The AmpliChip CYP450 Test: Principles, Challenges, and Future Clinical Utility in Digestive Disease

Transcription

1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2006;4: CLINICAL GENOMICS The AmpliChip CYP450 Test: Principles, Challenges, and Future Clinical Utility in Digestive Disease BRIAN D. JURAN,* LAURENCE J. EGAN, and KONSTANTINOS N. LAZARIDIS* *Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota; and Department of Pharmacology and Therapeutics, National University of Ireland, Galway, Ireland Understanding genetically encoded inherited differences in drug metabolism and targets (ie, receptors, transporters) offers the promise of minimizing adverse drug reactions and improving therapies. Among the enzymes involved in drug metabolism, the cytochromes P450 (CYP450) hold a central position. In fact, CYP450 are involved in the biotransformation of most drugs used in clinical practice. Recent advances in the development of DNA-based diagnostics, coupled with a better understanding of genetic polymorphisms in influencing pharmacologic responses, have provided the foundation for novel in vitro tests that may predict side effects and/or therapeutic responses. The AmpliChip CYP450 test was developed as a clinical test to evaluate an individual s metabolic capacity for certain drugs by identifying polymorphisms of 2 CYP450 enzymes (ie, CYP2D6 and CYP2D19). Even though the AmpliChip CYP450 has been approved by the US Food and Drug Administration, its practical clinical utility has not yet been determined, and there is a paucity of data related to gastrointestinal and liver diseases. An understanding of the principles and opportunities provided by this new category of diagnostic test is key before planning the necessary studies to evaluate the usefulness of AmpliChip CYP450 in gastroenterologic clinical practice. Successful pharmacologic treatment implies effective therapy without the development of adverse drug reactions (ADRs). Effective therapy entails a beneficial response to treatment despite the inherited and acquired variations in drug bioavailability and targeting that often lead to treatment failures (TFs), even for the compliant patient. ADRs and TFs represent a significant burden to overall health costs; indeed, more than 2 million serious ADRs occur annually in the United States, resulting in more than 100,000 deaths 1 and costing the economy an estimated $100 billion. 2 Moreover, many patients receiving prescription drugs fail to fully respond to first-line treatment and require additional pharmacologic intervention, further escalating these costs. Factors contributing to ADRs and TFs include drug drug interactions, physician patient miscommunication regarding proper drug administration, physician prescription error, and significant differences in how individuals metabolize drugs. Genetically encoded variation in the activity of drug-metabolizing enzymes has emerged as a potential tool for predicting drug response and ADRs in populations. Understanding the links between genetic polymorphisms of drug-metabolizing enzymes and responses to therapy may lead to individualized medicine. Among the drug-metabolizing pathways, the CYP450 system represents the main component of phase I metabolism (the enzymatic modification of functional groups). Genes encoding drug-metabolizing CYP450 enzymes, such as CYP2C19 and CYP2D6, are highly polymorphic. Prediction of an individual s capacity to metabolize pharmaceuticals based on the genotype of the polymorphic CYP450 enzymes is now possible. This has the potential to significantly decrease ADRs and TFs in the future. However, introduction of this approach into clinical practice requires robust, affordable, and accessible genotyping technologies that can be easily performed by most clinical laboratories and can be applied to large populations undergoing therapy. The AmpliChip CYP450 test is the first microarraybased assay to be approved for diagnostic use in the clinical setting. Based on the Affymetrix GeneChip platform, the AmpliChip CYP450 test assesses phenotype of Abbreviations used in this paper: ADR, adverse drug reaction; IBS-C, constipation-predominant irritable bowel syndrome; CYP450, cytochrome P450; IBS-D, diarrhea-predominant irritable bowel syndrome; EM, extensive metabolizer; GERD, gastroesophageal reflux disease; IBS, irritable bowel syndrome; IM, intermediate metabolizer; MALT, mucosa-associated lymphoid tissue; PM, poor metabolizer; PPI, proton pump inhibitor; SNP, single nucleotide polymorphism; TF, treatment failure; UM, ultra-rapid metabolizer by the American Gastroenterological Association Institute /06/$32.00 doi: /j.cgh

2 July 2006 THE AMPLICHIP CYP450 TEST 823 Table 1. Common Pharmaceutical Substrates of CYP2D6 and CYP2C19 CYP2C19 Substrates PPIs Omeprazole Esomeprazole Lansoprazole Pantoprazole Rabeprazole Tricyclic antidepressants Amitriptyline Clomipramine Antiepileptics Diazepam Phenytoin Phenobarbitone Others Cyclophosphamide Progesterone CYP2D6 Substrates Antipsychotics Fluoxetine Haloperidol Paroxetine Thioridazine Venlafaxine Tricyclic antidepressants Amitriptyline Desipramine Imipramine Nortriptyline Beta-blockers Alprenolol Carvedilol Metoprolol Propranolol Timolol Others Codeine Dextromethorphan Flecainide Tamoxifen drug metabolism capacity for the CYP450 genes CYP2D6 and CYP2C19, which are involved in the metabolism of several commonly prescribed drugs (Table 1). 3,4 Both the AmpliChip test and the Affymetrix platform received US Food and Drug Administration (FDA) approval under the newly created generic heading , Drug-Metabolizing Enzyme Genotyping System. 5 The AmpliChip CYP450 test may allow pre-prescription decision making based on a patient s capacity to metabolize certain drugs, and may help prevent ADRs and TFs by providing a basis for adjusting dosages or prescribing alternate therapies when available. But the AmpliChip CYP450 test is applicable to only a select list of drugs (Table 1), representing 25% of currently prescribed medications. Perhaps the most exciting and potentially important aspect of this newly approved technology is the FDA s approval of the Affymetrix platform on which the AmpliChip test is run. The Affymetrix system is capable of running any number of specific chip-based assays and is highly scalable, providing the means for future versions of the AmpliChip to assess variation in a greater number of drug-metabolizing genes. The tests are simplified, semiautomated, 6 and applicable in small laboratories, obviating the need for specialized molecular genetics laboratories. In this review, we describe the principles, discuss the challenges, and explore the potential uses of the Ampli- Chip CYP450 test. Enzymes of Drug Metabolism Many of the enzymes important to drug metabolism harbor common polymorphisms at the genetic level that potentially affect enzymatic activity and, ultimately, response to treatment (Figure 1). Drug metabolism entails phase I reactions, generally the enzymatic modification of functional groups, setting the stage for phase II reactions involving conjugation with endogenous compounds and facilitating excretion from the Figure 1. Polymorphic drug-metabolizing enzymes. Many drug-metabolizing enzymes responsible for phase I (modification of functional groups) or phase II (conjugation with endogenous compounds) reactions harbor common genetic polymorphisms that potentially affect response to therapy. Those enzymes for which polymorphisms have been shown to alter drug effects are separated from the pie charts. The contribution to phase I and phase II metabolism of drugs of each enzyme is estimated by the relative size of each section. Phase I: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; CYP, cytochrome P450; DPD, dihydropyrimidine dehydrogenase; NQO1, NADPH:quinine oxidoreductase or DT diaphorase. Phase II: COMT, catechol O-methyltransferase; GST, glutathione S-transferase; HMT, histamine methyltransferase; NAT, N- acetyltransferase; STs, sulfotransferases; TPMT, thiopurine methyltransferase; UGTs, uridine 5=-triphosphate glucuronosyltransferases. (Reprinted with permission. 43 Copyright 1999 AAAS.)

3 824 JURAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 4, No. 7 body. 7 Phase I drug metabolism is largely dependent on the CYP450 enzymes, 8 2 of which are assessed by the AmpliChip CYP450 test. Although there are numerous other important drug-metabolizing enzymes besides the CYP450 enzymes (see Figure 1), a review of these is beyond the scope of this article. The CYP450 Genes The CYP450 gene superfamily encodes enzymes responsible for the metabolism of exogenous dietary and environmental chemicals (ie, xenobiotics), 9 including most of the pharmaceutical agents currently prescribed. Moreover, it plays a key role in the biosynthesis and degradation of many endogenous compounds, including bile acids, cholesterol, and steroids. 10 CYP450 genes are found in nearly all life forms and have existed for more than 3 billion years, 11 pointing to the importance of detoxifying environmental chemicals. The diversity of this gene family among organisms is great, with plants generally having many more CYP450 genes than vertebrates, due to the immobile plants need for specialized metabolic functions to thrive in the face of challenges posed by the environment and predators. 12 Humans have 57 CYP450 genes coding for functional enzymes, as well as 58 nonfunctional pseudogenes. 13 These genes have been organized into 18 families and 43 subfamilies 13 based on similarities of amino acid sequence. Although mice have nearly twice as many CYP450 genes as humans, the same 18 families are represented in both, many of which are highly conserved. 13 The large difference in the overall number of CYP450 genes between humans and mice is due primarily to the expansion of a few xenobiotic metabolizing CYP450 subfamilies in mice, 13 likely the result of dietary selection. CYP450 genes are quite polymorphic, leading to significant differences in the enzymatic activity of their encoded proteins and the rate at which individuals metabolize substrates for these enzymes. 8,10 This has been associated with marked effects on the bioavailability of active drugs, which can lead to ADRs or TFs. In addition to these inherited polymorphisms, many of the drugmetabolizing CYP450s are both positively and negatively affected by numerous exogenous dietary compounds, 3,11 complicating treatment and potentially leading to ADRs or TFs due to drug diet or drug drug interactions. Structurally, the drug-metabolizing CYP450s are membrane-bound, heme-containing proteins. 9 These enzymes are located in the microsomes of the endoplasmic reticulum in many cell types of the liver, kidney, small intestine, lung, and brain. Acting primarily as monooxygenases, they catalyze the modification of xenobiotics through hydroxylation in phase I drug metabolism. 7 This sets the stage for further phase II conjugation and the eventual excretion of the substance from the body. 7 Phase I reactions can also catalyze conversion of inactive prodrugs to their active form, such as in the case of amitriptyline conversion to nortriptyline, catalyzed primarily by CYP2C These drug-metabolizing enzymes belong to the first 3 CYP450 families (ie, CYP1, CYP2, and CYP3) and are responsible for mediating the phase I metabolism of many clinically prescribed drugs. 11 The most abundant CYP450 enzyme expressed in the liver is CYP3A4, which accounts for approximately 40% of phase I drug metabolism. 8 Although there is high variability in the activity of this enzyme between individuals, the protein-coding region of CYP3A4 is highly conserved in humans; indeed, no common variations altering enzyme functionality have been observed. 8 The variability of CYP3A4 activity is thought to be genetic, 8 but the mechanisms behind this variability have not been elucidated. It is possible that genetic variation affecting CYP3A4 gene expression, acting in concert with differences in lifestyle, diet, and environmental exposure, may modulate the activity of this enzyme. The effect of CYP450 genetic polymorphism on drug-metabolizing activity is most pronounced in 3 of the CYP450 genes (CYP2C9, CYP2C19, and CYP2D6), which together account for 35% 45% of CYP450-mediated phase I drug metabolism. 15 Because the polymorphic CYP450 genes are most amenable to predicting individual metabolic response by genotyping, and because 2 of these genes (CYP2C19 and CYP2D6) are assessed by the AmpliChip CYP450 test, here we provide a brief overview of these CYP450 genes. CYP2C9. The second most abundant CYP450 enzyme in the liver (after CYP3A4), CYP2C9 is involved in the phase I metabolism of 10% 15% of prescribed medications, including the nonsteroidal anti-inflammatory drugs, anticonvulsant agents, and anticoagulant drugs, such as warfarin and coumarols. 16,17 A large number of polymorphisms have been described in this gene, but only 2 alleles (CYP2C9*2 and CYP2C9*3) have been shown to affect the drug-metabolizing function of a significant portion of the population. 16,17 Approximately 35% of Caucasians carry at least 1 of the variant alleles, with CYP2C9*2 twice as prevalent as CYP2C9*3 (allele frequencies of 11% and 7%, respectively). The prevalence of CYP2C9 variants is around 13% in Africans and only 3.5% in Asians, who do not seem to carry the CYP2C9*3 allele. 17 The presence of variant CYP2C9

4 July 2006 THE AMPLICHIP CYP450 TEST 825 Figure 2. Genomic structure of CYP2C19. The CYP2C19 gene, located on chromosome 10 (10q23.33), spans 90,209 base pairs and contains 9 exons (shaded boxes), covering 1473 base pairs and coding for a 491 amino acid peptide. Intervening intron sequences are represented by broken connecting lines, and size is not in scale with the exons. The 2 major allelic variants are shown. The CYP2C19*2 allele harbors the 681 G A variant, causing a splicing defect resulting in frame shift and premature termination. The CYP2C19*3 allele harbors the 636 G A variant, causing the creation of a stop codon leading to a truncated protein. alleles generally results in reduced ability to clear the targeted compounds, an effect that is more pronounced in persons who are homozygous for the variant alleles than in those who carry just 1 variant. 17 The inadequate drug clearance, coupled with the narrow therapeutic index and high potential for ADRs, of many of these compounds suggests that dosage adjustments based on the results of CYP2C9 genotyping could have significant clinical benefit. But CYP2C9 activity overlaps with other CYP450 enzymes, as well as with non-cyp450 enzymes involved in metabolism, 17 and at present it is unclear whether checking the CYP2C9 genotype will prove to be clinically important or be widely applied in the clinical setting. Currently, the AmpliChip CYP450 test does not assess for the CYP2C9 genotype. CYP2C19. CYP2C19 plays a primary role in the metabolism of many proton pump inhibitors (PPIs), tricyclic antidepressants, and antiepileptics, 4,18 as listed in Table 1. The genomic structure of CYP2C19 comprises 9 exons and 8 intervening introns (Figure 2), spanning more than 90 kb of genomic sequence on chromosome 10 (10q23.33). To date, some 15 variant alleles of CYP2C19 have been identified and posted to the CYP450 allele nomenclature website ( However, only 2 variants, CYP2C19*2 and CYP2C19*3, have been shown to affect the drug metabolism of a significant percentage of the population. 4 The CYP2C19*2 variant contains a G A single nucleotide polymorphism (SNP) at position 681 in exon 5, resulting in a splicing defect and premature termination. 4,19 The CYP2C19*3 variant contains a G A SNP at position 636 in exon 4, directly resulting in a stop codon and a truncated protein. 4,20 The genomic structure and relative location of these SNPs is shown in Figure 2. Both of these variant alleles produce an inactive form of the CYP2C19 enzyme, 19,20 and so the genotype of CYP2C19 can be used to predict a phenotype of metabolic response. Individuals who harbor 2 wild-type CYP2C19 alleles (CYP2C19*1) are classified as extensive metabolizers (EMs), whereas those with 2 variant alleles (and thus no CYP2C19 activity) are classified as poor metabolizers (PMs). 4,18 Individuals with 1 wild-type and 1 variant allele are known as heterozygous extensive metabolizers (het-ems). In the AmpliChip CYP450 test, these individuals are classified as EMs, because CYP2C19 metabolism by het-ems is often closer to that of homozygous EMs than that of PMs. The CYP2C19 PM phenotype is present in nearly 20% of Asians, but in only 2% 3% of Caucasians and 4% of Africans, 10 illustrating the inherent racial differences in drug metabolism. The clinical usefulness and cost-effectiveness of the AmpliChip CYP450 test in CYP2C19 genotyping have yet to be determined, primarily because the 2 variant alleles for which it tests (CYP2C19*2 and CYP2C19*3) are easily assessed by simple, less expensive polymerase chain reaction restriction fragment length polymorphism assays. 19,20 But these assays have not been commercialized in a form suitable for use by smaller clinical laboratories. Finally, interpretation of the clinical impact of the CYP2C19 genotyping is complicated by certain nongenetic factors, such as enzyme inhibition, old age, and liver cirrhosis, which can modulate CYP2C19 activity. 4 CYP2D6. The most extensively studied drug-metabolizing CYP gene, CYP2D6 is highly polymorphic and thus displays a wide spectrum of activity in humans. 3,21 The 9 exons and 8 intervening introns of CYP2D6 span approximately 4.4 kb of the genome at chromosome 22 (22q13.2) (Figure 3), coding for a protein of 498 amino acids. Although comprising only a small amount of the total CYP enzyme content of the liver, CYP2D6 is involved in the phase I metabolism of approximately 20% of all prescribed drugs, including many widely prescribed antidepressants, antihypertensives, and beta-blockers, 3,21 some of which are listed in Table 1. Interindividual variability of the CYP2D6 gene is quite high; nearly 100 variant alleles have been described (see many of which lead to significant or complete loss of metabolic function. 3,21 Extensive study of the relationship between CYP2D6 allelic variation and enzymatic activity has led to the Figure 3. Genomic structure of CYP2D6. The CYP2D6 gene located on chromosome 22 (22q13.2) spans 4379 base pairs and contains 9 exons (shaded boxes), covering 1655 base pairs and coding for a 498 amino acid peptide. Intervening intron sequences are represented by connecting lines, and size is shown in scale with the exons.

5 826 JURAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 4, No. 7 Figure 4. AmpliChip CYP450 predictive phenotypes for CYP2D6. Predicted CYP2D6 metabolic phenotypes based on allelic composition. U: UMs have multiple copies (3 or more) of fully functional alleles (1, 2, or 35) and exhibit excessive metabolic activity. E: EMs carry 1 or 2 fully functional alleles. I: IMs have 1 reduced-activity allele (9, 10, 17, 29, 36, or 41) and 1 null allele (3 8, 11, 14A, 19, 20, or 40). P: PMs carry 2 null alleles and exhibit no CYP2D6 enzymatic activity. ability to predict a metabolic phenotype for an individual based on the CYP2D6 genotype. 3,21 At one extreme is the CYP2D6 PM phenotype, which results from carrying 2 alleles coding for inactive protein. Individuals with this phenotype have no ability to catalyze CYP2D6- dependent drug metabolism and thus are at increased risk of ADRs due to toxicity because of their inability to eliminate the drug, or of TFs because of their inability to convert a prodrug to its active form. The PM phenotype is present in 5% 10% of Caucasians but in only 1% 2% of Asians. 10 Conversely, some individuals harbor alleles containing multiple copies of functional gene sequences and exhibit significantly increased CYP2D6 metabolic activity, 22 classifying the ultra-high metabolizer (UM) phenotype. 22,23 Present primarily in African (30%) 24 and African admixed European populations, such as Italy and Greece (10%), 3 individuals with the UM phenotype are particularly susceptible to TFs at standard doses of prescription drugs due to rapid elimination and inability to sustain therapeutic drug levels. 23 The EM phenotype is considered normal; these individuals carry at least 1, but not more than 2, functional CYP2D6 alleles. A fourth phenotype of CYP2D6 metabolism is the intermediate metabolizers (IMs), who lack the metabolic capacity of the EMs, but do have some enzymatic capacity because they carry 1 allele associated with reduced function. 21 The predictive phenotypes for CYP2D6 based on the AmpliChip CYP450 test are shown in Figure 4. Despite the wide spectrum of CYP2D6 activity in the population, no discernible phenotype has been associated with absent or severely increased function, suggesting that CYP2D6 plays little if any role in the metabolism of endogenous substrates. 3 CYP2D6 activity serves primarily in the detoxification of exogenous compounds, typically medications. Interestingly, the CYP2D subfamily is one of those expanded in mice, who express 9 active CYP2D enzymes compared with the 1 enzyme expressed by humans, 13 suggesting that this enzyme subfamily is heavily influenced by dietary selection. The high degree of polymorphism in the human CYP2D6 gene and the lack of other CYP2D family members suggests that CYP2D6 may be slowly disappearing in humans due to the lack of selective pressure to maintain the gene. However, the high prevalence of the UM phenotype in Ethiopian Africans (30%) has led the Ingelman-Sundberg group to postulate that a population expansion in Figure 5. Prediction of drug response in the population based on genetic variation. Simple genetic variation in the form of a variant SNP can be used to discriminate between the individuals who will respond favorably to pharmaceutical intervention (black) and those who will have a minor (tan) or major (red) adverse reaction to the treatment. This ability to leverage genetic information to predict ADR will allow physicians to prescribe alternate therapies when available and to identify those in need of more rigorous monitoring when not.

6 July 2006 THE AMPLICHIP CYP450 TEST 827 Ethiopia coupled with severely limited dietary resources some 10,000 20,000 years ago may have led to positive selection of the alleles carrying CYP2D6 gene duplications. 3 The AmpliChip CYP450 Test The AmpliChip CYP450 test is built around the well-proven Affymetrix GeneChip platform. Briefly, highly dense fields of oligonucleotides are chemically synthesized on glass chips using a series of photolithographic activation steps, a technology adapted from the semiconductor industry. The sample is hybridized to these fields of oligonucleotides, and results are obtained using an automated scanner and displayed on a computer screen. The first application of the Affymetrix technology was in the quantitation of large numbers of expressed RNA transcripts using in vitro transcription to produce suitably labeled targets (ie, gene expression profiling). But the AmpliChip CYP450 test uses the much more stable nucleic acid DNA as a template for labeling and involves a polymerase chain reaction based on Roche s well-proven platform. The AmpliChip CYP450 test provides predictive phenotypes of the drug-metabolism capacity of the CYP450 genes CYP2D6 and CYP2C19. To do this, it analyzes 29 polymorphisms and variations in the highly polymorphic CYP2D6 gene and 2 polymorphisms in CYP2C19. Applicability of the AmpliChip CYP450 Array in Gastrointestinal Disease Numerous genetic and nongenetic factors can affect drug response in an individual. Indeed, inherited differences in the way a person metabolizes drug(s) may afford a basis on which to predict treatment response (Figure 5). For example, ADRs and TFs often occur when the therapeutic window is narrow, and thus only a small margin exists between effective and toxic doses. Knowledge of an individual s capacity to metabolize such drugs before initiation of therapy could provide a way to tailor medication doses to achieve the optimal effect while avoiding toxicity. Alternatively, this knowledge may suggest the use of alternate therapies, if available. Clinical relevance transpires when variation in human CYP450 metabolism is relatively abundant and individuals are treated with commonly used medications. In such cases, the potential for notable clinical consequences due to wide variation in metabolic capacity exists, as shown in Table 2. In addition to the clinical consequences of varying CYP450 metabolic capacity in the population, the resulting ADRs and TFs can have a significant impact on treatment costs. For example, a study of psychiatric patients found that the annual cost of therapy was $4000 $6000 higher in those with PM and UM phenotypes of CYP2D6 than in those with EM and IM phenotypes of the same enzyme. 25 Despite this finding, the costs associated with genotyping (using the Ampli- Chip CYP450 test) large numbers of patients must be taken into account. Here we provide an overview of published reports regarding the effects of the CYP2C19 and CYP2D6 genotypes on the treatment of selected digestive diseases, and address the challenges and clinical utility of applying the AmpliChip CYP450 test to these genotypes. PPIs and Helicobacter pylori Eradication Most studies of the effect of CYP2C19 polymorphisms on drug metabolism have focused on the PPIs, because these are among the most widely used medications metabolized by this enzyme. Traditional triple therapy for H. pylori eradication involves administration of a PPI, such as omeprazole or lansoprazole, in combination with 2 antibiotics (generally amoxicillin and clarithromycin). This treatment is widely successful, with complete eradication achieved in 80% of individuals Interestingly, studies have shown near-complete H. pylori eradication in patients with CYP2C19 PM phenotypes using omeprazole in combination with dualor triple-therapy schemes, suggesting that clarithromycin may not be needed as a first-line treatment in these individuals. 29 Individuals who are homozygous for the wild-type CYP2C19*1 allele (ie, EM phenotype) are most likely to fail this first-line treatment effort using standard approaches, because the PPI is quickly metabolized and sufficient acid inhibition is not maintained. Recent studies have demonstrated improved treatment efficacy in this EM group from various alternative therapies, including the concomitant dosing of 2 PPIs 32 and administration of high-dose (40 mg) esomeprazole, 33 both in combination with standard antibiotic regimens. Although these studies both showed significant improvement over the control regimens (lansoprazole only and 20 mg esomeprazole, respectively) for the homozygous EM Table 2. Features of Drug Metabolism in CYP2D6 Poor Metabolizers and Ultra-rapid Metabolizers CYP2D6 PM No enzymatic activity million subjects CYP2D6 UM Gene duplications million subjects Slow drug metabolism High drug levels at usual dose High risk for ADRs Rapid drug metabolism No drug response at usual dose High risk of TFs

7 828 JURAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 4, No. 7 phenotype, they found no difference in treatment efficacy in either the het-em group or the PM group. Because PPIs have been shown to be safe and well tolerated, and because H. pylori eradication regimens are short (7 14 days), minimization of ADRs by CYP2C19 genotyping for these protocols is of little concern. However, avoidance of TFs could have a significant clinical impact by preventing complications of prolonged H. pylori infection (eg, peptic ulcer, gastric cancer, or mucosa-associated lymphoid tissue [MALT] lymphoma) and minimizing the development of drugresistant strains from excessive use of broad-spectrum antibiotics. 34 Although the CYP2C19 genotype does seem to effect the outcome of PPI-based H. pylori treatment, much is left to be proven and learned before wide-scale CYP2C19 genotyping can be used to provide more individualized eradication regimens. For example, polymorphisms of CYP3A4 have recently been implicated as contributing to successful treatment in CYP2C19 EMs and het- EMs, 30 suggesting that additional metabolic factors are at work in determining the ultimate therapeutic response. Further evidence for this is provided by the fact that a high percentage of CYP2C19 EMs and het-ems do achieve complete H. pylori eradication on the current standard regimens. Thus, at present, CYP2C19 genotyping would identify a small portion of the total population ( 5% of Caucasians and Africans and 20% of Asians 10 ) who will undoubtedly respond favorably to treatment that is, the PMs, who could potentially receive a more conservative treatment course. Stratification of the CYP2C19 EMs and het-ems may potentially affect treatment decisions, with the alternative therapies mentioned earlier selected for the EMs. 32,33 However, the AmpliChip CYP450 test currently fails to distinguish between these groups of EMs and het-ems, and thus it is not useful for use in tailoring treatment choices. Finally, the 20% of individuals who ultimately fail initial therapy for H. pylori eradication would not be identified by CYP2C19 genotyping before treatment. Although the AmpliChip CYP450 test shows promise, much work is needed before genotyping-based individualized drug therapy for H. pylori eradication becomes practical. Treatment of Gastroesophageal Reflux Disease PPIs also play a primary role in the treatment of gastroesophageal reflux disease (GERD), a very common disorder. In contrast to H. pylori eradication, treatment of GERD with PPIs, such as omeprazole, is long-term and has been proven to be quite efficacious and safe. 35,36 A recent study of omeprazole tolerance in Japanese patients (with a high prevalence of CYP2C19 PMs) treated for GERD found no difference in the seriousness or frequency of ADRs among the EM, het-em, and PM groups, 37 suggesting that dosage adjustment based on CYP2C19 genotype is not necessary. Thus, the potential clinical benefit of wide-scale CYP2C19 genotyping for treating GERD with PPIs is likely quite small and, based on currently available data, would not appear to justify the additional costs involved. However, it is possible that a significant reduction in overall drug use and costs could be realized if dose adjustments can be introduced while maintaining therapeutic results based on CYP2C19 (and possibly other CYP450) genotypes, especially in those populations with a high prevalence of variant alleles. For example, it may be possible to use lower doses of PPIs in Asians with the CYP2C19 PM genotype. Although the current cost of CYP450 genotyping appears to preclude its useful application to this and many other individual diseases, it is conceivable that someday the cost of genotyping could be justified based on its wide applicability to many drugs and the treatment of many diseases. At a minimum, extensive study is needed to establish such global approaches toward maximizing therapeutic intervention. Treatment of Irritable Bowel Syndrome Of all the conditions treated by gastroenterologists, irritable bowel syndrome (IBS) is quite common and has a significant impact on those afflicted and on society at large. The symptoms and severity of IBS range widely, as do the current approaches to treatment. 38 Agents directly affecting serotonin have recently been used and show some benefit for both IBS-D (alosteron) 39 and IBS-C (tegaserod) 40 patients. However, ADRs limit application of these drugs to severe cases of IBS. 38,41 Alosetron is metabolized primarily by CYP2C9 and CYP3A4, 39 but there have been no reports on treatment outcome based on these CYP450 genotypes, and the AmpliChip CYP450 test does not assess variations of these enzymes. The metabolism of tegaserod is not mediated by the CYP450s. 40 Antidepressants, including serotonin reuptake inhibitors (eg, paroxetine, fluoxetine) and tricyclic antidepressants (eg, desipramine, imipramine), are commonly used to treat IBS and appear to relieve pain and increase quality of life. 38 ADRs are a major problem with the antidepressants and lead to high dropout rates in studies, possibly masking the benefits of these drugs. 38 Interestingly, the serotonin reuptake inhibitors and tricyclic antidepressants are metabolized primarily by CYP2D6; 3,4 however, no studies on the influence of CYP2D6 on outcomes of

8 July 2006 THE AMPLICHIP CYP450 TEST 829 Table 3. Approximate Dose Adjustments of Selected Antidepressants for Various CYP2D6 Phenotypes Drug PM IM EM UM Imipramine Nortriptyline Amitriptyline NOTE: Dosage adjustments expressed as percent of standard dose. (Modified and reprinted with permission. 3 ) antidepressant treatment in IBS have been published to date. Assessment of CYP2D6 metabolizer phenotype (ie, PM, IM, EM, and UM) is a major strength of the Ampli- Chip CYP450 test; alternative methods of typing this gene are quite laborious. Thus, using the AmpliChip CYP450 test to determine CYP2D6 metabolic phenotype may provide a sound basis for further study of the role of genetic factors in determining the efficacy of antidepressants in treating IBS. This test may also indicate fairly significant dosage adjustments of selected antidepressants to achieve the desired therapeutic efficacy, as suggested from the results of a meta-analysis (Table 3). 3,42 Conclusions ADRs and TFs are frequently encountered in clinical practice. Among the factors influencing these events, genetically predisposed variation in the ability to metabolize certain pharmaceutical compounds is extremely important, because it profoundly influences an individual s response to treatment. The significance of polymorphic CYP450 enzymes in the metabolism of a notable number of medications has been known for decades. Recent regulatory approval of the AmpliChip CYP450 assay provides an opportunity to predict the metabolic function of 2 important CYP450 enzymes in the clinical setting, offering a possible pathway to more individualized drug therapy. However, significant obstacles need to be overcome before this new test can be widely applied and have an impact on reducing ADRs and TFs in gastroenterologic practice. The present AmpliChip CYP450 assay is practical only when applied to drugs metabolized at least in part by CYP2D6; CYP2C19 variation can be readily determined by less expensive means. Based on data available at present, this reduces the usefulness of the test for improving the efficacy of H. pylori and GERD treatment through optimization of PPI dosing. This recommendation may change with the availability of an extensive study that demonstrates otherwise. On the other hand, the AmpliChip CYP450 test certainly could affect the treatment of IBS with antidepressant medications. Dosage adjustments based on the CYP2D6 metabolic phenotypes have already been established for some these drugs and are beginning to be applied in the field of psychiatry. Nevertheless, considerable effort is needed to demonstrate a benefit of antidepressants in treating IBS before the AmpliChip CYP450 test can be routinely used. Indeed, a more effective way to prove the efficacy of antidepressants and utility of the AmpliChip CYP450 test would be to perform a randomized controlled trial, with selection of doses based on CYP2D6 metabolic phenotype. In closing, FDA approval of the AmpliChip CYP450 test and the Affymetrix platform used to perform it is a significant step toward a more individualized approach to pharmacologic therapy. However, further clinical studies and expansion of the test to include many additional polymorphic enzymes involved in drug metabolism are needed before this technology can be widely applied and have a true impact on society at large. References 1. Lazarou J, Pomeranz BH, Corey PN. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA 1998;279: Ingelman-Sundberg M. Pharmacogenetics: an opportunity for a safer and more efficient pharmacotherapy. J Intern Med 2001; 250: Ingelman-Sundberg M. Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenom J 2005;5: Desta Z, Zhao X, Shin JG, et al. Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet 2002;41: US Food and Drug Administration. Medical devices; clinical chemistry and clinical toxicology devices; drug metabolizing enzyme genotyping system: final rule. Fed Regist 2005;70: Chou WH, Yan FX, Robbins-Weilert DK, et al. Comparison of two CYP2D6 genotyping methods and assessment of genotype phenotype relationships. Clin Chem 2003;49: Nelson DR. Cytochrome P450s in humans. Available at drnelson.utmem.edu/p450lect.html. Accessed January Ingelman-Sundberg M. Pharmacogenetics of cytochrome P450 and its applications in drug therapy: the past, present and future. Trends Pharmacol Sci 2004;25: Estabrook RW. A passion for P450s (rememberances of the early history of research on cytochrome P450). Drug Metab Dispos 2003;31: Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med 2005;352: Danielson PB. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab 2002;3: Nelson DR. Cytochrome P450 and the individuality of species. Arch Biochem Biophys 1999;369: Nelson DR, Zeldin DC, Hoffman SM, et al. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 2004; 14: Steimer W, Zopf K, von Amelunxen S, et al. Amitriptyline or not, that is the question: pharmacogenetic testing of CYP2D6 and

9 830 JURAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 4, No. 7 CYP2C19 identifies patients with low or high risk for side effects in amitriptyline therapy. Clin Chem 2005;51: Caraco Y. Genes and the response to drugs. N Engl J Med 2004;351: Rettie AE, Jones JP. Clinical and toxicological relevance of CYP2C9: drug drug interactions and pharmacogenetics. Annu Rev Pharmacol Toxicol 2005;45: Kirchheiner J, Brockmoller J. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther 2005;77: Blaisdell J, Mohrenweiser H, Jackson J, et al. Identification and functional characterization of new potentially defective alleles of human CYP2C19. Pharmacogenetics 2002;12: de Morais SM, Wilkinson GR, Blaisdell J, et al. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J Biol Chem 1994;269: de Morais SM, Wilkinson GR, Blaisdell J, et al. Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese. Mol Pharmacol 1994;46: Zanger UM, Raimundo S, Eichelbaum M. Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry. Naunyn Schmiedebergs Arch Pharmacol 2004;369: Johansson I, Lundqvist E, Bertilsson L, et al. Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultra-rapid metabolism of debrisoquine. Proc Natl Acad SciUSA1993;90: Bertilsson L, Dahl ML, Sjoqvist F, et al. Molecular basis for rational megaprescribing in ultrarapid hydroxylators of debrisoquine. Lancet 1993;341: Aklillu E, Persson I, Bertilsson L, et al. Frequent distribution of ultra-rapid metabolizers of debrisoquine in an Ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles. J Pharmacol Exp Ther 1996;278: Chou WH, Yan FX, de Leon J, et al. Extension of a pilot study: impact from the cytochrome P450 2D6 polymorphism on outcome and costs associated with severe mental illness. J Clin Psychopharmacol 2000;20: Lind T, Veldhuyzen van Zanten S, Unge P, et al. Eradication of Helicobacter pylori using one-week triple therapies combining omeprazole with two antimicrobials: the MACH I Study. Helicobacter 1996;1: Lind T, Megraud F, Unge P, et al. The MACH2 study: role of omeprazole in eradication of Helicobacter pylori with 1-week triple therapies. Gastroenterology 1999;116: Katelaris PH, Forbes GM, Talley NJ, et al. A randomized comparison of quadruple and triple therapies for Helicobacter pylori eradication: The QUADRATE Study. Gastroenterology 2002;123: Tanigawara Y, Aoyama N, Kita T, et al. CYP2C19 genotype-related efficacy of omeprazole for the treatment of infection caused by Helicobacter pylori. Clin Pharmacol Ther 1999;66: Sapone A, Vaira D, Trespidi S, et al. The clinical role of cytochrome p450 genotypes in Helicobacter pylori management. Am J Gastroenterol 2003;98: Aoyama N, Tanigawara Y, Kita T, et al. Sufficient effect of 1-week omeprazole and amoxicillin dual treatment for Helicobacter pylori eradication in cytochrome P450 2C19 poor metabolizers. J Gastroenterol 1999;34: Okudaira K, Furuta T, Shirai N, et al. Concomitant dosing of famotidine with a triple therapy increases the cure rates of Helicobacter pylori infections in patients with the homozygous extensive metabolizer genotype of CYP2C19. Aliment Pharmacol Ther 2005;21: Sheu BS, Kao AW, Cheng HC, et al. Esomeprazole 40 mg twice daily in triple therapy and the efficacy of Helicobacter pylori eradication related to CYP2C19 metabolism. Aliment Pharmacol Ther 2005;21: Lehmann DF, Medicis JJ, Franklin PD. Polymorphisms and the pocketbook: the cost-effectiveness of cytochrome P450 2C19 genotyping in the eradication of Helicobacter pylori infection associated with duodenal ulcer. J Clin Pharmacol 2003;43: Solvell L. The clinical safety of omeprazole. Digestion 1990;47: Joelson S, Joelson IB, Lundborg P, et al. Safety experience from long-term treatment with omeprazole. Digestion 1992;51: Ohkusa T, Maekawa T, Arakawa T, et al. Effect of CYP2C19 polymorphism on the safety and efficacy of omeprazole in Japanese patients with recurrent reflux oesophagitis. Aliment Pharmacol Ther 2005;21: Tillisch K, Chang L. Diagnosis and treatment of irritable bowel syndrome: state of the art. Curr Gastroenterol Rep 2005;7: D Souza DL, Dimmitt DC, Robbins DK, et al. Effect of alosetron on the pharmacokinetics of fluoxetine. J Clin Pharmacol 2001; 41: Appel-Dingemanse S. Clinical pharmacokinetics of tegaserod, a serotonin 5-HT 4 receptor partial agonist with promotile activity. Clin Pharmacokinet 2002;41: Hadley SK, Gaarder SM. Treatment of irritable bowel syndrome. Am Fam Physician 2005;72: Kirchheiner J, Nickchen K, Bauer M, et al. Pharmacogenetics of antidepressants and antipsychotics: the contribution of allelic variations to the phenotype of drug response. Mol Psychiatry 2004;9: Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999;286: Address requests for reprints to: Dr. Konstantinos N. Lazaridis, Center for Basic Research in Digestive Diseases, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, Minnesota lazaridis.konstantinos@mayo.edu