gene duplications or multiple duplications of functional alleles resulting in increased enzyme activity. These genotypes are rare in Caucasians and Asians but are common in Ethiopians and Saudi Arabians. Initial studies suggest that women with decreased CYP2D6 activity had poorer clinical outcomes when treated with tamoxifen, both in the adjuvant and chemoprevention settings. For example, in a large retrospective analysis of German and US patients, Schroth et al showed that the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers. Ultra rapid metabolizers, and possibly extensive metabolizers, may potentially benet from extended tamoxifen use (up to 5 years) before switching to AI. However, retrospective analyses of 2 large adjuvant breast cancer trials, ATAC and BIG1-98, were presented recently, and they failed to establish a relationship between CYP2D6 polymorphisms and treatment outcome in patients treated with tamoxifen. Although only in abstract form, these results highlight the uncertainty surrounding genetic testing for CYP2D6 to guide tamoxifen therapy currently. Administration of CYP2D6 inhibitors may affect tamoxifen-related breast cancer outcomes by converting an extensive metabolizer to a phenotypic poor metabolizer and should be avoided. Potent CYP2D6 inhibitors include antidepressants such as uoxetine and paroxetine, whereas moderate/weak inhibitors include cimetidine, amiodarone, ticlopidine and haloperidol. An FDA approved microarray-based pharmacogenetic test, the AmpliChip CYP450R test, is available for pretreatment testing of CYP2D6. It can detect 27 variant alleles of CYP2D6 and three variants of CYP2C19 using an array of over 15,000 probes. The variants tested include nonfunctional variants (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*6), and those that are more commonly found in East Asians (CYP2D6*10), African Americans (CYP2D6*17),
39

and Middle Eastern and North African populations (CYP2D6*2XN). At this point, expert opinion is that there is currently no strong evidence to routinely recommend CYP2D6 testing prior to tamoxifen prescription, and more studies are required. While CYP2D6 is important, it may not be the only determinant of tamoxifen outcome. Other determinants may include other metabolizing enzymes, complex interplay of different active metabolites in addition to endoxifen, and possibly concomitant medications. It is still recommended that patients who are on tamoxifen avoid potent CYP2D6 inhibitors. Major drawbacks/limitations: Inherited component of the response to drugs is often polygenic. Furthermore, the drug response is probably affected by multiple genes, each genes with multiple polymorphisms distributed in the general population. Racial differences add further confounding factors. Drug response might be predicted from a certain pattern of polymorphisms rather than only a single polymorphism.this could prevent predictions about drug responses across the general patient population, and it emphasizes the need to stratify clinical pharmacogenomics studies. SNP maps and candidate-gene stratigies are based on existing knowledge of a medications mechanism of action and pathways of metabolism and disposition. The candidate-gene strategies has the advantage of focusing resources on a manageable number of genes and polymorphisms that are likely to be important but the limitations limitations are the incompleteness of knowledge of a medications pharmacokinetics and mechanisms of action. The dynamic complexity of the human genum, involvement of multiple genes in drug responses, and racial differences in the prevalence of gene variants impede effective genumewide scanning and progress towards practical clinical applications. Genomic technologies are still evolving rapidly, at an exponential pace similar to the development of computer technology over the past 20 years. Finding the gene variations that are responsible for a particular drug response is very complex. This is so as the human genome is very large and consists of 3 billion bases and single nucleotide polymorphisms occur every 100 to 300 bases. With this being complex it means that limited information about which all genes that are responsible for each drug response is available, serving to only further complicate things. As a result, for persons whose gene
40

variations prevent them from tolerating one or two approved medicines, wouldnt have any drug alternative available to them. The use of pharmacogenomics has been limited since there is a lack of scientific evidence for improved patient care. Although it is used for treating diseases such as cancer, HIV and cardiovascular diseases among others, it is done so to a limited extent. Variations in mutations with drug metabolism and drug response may arise due to persons varying lifestyle, diet, age, environment and overall health condition. Although pharmacogenomics has the potential to reduce health care costs and yield economic benefits, it is very costly to introduce a new drug to the market. This coupled with the fact that this new drug only suits a fraction of the population, discouraging pharmaceutical companies to make such an investment. As a result that fraction with rare or complex genetic conditions or those who are not responding to any known treatment will be deprived of effective treatments. Pharmacogenomics has to deal with the issues of privacy and ultimately genetic discrimination which can affect access to life and health insurance. In addition, patients psyche can be affected since they may be concerned about being labeled as either a responder or a non-responder, to a given therapy (Squassina et al., n.d.). With pharmacogenomics being fairly new phenomena, medical practitioners and primary health care providers lack adequate knowledge and preparation about pharmacogenetic testing and personalized medicine. This makes it difficult for them to actually carry out pharmacogentic tests, interpret their results and effectively utilize it in clinical practice. Pharmacogenomics is the branch of pharmacology that deals with the influence

of genetic variation on the drug response in patients. The limitations of pharmacogenomics could be the requirement for behavioral, changes to prevent disease and adverse reactions. Many genes are likely to be involved in how someone reacts to a drug. It means that targeting different drugs may be very complex. Everyone has small variations in their genes that do not cause any problem with the way that the gene works. Since these differences may influence drug metabolism or how the condition develops, the variations would need to be identified. This process is very difficult and time consuming. In addition other factors may influence a specific drug reaction such as interactions with other drugs and environmental factors. The

41

influence of these factors will need to be determined before any conclusions are made about the genetic influence on how the drug is working. Genetic tests are very expensive if done on a case-by-case basis. Ethical Consideration Over the past several years, as a result of developments from the Human Genome Project (HGP), there have been unprecedented advances in our understanding of the role of genetic polymorphisms in the response to therapeutic drugs. Pharmacogenomics is a relatively new field that was spawned by these advances, which includes the study of genetic variations or polymorphisms between individuals, and how these variations influence responses to therapy. Although pharmacogenomics and the older term pharmacokinetics are often used interchangeably, pharmacogenomics is broader in scope, and refers to the complex interactions of genes across the genome. Pharmacogenomics includes identifying candidate genes and polymorphisms, correlating these polymorphisms with possible therapies, predicting drug response and clinical outcomes, reducing adverse events and selection, and selecting dosing of therapeutic drugs on the basis of GENOTYPE4. Individual variations in the response to drugs and drug toxicity are a common occurrence in the clinical setting and in drug development research protocols. Indeed, individual differences in drug response contribute to several adverse events that have long been recognized as benign important clinical problem. A predictive drug-testing scheme based on the supposition that there is a genetic polymorphism of drug metabolism might include the following steps: (1) The chemical configuration of a new (or known) compound may suggest the most probable aim sources of metabolism; (2) Genetic testing and/or sequencing of genetic mutations or polymorphisms with high correlation to drug metabolism function; (3) Analysis of phenotypic expression The Pharmacogenomics Journal of a given polymorphic gene and assignment of the protein to a biochemical drug-metabolizing pathway; (4) Investigation of drug metabolism in vitro with: (a) human microtonal liver preparations of known phenotypes/ genotypes from a patient with a given polymorphism; (b) human erythrocytes of known phenotypes/genotypes from a patient with a given polymorphism (c) eukaryotic cells carrying expression vectors incorporating known human genes compared to
42

cells carrying expression vectors incorporating the polymorphic homologues of the normal gene; (d) as (c) with inhibitors or previously recognized polymorphic substrates; and (e) as (c) with antibodies to known polymorphic enzymes; (5) Perform animal studies (in more than one species) to identify the main routes of metabolism and toxicological aspects; (6) Investigation in a few humans in vivo to confirm results of animal studies; (7) If the existence of biotransformation polymorphism is substantiated, begin sting on typed panels of healthy individuals of different genetic backgrounds, who represent the different phenotypes of the polymorphisms; (8) If the polymorphism is confirmed, investigate single dose and multiple dose kinetics in relation to the pharmacological effect: (a) in phenotyped healthy persons of different backgrounds; and (b) in phenotyped patients suffering from the conditions for which the drug is being developed; and (9) Formulate appropriate policies and procedures: (a) information about the polymorphism and its implications included in drug labeling; and (b) advocate phenotyping and DNA genotyping before starting treatment with known or newly approved drugs. Much of the discussion about ethical and legal issues relating to pharmacogenetics is centered on the issue of genetic testing, a topic that has recently been the focus of a number of guidelines, advisories, white papers, etc., issued by a number of committees in both Europe and the United States. It is interesting to note that the one characteristic that almost all these documents share is a studious avoidance of defining exactly what a genetic test is. Where definitions are given, they tend to be very broad, including not only the analysis of DNA but also of transcription and translation products affected by inherited variation. As much as the most sensible solution to this dilemma would be a consensus to The Role of Genotyping in Pharmacological Therapy treat all personal medical data in a similar fashion regardless of the degree to which DNA-encoded information affects it (noting that there really is not any medical data that are not to some extent affected by intrinsic patient properties), it may, for the time being, be helpful to let the definition of what constitutes genetic data be guided by the public perception of genetic data, in as much as the whole discussion of this topic is prompted by these public perceptions. In the public eye, a genetic test is usually understood either (1) as any kind of test that establishes the diagnosis (or predisposition) of a classic monogenic,
43

heritable disease or (2) as any kind of test based on nucleic acid analysis. This includes the (non-DNA-based) Guthrie test for phenylketonuria as well as forensic and paternity testing and the DNA-based test for Lp(a), but not the plasmaprotein- based test for the same marker (even though the information derived isidentical). Since monogenic disease is, in effect, excluded from this discussion, it stands to reason to restrict the definition of genetic testing to the analysis of (human) DNA sequence. Based on the perceived particular sensitivity of genetic data, institutional review-boards commonly apply a specific set of rules for granting permission totest for DNA-based markers in the course of drug trials or other clinical research, mization of samples and data, specific stipulations about availability of genetic counseling, provision to be able to withdraw samples at any time in the future, etc. Arguments have been advanced (Roses 2000) that genotype determinations for pharmacogenetic characterization, in contrast to genetic testing for primary disease risk assessment, are less likely to raise potentially sensitive issues with regard to patient confidentiality, the misuse of genotyping data or other nucleicacidderived information, and the possibility of stigmatization. While this is certainly true when pharmacogenetic testing is compared to predictive genotyping for highly penetrant mendelian disorders, it is not apparent why in common complex disorders, issues surrounding predictors of primary disease risk would be any more or less sensitive than those pertaining to predictors of likely treatment success or failure. Indeed, two lines of reasoning may actually indicate an increased potential for ethical issues and complex confrontations among the various stakeholders to arise from pharmacogenetic data. First, while access to genotyping and other nucleic acid-derived data related to disease susceptibility can be strictly limited, the very nature of pharmacogenetic data calls for a rather more liberal position regarding use: if this information is to serve its intended purpose, i.e., improving the patients chance for successful treatment, then it is essential that it is shared among at least a somewhat wider circle of participants in the health care process. Thus, the prescription for a drug that is limited to a group of patients with a particular genotype will inevitably disclose those patients genotype to anyone of a large number of individuals involved in the care of those patients at the medical and administrative level. The only way to limit this quasi-public disclosure of this type of patient genotype data would be if he or she were to sacrifice the benefits of the indicated treatment for the sake of data confidentiality. Second, patients profiled to carry a high disease probability along with a high likelihood for treatment response may be viewed, from the standpoint of insurance risk, for example, as comparable to patients displaying the opposite profile, i.e., a low risk to develop the disease,
44

but having a high likelihood not to respond to medical treatment, if the disease indeed occurs. For any given disease risk, then patients less likely to respond to treatment would be seen as a more unfavorable insurance risk, particularly if non-responder status is associated with chronic, costly illness rather than with early mortality, the first case having much more far-reaching economic consequences. The pharmacogenetic profile may thus, under certain circumstances, become a more important (financial) risk-assessment parameter than primary disease susceptibility, and would be expected, in as much as it represents but one stone in the complex disease mosaic, to be treated with similar weight, or lack thereof, as other genetic and environmental risk factors. Practically speaking, the critical issue is not only, and perhaps not even predominantly, the sensitive nature of the information and how it is disseminated and disclosed, but how and to what end it is used. Obviously, the generation and acquisition of personal medical information must always be contingent on the individuals free choice and consent, as must be all the application of such data for specific purposes. Beyond this, however, there is today an urgent need for the requisite dialogue and discourse among all stakeholders within society to develop and endorse a set of criteria by which the use of genetic, and indeed of all personal medical information, should occur. It will be critically important that society as a whole endorses, in an act of solidarity with those destined to develop a certain disease, guidelines that support the beneficial and legitimate use of the data in the patients interest while at the same time prohibiting their use in ways that may harm the individual, personally, financially, or otherwise. As long as we trust our political decision processes to reflect the consensus of society, and as long as such consensus reflects the principles of justice and equality, the resulting set of principles should assert such proper use of medical information. Indeed, both aspects, data protection and patient/subject protection, are seminal components of the mandates included in the WHOs Proposed International Guidelines on Ethical Issues in Medical Genetics and Genetic Services which mandate autonomy, beneficence, no maleficence, and justice.

45

References 1. Lumelsky, A., 2003. Genetic Testing and Government Regulation: The Growing Significance of Pharmacogenomics
2. Clarke, W. and McMillan, G., 2006. Personalized Medicine: Emerging Strategies for

Therapeutic Drug Management. Clinical Laboratory News.

3. Mallal, S. and Phillips, E., 2007. The introduction of Phamacogenetic Screening to HIV

Clinical Practice, Potential Benefits and Challenges. European Infectious Disease. Pp.14-17.
4. Yong, W.P., and Innocenti, F., 2007. Translation of Pharmacogenetic Knowledge into

Cancer Therapeutics. Clinical Advances in Hematology & Oncology. 5(9), pp.699-701.

5. Laurence L. Burton., ed., Goodman & Gilmans The Pharmacological Basis of

Therapeutics. 12th edition. McGraw Hill: New York

6. Polymorphic Drug Metabolising Enzymes: Assessment of Activities by Phenotyping

and Genotyping in Clinical Pharmacology

7. U.S. Food and Drug Administration, 2006. Guidance for Industry and FDA Staff:

Pharmacogenetic Tests and Genetic Tests for Heritable Markers [pdf] Available at: <http://www.fda.gov/cber/guidelines.htm> 8. indpaintner, K., The Role of genotyping in Pharmacological Therapy.pp16-19
9. Honda, M., Nozawa, T. and Igarashi, N. 2005. Effect of CYP2D6*10 on the

Pharmacokinetics of R- and S-Carvedilol in Healthy Japanese Volunteers. Biol. Pharm. Bull. 28(8) p1477.
10. Current Applications of Clinical Genetic Testing for Psychiatric Practice. 2012.

Current Applications of Clinical Genetic Testing for Psychiatric Practice. [ONLINE] Available razekJanuary2007.aspx. [Accessed 19 May 2012] at: http://www.minnesotamedicine.com/PastIssues/PastIssues2007/January2007/ClinicalM

46

11. Estimation of the Warfarin Dose with Clinical and Pharmacogenetic Data. 2012.

Estimation of the Warfarin Dose with Clinical and Pharmacogenetic Data. [ONLINE] Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722908. [Accessed 19 May 2012].
12. IP Soh, T & Yong, W (2011) Germline Genetic Testing to Predict Drug Response and

Toxicity in Oncology Reality or Fiction?. Annals Academy of Medicine. 40 (8) p350353

13. IFCC - Application of pharmacogenetics in dose individualization in diabetes

psychiatry cancer and card. 2012. IFCC - Application of pharmacogenetics in dose individualization in diabetes psychiatry cancer and card. [ONLINE] Available at: http://www.ifcc.org/ifcc-communications-publications-division-%28cpd%29/ifccpublications/ejifcc-%28journal%29/e-journal-volumes/vol-19-n%C2%B01/application-of-pharmacogenetics-in-dose-individualization-in-diabetes-psychiatrycancer-and-card. [Accessed 19 May 2012].
14. Pharmacogenetic Application in Drug Development and Clinical Trials. 2012.

Pharmacogenetic Application in Drug Development and Clinical Trials. [ONLINE] Available at: http://dmd.aspetjournals.org/content/29/4/591.full. [Accessed 19 May 2012].
15. Textbook of Personalized Medicine - Kewal K. Jain - Google Books. 2012. Textbook of

Personalized Medicine - Kewal K. Jain - Google Books. [ONLINE] Available at: http://books.google.com.bd/books? id=_yIvaMp5nzkC&pg=PA101&lpg=PA101&dq=limitations+of+pharmacogenetics&s ource=bl&ots=eIVJD3JsZW&sig=d_yL0pagFbswk7o2JOQUld16Bg&hl=en&sa=X&ei=V863T4jQG8ntrQeI9P35Bw&sqi=2& redir_esc=y#v=onepage&q=limitations%20of%20pharmacogenetics&f=false. [Accessed 20 May 2012].
16. Sgugenetics / Limitations of Pharmacogenomics. 2012. sgugenetics / Limitations of

Pharmacogenomics.

[ONLINE]

Available

at:

http://sgugenetics.pbworks.com/w/page/47492447/Limitations%20of %20Pharmacogenomics. [Accessed 20 May 2012].

47

17. 2012. [ONLINE] Available at: http://www.genetics.edu.au/Information/Genetics-Fact-

Sheets/PharmacogeneticsPharmacogenomicsFS25. [Accessed 20 May 2012].

48