Cleveland Clinic Laboratories

Genetic Test of Cytochrome P450 2C19 (CYP2C19) for Clopidogrel Therapy

Background Information Clinical Significance of Genetic Testing Clopidogrel is an oral antiplatelet drug used to treat acute Clopidogrel is converted into an active metabolite in the coronary syndromes (ACS) including patients undergoing liver by several CYP enzymes including CYP1A2, CYP2B6, percutaneous coronary intervention (PCI), myocardial CYP2C9, CYP2C19, CYP3A4 and CYP3A5 (Figure 1). Of infarction (MI), cerebrovascular disease and peripheral these, CYP2C19 plays a major role in the generation of arterial disease. Clopidogrel is typically prescribed at a daily active metabolite. There is a wide variability observed among dosage of 75 mg with or without an initial loading dose of patients in response to clopidogrel therapy that is attributed 300-600 mg, as needed. In combination with a daily dose to at least three common variants in CYP2C19 gene. The of 75-325 mg aspirin, it is used at 75 mg dosage to prevent wild-type form of the CYP2C19 gene (*1) encodes an enzyme stent-thrombosis following PCI. with normal activity (extensive metabolizer) that current drug dosing recommendations are based on. Two variants (*2 Clopidogrel administered as a pro-drug is largely (~85%) and *3) encode enzymes with non-functional activity (poor hydrolyzed by esterases into inactive metabolites, and only metabolizer). In contrast, a third variant (*17) encodes an approximately 15% is converted into an active form by hepatic enzyme with increased activity (ultra metabolizer). Cytochrome P450 (CYP) enzymes, primarily by isoenzyme CYP2C19. The active metabolite binds irreversibly to the As a result, CYP2C19*2 and CYP2C19*3 variants confer platelet adenosine diphosphate (ADP) receptor P2RY12 to non-functional or markedly reduced metabolic status and inhibit platelet aggregation and consequent thrombosis have been associated with non-responsiveness to clopidogrel (Figure 1). The antithrombotic effect of clopidogrel is not reflected in reduced platelet inhibition and poor outcomes optimal in all patients, and up to 30% do not benefit from including increased risk for stent thrombosis, MI, stroke and the therapy as determined from the measurement of residual death. On the other hand, CYP2C19*17 is associated with platelet reactivity. Non-response or sub-optimal response to an increased response to clopidogrel and risk for bleeding. therapy can stem from pharmacokinetics and pharmaco- Accordingly, while sub-optimal response to clopidogrel can dynamics, co-administration of other drugs and predisposition lead to thrombosis, super-optimal response can result in due to genetic, clinical and cellular factors. bleeding. Thus, the genetic testing of patients to determine CYP2C19 gene variants can enable individualized antithrom- Among the CYPs, certain variations in the gene encoding botic therapy. However, this genotyping assay should be CYP2C19 enzyme have been associated with poor metabo- used in conjunction with the aspirin/clopidogrel aggregation lism of clopidogrel, and subsequent decreased formation of assay to determine platelet response to clopidogrel. active metabolite. The Food and Drug Administration (FDA) has issued a black box warning about the reduced effective- This genetic test is used to identify patients at risk for adverse ness of clopidogrel in patients who are poor metabolizers of events due to impaired clopidogrel metabolism by: the drug. The FDA has indicated the availability of tests to Detecting two variants of CYP2C19 gene responsible for identify genetic differences in CYP2C19 function, and advised non-functional activity. healthcare professionals to consider alternate therapy or dosing strategies for poor metabolizers. • CYP2C19*2 and CYP2C19*3 Detecting one variant of CYP2C19 gene responsible for increased functional activity. • CYP2C19*17

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> > > Enzyme Activity *2 *3 12 Enzyme Activity Active Metabolite Enzyme Activity Active Metabolite Unknown UM / Increased function UM / Increased function PM / Non-function PM / Non-function PM / Non-function IM / Decreased function IM / Decreased function EM / Normal function Phenotype/ Alternate Dosing > > Alternate Therapy *17

> *1 variant alleles designated *17 variant alleles designated Individuals with CYP2C19 activity compared to *1 (wildtype)have increased enzyme increased levels of active metaboliteindividuals, leading to maintenance doses of clopidogrel. and may require lower of either genotype comprised Individuals with a CYP2C19 allele with combination of *4,*6- *2 or *3 and *17, and unknown metabolizer phenotype. *10 or *17 have an frequency of these alleles, the Due to the low population based on level of CYP2C19 activity cannot be predicted genotype. CYP2C19 2. 3. > > > > Hepatic Metabolism > Antithrombotic Effect Active Metabolite CYP1A2 CYP2B6 CYP2C9 CYP3A4 CYP3A5 ~ 15% Inhibition of Platelet Aggregation Clopidogrel (Plavix; Pro-Drug) 681 G>A or 636 G>A / -806 C>T / 681 G>A or 636 G>A -806 C>T/-806 C>T wild type/-806 C>T 636 G>A/636 G>A 681 G>A/636 G>A 681 G>A/681 G>A wild type/636 G>A wild type/681 G>A wild type/wild type Allele Irreversible Binding to Platelet ADP Receptor P2RY Irreversible Binding to Platelet ADP Receptor

> pidogrel abolism and mechanism of action of Clo Esterases > Intestinal Absorption ~ 85% Inactive Metabolite are shown in Table 1. CYP2C19 are shown in Table Figure 1. Met CYP2C19*2 or *3/*17 CYP2C19*17/*17 CYP2C19*1/*17 CYP2C19 *3/*3 CYP2C19 *2/*3 CYP2C19 *2/*2 CYP2C19 *1/*3 CYP2C19 *1/*2 CYP2C19 *1/*1 Genotype EM (Extensive Metabolizer); IM (Intermediate Metabolizer); PM (Poor Metabolizer); UM (Ultra Metabolizer) Metabolizer); PM (Poor Abbreviations: EM (Extensive Metabolizer); IM (Intermediate pidogrel hrome P450 2C19 genotype and phenotype for Clo 1. Cytoc Table variant alleles designated *2 variant alleles designated Individuals with CYP2C19 enzyme activity compared to and *3 have non-functional leading to absent or markedly *1 (wild-type) individuals, decreased metabolite that may require increased maintenance doses of clopidogrel. Interpretation is based on the presence of specificInterpretation of the assay alleles . The genotypes including genetic variants of CYP2C19 and phenotypes of 1. 4. Prevalence of CYP2C19 gene variants differs depending References on racial and ethnic background. The frequency of allele 1. Collet JP, Hulot JS, Pena A, Villard E, Esteve JB, Silvain CYP2C19*2 has been reported as approximately 30-35% J, Payot L, Brugier D, Cayla G, Beygui F, Bensimon G, of Asians, and 15-26% of Caucasian and African Ameri- Funck-Brentano C, Montalescot G. Cytochrome P450 cans. The frequency of allele CYP2C19*3 has been 2C19 polymorphism in young patients treated with reported as approximately 10% of Asians, and less than clopidogrel after myocardial infarction: a cohort study. 2% of Caucasian and African Americans. Lancet. 2009;373:309-317. 5. This genotyping assay should be used in conjunction with 2. Desta Z, Zhao X, Shin JG, Flockhart DA (2002). “Clinical the aspirin/clopidogrel aggregation assay as a functional significance of the cytochrome P450 2C19 genetic screen to determine platelet response to clopidogrel. polymorphism”. Clin Pharmacokinet. 2002;41:913-58. 6. Genotype-phenotype interpretation should be made in the 3. Ellis KJ, Stouffer GA, McLeod HL, Craig CR. Clopidogrel context of a patient’s clinical condition and concomitant pharmacogenomics and risk of inadequate platelet medications, which may be substrates, inhibitors and inhibition: US FDA recommendations. Pharmacogenomics. inducers of CYP2C19. 2009;10:1799-1817. Methodology 4. Flockhart, DA (2007). “Drug Interactions: Cytochrome An array-based test kit employing Infiniti analyzer (Auto- P450 Drug Interaction Table”. Indiana University School Genomics Inc., CA) is used for genotyping CYP2C19 variants. of Medicine. http://medicine.iupui.edu/flockhart/table. The assay involves a multiplex PCR amplification of genomic htm. Accessed on April 6, 2011. DNA followed by allele-specific primer extension using fluores- 5. Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, cent labeled dCTP and hybridization on to a micro-array Brandt JT, Walker JR, Antman EM, Macias W, Braunwald coated with capturing oligonucleotides, which are specific for E, Sabatine MS. Cytochrome p-450 polymorphisms and complementary oligonucleotides linked to the allele specific response to clopidogrel. N Engl J Med. 2009;360:354- primer-extended products. 362. A built-in confocal microscope is enabled to capture fluores- 6. Sangkuhl K, Klein TE, Altman RB. Clopidogrel pathway. cent signal from the pre-determined hybridization spots Pharmacogenetics and Genomics. 2010;20:463-465. corresponding to specific products and genotypes deciphered 7. Shuldiner AR, O’Connell JR, Bliden KP, Gandhi A, Ryan from signal ratio. K, Horenstein RB, Damcott CM, Pakyz R, Tantry US, Limitations of the Assay Gibson Q, Pollin TI, Post W, Parsa A, Mitchell BD, Faraday N, Herzog W, Gurbel PA. Association of cytochrome Analysis for specific genetic variants detected in this test does P450 2C19 genotype with the antiplatelet effect and not rule out the possibility of the presence of other variant clinical efficacy of clopidogrel therapy. JAMA. alleles that may influence drug effect and metabolism. 2009;302:849-857. CYP2C19 variant alleles are important in the metabolism of drugs other than clopidogrel including but not limited to 8. Sibbing D, Koch W, Gebhard D, Schuster T, Braun S, anticonvulsants, antidepressants, antiulcer and antimalarial Stegherr J, Morath T, Schömig A, von Beckerath N, drugs, and proton pump inhibitors. Co-administration of drugs Kastrati A. Cytochrome 2C19*17 allelic variant, platelet metabolized by CYP2C19 may increase or decrease the aggregation, bleeding events, and stent thrombosis in CYP2C19 activity. A *2 or *3 result for CYP2C19 is associated clopidogrel-treated patients with coronary stent placement. with a poor metabolizer phenotype for all drugs metabolized Circulation. 2010;121:512-518. by CYP2C19. Non-genetic factors such as concurrent medica- 9. Simon T, Verstuyft C, Mary-Krause M, Quteineh L, Drouet tions, impaired hepatic function, obesity, insulin resistance E, Méneveau N, Steg PG, Ferrières J, Danchin N, Becque- and non-compliance can also affect CYP2C19 metabolism. mont L; French Registry of Acute ST-Elevation and Non- These can lead to an increase or decrease in function relative ST-Elevation Myocardial Infarction (FAST-MI) Investigators. to the predicted genotype. Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med. 2009;360:363-375. Cleveland Clinic Laboratories

Test Overview Test Name Genetic Test of Cytochrome P450 2C19 (CYP2C19) for Clopidogrel Therapy

Methodology Multiplex PCR and array hybridization assay

Specimen Requirements Testing Volume/Size: 3 mL; Type: Whole blood; Tube/Container: EDTA (Lavender); Collection Temperature: Refrigerated

Reference Range An interpretive report will be provided.

Ordering Mnemonic 2C19CL

Billing Code 88362

CPT Codes 83891, 83900, 83901, 83914 x6, 83912

Technical Information Contacts: Scientific Information Contacts: Kelly Lyon, BS Kandice Kottke-Marchant, MD, PhD Gurunathan Murugesan, PhD 216.444.8283 216.444.2484 216.444.9484 [email protected] [email protected] [email protected]

James Pettay, MT(ASCP) Joyce Heesun Rogers, MD, PhD 216.440.2130 216.445.2719 [email protected] [email protected]

201112.055 (8.27.13 rev)