Effectiveness of Clopidogrel Dose Escalation to Normalize Active Metabolite Exposure and Antiplatelet Effects in CYP2C19 Poor Metabolizers

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1 Special Section: Clopidogrel Editor's Choice Effectiveness of Clopidogrel Dose Escalation to Normalize Active Metabolite Exposure and Antiplatelet Effects in CYP2C19 Poor Metabolizers The Journal of Clinical Pharmacology 54(8) Published 14 The Authors The Journal of Clinical Pharmacology published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology DOI:.02/jcph.293 Richard B. Horenstein, MD 1, Rajnikanth Madabushi, PhD 2, Issam Zineh, PharmD, MPH 2, Laura M. Yerges-Armstrong, PhD 1, Cody J. Peer, PhD 3, Robert N. Schuck, PharmD, PhD 2, William Douglas Figg, PharmD 3, Alan R. Shuldiner, MD 1,4, and Michael A. Pacanowski, PharmD, MPH 2 Abstract Carriers of two copies of the loss-of-function CYP2C19*2 variant convert less clopidogrel into its active metabolite, resulting in diminished antiplatelet responses and higher cardiovascular event rates. To evaluate whether increasing the daily clopidogrel dose in poor metabolizers (PM) overcomes the effect of the CYP2C19 * 2 variant, we enrolled 18 healthy participants in a genotype-stratified, multi-dose, three-period, fixed-sequence crossover study. Six participants with the *1/*1 extensive (EM), *1/*2 intermediate (IM), and *2/*2 poor metabolizer genotypes each received,, and each for 8 days. In each period, maximal platelet aggregation 4 hours post-dose (MPA4) and active metabolite area under the curve (AUC) differed among genotype groups (P <.05 for all). At day 8, PMs needed daily and IMs needed daily to attain a similar MPA4 as EMs on the dose (32.6%, 33.2%, 31.3%, respectively). Similarly, PMs needed daily to achieve active metabolite concentrations that were similar to EMs on (AUC 37.7 and 33.5 ng h/ml, respectively). These results suggest that quadrupling the usual clopidogrel dose might be necessary to overcome the effect of poor CYP2C19 metabolism. Keywords pharmacogenomics, clopidogrel, CYP2C19, pharmacokinetics Clopidogrel is an antiplatelet drug used to prevent cardiovascular events in patients with acute coronary syndromes or a history of stroke, myocardial infarction, or peripheral arterial disease. Antiplatelet responses, as measured by ex vivo platelet function testing or aggregometry, are small or absent in approximately one-third of patients. 1 CYP2C19, a genetically polymorphic drug metabolizing enzyme, is one of several cytochrome P4 drug metabolizing enzymes that are involved in the two oxidative steps that generate the active metabolite of clopidogrel. 2,3 Based on CYP2C19 genotype, patients can be classified as ultrarapid (UM), extensive (EM), intermediate (IM), or poor clopidogrel metabolizers (PM). Although studies evaluating the interaction between pharmacological inhibitors of CYP2C19 and clinical outcomes in patients receiving clopidogrel have produced mixed results, 4 CYP2C19 genotype has emerged as a reproducible genetic predictor of clopidogrel response, particularly in patients undergoing percutaneous coronary intervention (PCI). 5 Individuals with reduced function CYP2C19 alleles, particularly CYP2C19*2 homozygotes (PMs), have lower active 1 Program in Personalized and Genomic Medicine, Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, Baltimore, MD, USA 2 Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA 3 Clinical Pharmacology Program, National Cancer Institute, Bethesda, MD, USA 4 Geriatric Research and Education Clinical Center, Veterans Administration Medical Center, Baltimore, MD, USA Published 14. This article is a U.S. Government work and is in the public domain in the USA Submitted for publication 8 January 14; accepted 21 March 14. Corresponding Author: Michael A. Pacanowski, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 903 New Hampshire Avenue, White Oak Building 51, Room 3112, HFD8, Silver Spring, MD , USA michael.pacanowski@fda.hhs.gov Disclaimer: The opinions expressed in the article are those of the authors. No official FDA or NIH policy or endorsement is intended nor should be inferred. Clinical Trial Registration: NCT0134.

2 866 The Journal of Clinical Pharmacology / Vol 54 No 8 (14) metabolite concentrations, which results in diminished antiplatelet responses and higher cardiovascular event rates. 6 8 The significantly diminished active metabolite concentrations, attenuated antiplatelet effect, and increased cardiovascular outcome rates in PMs led to placement of a Boxed Warning in the clopidogrel drug label for prescribers to consider alternative treatments or treatment strategies in PMs. 9 Dosing regimens to overcome the diminished responses have the potential to improve clinical outcomes for patients predicted by genotype to be likely clopidogrel non-responders. Administering clopidogrel loading doses of 0 mg or higher (without respect to genotype), instead of, has become a common practice that has been shown to result in higher antiplatelet responses and improved outcomes.,11 Similarly, in studies, higher maintenance doses lead to greater protection from stent thrombosis, although bleeding rates are also higher. Considering the redundancy in the metabolic activation pathway of clopidogrel, which includes CYP1A2, 2B6, 2C9, 3A4, and 3A5, in addition to CYP2C19, higher loading and maintenance doses may be able to compensate for the lower active metabolite exposures resulting from CYP2C19 poor metabolism. However, the optimal dose to normalize exposures and antiplatelet responses across CYP2C19 genotype groups has not been determined, despite the initiation of clinical trials designed to evaluate the clinical utility of higher clopidogrel doses Targeting dose adjustments to those who are most likely to have reduced antiplatelet responses could improve outcomes and minimize the bleeding risks associated with nontargeted dose increases. The objective of this prospective, CYP2C19 genotypestratified, dose-escalation study of clopidogrel was to determine whether increasing clopidogrel maintenance doses up to daily increases active metabolite exposure and antiplatelet responses in subjects who are CYP2C19 IMs and PMs, providing evidence for the feasibility of dose-adjustment strategies to normalize the active metabolite and antiplatelet responses. Methods Subjects Eighteen healthy, white, Amish men and women between the ages of and years who had participated previously in the Pharmacogenomics of Antiplatelet Intervention (PAPI) study were recruited. 8 Six subjects from each of the CYP2C19 genotype groups [*1/*1 (EM), *1/*2 (IM), and *2/*2 (PM)] were enrolled. EM and IM subjects were matched by age and sex to PM subjects. Subjects were excluded if the following were present: blood pressure >1/95 mmhg, malignancy, creatinine>2.0 mg/dl, AST or ALT > 2 times the upper limit of normal, hematocrit <32% or >%, TSH < 0.4 or >0.5 miu/l, history of bleeding disorder or gastrointestinal bleed, history of cardiovascular disease or type 2 diabetes, platelet count <1,000 or >0,000, surgery within 6 months, allergy to clopidogrel, breast feeding. All participants were healthy subjects who were not taking concurrent medications, and all nutritional supplements, including multivitamins, were discontinued at least 7 days prior to the initial study visit through the end of the entire study period. Subjects provided written informed consent. The protocol was approved by the University of Maryland, Baltimore Institutional Review Board and the FDA Research Involving Human Subjects Committee. Study design The trial was a prospective, open-label, multiple-dose, fixed-sequence crossover study. All subjects initially received clopidogrel daily for 8 days (period 1). The dose of clopidogrel was increased in successive treatment periods to daily for 8 days (period 2) and daily for 8 days (period 3). There was a washout period between each treatment period of at least 7 days to allow platelet function to return to baseline; this washout period is commonly utilized for maintenancedose clopidogrel platelet function studies, consistent with platelet clearance. On the first day of each treatment period, subjects presented to the clinic and following baseline assessment of platelet function, the first dose of clopidogrel was administered under direct observation, after which blood samples were drawn for pharmacokinetic analysis and platelet aggregometry. Subjects were then discharged and instructed to take clopidogrel every day. Subjects returned to the clinic on day 8 of each treatment period, and platelet aggregometry was performed 4 hours after the last dose of each study period. Medication usage history and pill counts were performed to confirm adherence. All study procedures were carried out at the University of Maryland Amish Research Clinic in Lancaster, PA. Endpoints The primary endpoint was the difference in maximal platelet aggregation 4 hours post-dose (MPA4; units are a percentage) on day 8 of each treatment period (reflecting steady-state) between and the higher doses within each genotype group. Absolute MPA4 was selected rather than inhibition of platelet aggregation (IPA) in an effort to minimize the possibility of carry-over effects from each treatment period. Secondary endpoints compared by genotype groups (using EMs as the reference) and dosing periods (using as the reference) were as follows: MPA4 on day 1, relative IPA at 4 hours (IPA4) on days 1 and 8 (IPA4 ¼ [1 MPA4 on day 8/MPA0 on day 1 0]), area under the time concentration curve (AUC 0 4 ) and maximal concentration (C max ) for

3 Horenstein et al 867 clopidogrel active metabolite and parent compound on day 1 between and the higher doses within each genotype group. Genotyping Genotyping of the known common loss-of-function CYP2C19*2 variant (rs ) was performed using TaqMan 1 SNP genotyping assays (Applied Biosystems, Foster City, CA). Subsequent to the study, genotyping of the CYP2C19*17 variant (rs122485) and the CES1 G143E (rs ) SNP were performed using Taq- Man 1 SNP genotyping assays (Applied Biosystems). Sampling Blood samples were drawn at the beginning of each period prior to administering the clopidogrel to assess baseline platelet aggregation. Following the first dose, blood samples were drawn at 0.25, 0.5, 1, 2, and 4 hours for pharmacokinetic analyses. Blood samples for pharmacokinetic analysis of parent clopidogrel were collected in tubes containing EDTA, whereas blood samples for active metabolite were collected in tubes containing EDTA and the thiol protecting group (derivatizing agent) 2-bromo methoxyphenone (MPB; final concentration 2 mm in the 6-mL volume). Pharmacokinetic samples were drawn following the first dose because the active metabolite has a short half-life and does not accumulate with repeated dosing. In each period, blood samples for platelet aggregometry were drawn prior to dosing, 4 hours after the first dose, and 4 hours following the last dose (on day 8). Pharmacodynamic Assessments Platelet function was assessed by optical aggregometry. Venous blood was drawn into 3.2% citrate-anticoagulated tubes (Becton-Dickinson, Franklin Lakes, NJ). Platelet rich plasma was prepared from whole blood and platelet counts adjusted to 0,000 platelets/ml with platelet poor plasma. Platelet poor plasma was prepared individually for each participant using his own blood sample. Initially the specimen was spun at 8 rpm for minutes to obtain a sample of platelet rich plasma (approximately two-thirds of the sample), then the remaining one-third of the sample was centrifuged at 3,0 rpm for 15 minutes at room temperature to obtain the platelet poor plasma. Platelet aggregation was assessed in a PAP8E Aggregometer (Bio/Data Corporation, Horsham, PA) after stimulating samples with ADP mmol/l. Measurements were performed in duplicate and averaged to minimize variability. Pharmacokinetic Assessments Plasma concentrations of clopidogrel and its active metabolite were simultaneously assessed using a validated, rapid, and sensitive ultra HPLC-MS/MS assay, developed for this study as previously described, with a clopidogrel calibration range of 0.01 ng/ml and active metabolite calibration range of ng/ml. 18 The assay accuracy ranged from 11.7% to 2.37% with within-run precision less than 1.1% for clopidogrel and from 6.78% to 2.23% with within-run precision of less than 1.2% for the active metabolite. 18 Statistical Analyses Pharmacokinetic parameters for clopidogrel active metabolite and parent compound were estimated using standard non-compartmental methods with WinNonlin- Enterprise v5.2, Pharsight Corp. (Mountain View, CA). Plasma concentrations less than the lower limit of quantitation (LLOQ) were reported as zero and included as zero in calculation of PK parameters. The PK parameters calculated were C max,t max,t 1/2, and AUC 0 4. Descriptive statistics were calculated as medians and interquartile ranges (IQR), arithmetic means and standard deviation, or geometric means and coefficient of variation. Differences in platelet aggregation and pharmacokinetics among the three genotype groups were compared using the Kruskal Wallis test. Pairwise differences in platelet aggregation and pharmacokinetic parameters between and each of the higher doses and between genotypes were compared using the Wilcoxon signedrank test. Deviations from proportionality between dose and platelet aggregation or pharmacokinetic parameters (log-transformed) were evaluated using a mixed model with period and subject as random effects fit for each genotype stratum, or with a genotype treatment interaction term. Geometric mean ratios (GMR) of MPA4, C max, and AUC 0 4 (active metabolite and parent compound) were calculated for each of the higher doses relative to to assess the magnitude of change in exposure or response. The relationship between active metabolite exposure and response was further evaluated using linear regression. All statistical analyses were 2- sided at the 0.05 significance level, except GMR calculations used the typical 90% confidence interval (CI). The study was designed to enroll six subjects in each genotype group to provide % power to detect a % absolute mean difference in MPA4 between and each of the higher doses, assuming two-tailed a ¼ 0.05 and a standard deviation for the difference of 15%. Statistical analyses were performed using SAS version 9.2 or JMP 9.0 (Cary, NC). Results Disposition and Baseline Characteristics All 18 subjects completed all three periods of the protocol. The study population s mean (SD) age was 42 () years, 33% were women, and the mean (SD) body mass index was 27.7 (4.7) kg/m 2. One pretreatment sample from a

4 868 The Journal of Clinical Pharmacology / Vol 54 No 8 (14) MPA4 (%), Day IPA4 (%), Day EM IM PM EM IM PM Figure 1. Pharmacodynamic response to increasing doses of clopidogrel in CYP2C19 EMs, IMs, and PMs. MPA4 (left) and IPA4 (right) are shown by dosing period and CYP2C19 genotype. Boxplots represent median and interquartile range. Connecting lines represent group means. PM was hemolyzed during the period and was excluded from analysis. One IM and two EMs were carriers of the CYP2C19*17 variant, which is expected based on high linkage disequilibrium between the CYP2C19*17 and CYP2C19*2 variants (D 0 ¼ 1, r 2 ¼ 0.07) in the Amish population. 19 There were no adverse bleeding events in any of the participants. Mean pretreatment MPA did not differ by genotype in any of the treatment periods, and although a numeric decrease in pretreatment MPA from study period 1 to study period 3 was noted in EMs, no significant period effect was observed (e.g. for ADP mmol/l, period, EM 72%/IM 72%/PM 74%, P ¼.96; period EM 64%/ IM 74%/PM 67%, P ¼.36; period, EM 59%/IM 71%/PM %, P ¼.13). Platelet Aggregation Platelet aggregation results (MPA4 and IPA4) on day 8 of all three clopidogrel dosing periods are shown in Figure 1 by CYP2C19 genotype. At each dose level, MPA4 on day 8 differed significantly among the CYP2C19 genotype groups such that PMs had the lowest antiplatelet responses (i.e. highest MPA4; Table 1). Relative to EMs, MPA4 was approximately % higher in PMs (P ¼.02 in each dose period) and % higher in IMs (P >.05 for each dose period). The magnitude of the genetic effect was consistent across all dose levels. Compared to, increasing the daily clopidogrel dose resulted in greater platelet inhibition in all genotype groups (Figure 1 and Table 1). The incremental effect of increasing doses was similar across genotype groups (genotype dose interaction P ¼.5). Relative to, decreased MPA4 by 21% in EMs, 12% in IMs, and % in PMs, while decreased MPA4 by 39% in EMs, 32% in IMs, and 41% in PMs. The dose response relationship was not linear in any of the genotype groups; quadrupling the dose resulted in less than a doubling of response. Treatment of PMs with daily allowed them to achieve levels of platelet inhibition similar to in EMs (the reference dose and genotype; geometric mean MPA4 31% [95% CI 24 39%] for in PMs vs. 31% [95% CI 22 43%] for in Table 1. Steady-State MPA4 (Day 8) by CYP2C19 Genotype and Dose Parameter Group N Clopidogrel Dose GMR (90% CI) vs. vs. MPA4 (%) Overall (34) 32.2 (35) 24.4 (37) 0.82 ( ) 0.62 ( ) EM 6.6 (23) 24.3 (27) 18.6 (34) 0.79 ( ) 0.61 ( ) IM (23) 32.7 (19) 25.1 (25) 0.88 ( ) 0.68 ( ) PM (32) 42.2 (34) 31.3 (32) 0. ( ) 0.59 ( ) P-value * GMR, geometric mean ratio; CI, confidence interval; MPA4, maximal platelet aggregation at 4 hours post-dose; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer. Data presented as geometric mean (CV%). * Kruskal Wallis test for difference across genotypes. Signed-rank P <.05 versus within genotype stratum. Signed-rank P <.01 versus within genotype stratum.

5 Horenstein et al 869 Cmax (ng/ml) AUC0-4 (ng.hr/ml) EM IM PM EM IM PM Figure 2. Pharmacokinetics of clopidogrel active metabolite with increasing doses of clopidogrel in EMs, IMs, and PMs. Clopidogrel active metabolite C max (left) and AUC 0 4 (right) are shown by dosing period and CYP2C19 genotype. Boxplots represent median and interquartile range. Connecting lines represent group means. EMs). The results for MPA4 following the first dose on day 1 showed trends consistent with those on day 8 described above (not shown). IPA4 was more variable than MPA4. The findings for IPA4 were similar to MPA4, with IPA4 tending to be progressively lower in IMs and PMs compared to EMs. At a clopidogrel dose of, there was no significant difference in IPA4 among genotypes. Pharmacokinetics Clopidogrel active metabolite and parent compound pharmacokinetics (C max and AUC 0 4 ) on day 1 of all three dosing periods are shown in Figure 2 by CYP2C19 genotype. Consistent with the findings for antiplatelet responses, active metabolite C max and AUC 0 4 differed significantly among the genotype groups at all dose levels (Table 2). PMs had active metabolite exposures that were Table 2. Clopidogrel Parent and Active Metabolite Pharmacokinetics (Day 1) by CYP2C19 Genotype and Dose Parameter Group N Clopidogrel Dose GMR (90% CI) vs. vs. Active metabolite C max (ng/ml) Overall (39) 28.0 (57) 39.8 (58) 1.18 ( ) 1.68 ( ) EM (23).3 (42) 54.1 (24) 1.38 ( ) 1.86 ( ) IM ().8 (45) 44.4 (71) 1.05 ( ) 1.51 ( ) PM (18) 17.6 (44) 26.3 (44) 1.13 ( ) 1.69 ( ) P-value * Active metabolite AUC 0 4 (ng h/ml) Overall (42) 38.5 (48) 59.5 (54) 1.44 ( ) 2.23 ( ) EM (25) 53.6 (35).4 (24) 1.64 ( ) 2.46 ( ) IM (37) 43.8 (35) 73.7 (53) 1.32 ( ) 2.18 ( ) PM (19) 24.2 (26) 36 (35) 1.39 ( ) 2.07 ( ) P-value * Parent C max (ng/ml) Overall (158) 1.34 (164) 1.75 (173) 1.24 ( ) 2.18 ( ) EM (348) 1.73 (256) 2.92 (1) 1.89 ( ) 3.19 ( ) IM (3) 1. (157) 1.14 (1) 2.24 ( ) 1.96 ( ) PM (99) 1.07 (1.35) 1.57 (113) 1.24 ( ) 1.43 ( ) P-value * Parent AUC 0 4 (ng h/ml) Overall (287) 1.77 (254) 3.22 (199) 3.00 ( ) 5.14 ( ) EM (52) 2. (439) 4.91 (375) 3.47 ( ) 8.11 ( ) IM (134) 1.64 (378) 1.98 (1) 3.01 ( ) 3.65 ( ) PM (2) 1.63 (135) 3. (123) 2.51 ( ) 4.32 ( ) P-value * GMR, geometric mean ratio; CI, confidence interval; MPA4, maximal platelet aggregation at 4 hours post-dose; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; AUC, area under the time-concentration curve. Data presented as geometric mean (CV%). * Kruskal Wallis test for difference across genotypes. Signed-rank P <.05 versus within genotype stratum. Signed-rank P <.01 versus within genotype stratum.

6 8 The Journal of Clinical Pharmacology / Vol 54 No 8 (14) approximately half that of EMs (P <.01 at all dose levels). Active metabolite exposures in IMs were similar or only modestly reduced compared to EMs (average of % lower across dosing periods; P >.05 at all dose levels). The overall median T max was 1 hour and the overall median t 1/2 was 0.5 hours for the active metabolite; neither of these parameters differed by genotype in any of the dosing periods (not shown). Compared to daily, increasing the clopidogrel dose resulted in higher active metabolite exposure (C max and AUC 0 4 ) in all genotype groups (Table 2). Consistent with the findings for antiplatelet response, with increasing dose the increase in C max was less than dose proportional. Relative to, resulted in a 19% increase in active metabolite C max and 45% increase in AUC 0 4,on average across the genotype groups, while administering the quadruple dose resulted in a 69% increase in C max and 124% increase in AUC 0 4. At the 1 and dose levels, the absolute exposure increases tended to be greatest in EMs and IMs compared to PMs, but the relative increases in exposure were of similar magnitude across genotypes (genotype dose interaction P ¼.71 for C max and P ¼.56 for AUC 0 4 ). PMs treated with daily attained an AUC 0 4 similar to in EMs (geometric mean 36 ng h/ml [95% CI 25 52] for in PMs vs. 33 ng h/ml [95% CI 25 42] for in EMs). Similar findings were observed for C max. Clopidogrel parent compound exposures were highly variable. Consistent with the findings for the active metabolite, parent compound exposures increased with each successive dose increase (Table 2). Parent compound exposures did not differ according to CYP2C19 genotype, and the magnitude of change following each dose increase was similar across the genotype groups. Exposure-Response Relationship Active metabolite exposure was moderately correlated with antiplatelet responses in each dosing period as shown in Figure 3. Similar findings were observed for C max (not shown). Discussion Previous studies have demonstrated that individuals with high platelet reactivity after receiving standard doses of clopidogrel exhibit higher rates of cardiovascular events relative to those who have lower platelet reactivity.,21 Targeted strategies to minimize heterogeneity in antiplatelet responses, either by way of dose-adjustment or selecting patients for more potent antiplatelet therapies, have the potential to improve treatment outcomes. Common loss-of-function variants in CYP2C19 account for approximately 12% of the variation in residual platelet reactivity after standard-dose clopidogrel and are associated with poorer cardiovascular outcomes; 8 CYP2C19 MPA4, % 0 y = Ln(x) R 2 = EM- EM - EM - IM - IM - IM - PM - PM - PM AUC 0-4, ng.h/ml Figure 3. Exposure response relationship for clopidogrel active metabolite. MPA4 is plotted by AUC 0 4 according to CYP2C19 genotype and clopidogrel dose level. The line of best fit is plotted and the equation and R 2 value are provided. PMs consistently have low active metabolite exposure and antiplatelet responses. In this study, we demonstrated that increasing clopidogrel doses in CYP2C19 PMs overcomes the diminished antiplatelet responses and that active metabolite concentrations correlate with antiplatelet responses. Specifically, daily doses of were necessary in PMs to match the exposure and antiplatelet response observed with standard doses in EMs. The presence of increasing active metabolite concentrations with increasing doses suggests the possibility that other CYP enzymes, including CYP1A2, 2B6, 2C9, 3A4, and 3A5, which have been implicated in the conversion of clopidogrel to its active metabolite, take over the role of oxidizing the pro-drug sequentially to its active metabolite, particularly in CYP2C19*2 homozygotes (PMs) who lack all CYP2C19 activity. 2,3 Results from prior studies that tested the effectiveness of higher daily dosing of clopidogrel in IMs have been mixed. Simon et al 12 studied the effect of a 0 mg clopidogrel loading dose followed by 4 days of a maintenance dose in prospectively genotyped healthy subjects. The investigators found that the higher-dose regimen provided greater platelet inhibition and higher clopidogrel active metabolite concentrations in IMs, although not to the levels observed in EMs who received the standard loading dose and four days of clopidogrel. Similarly, other studies that used a maintenance dose in IMs have shown that such a dose adjustment did not, on average, reduce platelet reactivity to the levels observed with standard dosing in EMs 14,15,22,23 or with prasugrel, 24 despite providing additional platelet inhibition. The findings of our study are qualitatively consistent with these prior studies insofar as escalating clopidogrel doses helps increase the active metabolite concentrations and reduce platelet reactivity, and suggest that IMs require at least

7 Horenstein et al 871 double the approved daily clopidogrel dose to achieve similar levels of platelet inhibition to EMs. Importantly, our study shows that PMs taking the daily dose were able to achieve platelet inhibition on par with EMs taking the dose. In contrast, in ELEVATE-TIMI 56, the only other study in which a maintenance dose has been studied, this high-dose therapy was not able to lower on-treatment platelet reactivity in PMs to levels seen in EMs. 23 Also, in ELEVATE-TIMI 56, IMs receiving the 225 mg dose attained platelet reactivity measures that were similar to EMs taking daily, whereas we found that resulted in IM responses that were similar to EMs. While the doses identified in each study may differ somewhat, both our study and ELEVATE-TIMI 56 suggest that modifying the dose enhances response, and both studies report a nonlinear relation between increasing dose of clopidogrel and decreases in platelet aggregation at higher doses in the IM and PM groups, which is consistent with previous pharmacokinetic studies. 25 The differences between our findings may stem from the methodology. In particular, ELEVATE-TIMI 56 enrolled subjects with cardiovascular disease who were receiving aspirin and assessed on treatment platelet reactivity index determined through phosphorylation status of VASP and VerifyNow P2Y12, while we enrolled healthy subjects and evaluated ex vivo platelet aggregometry in response to ADP. Of particular relevance to ongoing and future trials, both studies suggest that daily may not be the most suitable dose to evaluate in response- or genotypeguided antiplatelet trials that include PMs. Our findings show that at least daily may be suitable for IMs, but PMs may require doses of of clopidogrel daily to reduce platelet aggregation and to attain concentrations of the active metabolite seen in EMs taking daily. To the extent that CYP2C19 IMs and PMs are enriched among the poor responders as determined by platelet function testing, available data suggest clopidogrel doses higher than daily may be necessary to reduce events in patients who appear to have resistance to clopidogrel therapy based solely on markers of platelet function. Importantly, the long-term safety with clopidogrel doses higher than daily has not been established. Moreover, the potential for accumulation of non-active metabolites and additional drug drug interactions that are not observed at lower clopidogrel doses must be considered. As such, these findings and the findings of ELEVATE-TIMI 56 raise an important issue about whether dose-adjusted strategies should be considered for PMs at all when alternative drugs that do not have a major CYP2C19 effect are available. Interestingly, although differences in active metabolite concentrations were observed across genotype groups in our study, parent compound concentrations did not significantly differ by genotype. These findings are consistent with the known metabolism of clopidogrel, in which approximately 85% of the parent compound is converted to non-active metabolites independent of CYP2C19, and only a minority is metabolized to the active metabolite by CYP2C19- dependent and -independent pathways. 26 Carboxylesterase 1 (CES1) is the primary enzyme responsible for converting clopidogrel into inactive carboxylic acid metabolites, while CYP1A2, 2B6, 2C9, 2C19, 3A4, and 3A5 all contribute to the formation of the active metabolite. 27 This complex metabolic process, with overlapping pathways of clopidogrel activation, has led to questions regarding the importance of any single metabolic enzyme in the production of the active metabolite. Moreover, drug drug interaction studies using CYP2C19 inhibitors (e.g. proton pump inhibitors), and studies evaluating CYP2C19 genotype, have not all demonstrated an association between CYP2C19 and clinical outcomes in clopidogrel-treated patients. 4,28 However, numerous pharmacokinetic and pharmacodynamic studies have established CYP2C19 genotype as an important factor in the activation and antiplatelet effects of clopidogrel. 26 Our findings provide additional data supporting the importance of CYP2C19 genotype in the activation of clopidogrel, and demonstrate that high doses of clopidogrel can overcome CYP2C19 metabolic deficiency, likely through activation of the parent compound via other metabolic pathways. Future studies are warranted to identify the mechanism by which parent clopidogrel is converted to its active metabolite in poor metabolizers. Our study has several limitations that are worth noting. First, the study was conducted in a small sample of healthy participants with no history of cardiovascular disease, and these participants were not taking aspirin. Thus, the study provides data which may not reflect the biological milieu of a patient on dual antiplatelet therapy following an acute event. However, this tailored design allowed us carefully to study the effect of escalating doses of clopidogrel in each genotype group, which were prospectively selected and matched for potential confounders, using a clopidogrel-specific response measure. Also, the magnitude of the pharmacokinetic and response differences between the genotype groups was similar to that observed in patients with cardiovascular disease taking aspirin. 23,29 Second, we evaluated only maintenance doses of clopidogrel and not loading doses that are commonly used in perioperative period for patients undergoing PCI. In addition, pharmacokinetic parameters were assessed following administration of only the first dose of clopidogrel; therefore, the possibility of a change in steady-state pharmacokinetic properties with high-dose clopidogrel administration cannot be dismissed. Third, the study was not designed to determine whether higher doses in IMs or PMs would carry similar risk to the

8 872 The Journal of Clinical Pharmacology / Vol 54 No 8 (14) dose in EMs for bleeding or other complications, which are a potential concern given the higher dose of parent drug. Nevertheless, the results from our analyses of the active metabolite concentrations and platelet aggregation suggest that PMs treated with daily would be at no increased risk for bleeding complications than would EMs treated with the approved dose. Fourth, we only enriched for the *2 loss of function variant in CYP2C19 and not other common or rare loss or gain of function CYP2C19 variants. In the current investigation one IM and two EMs were carriers of the CYP2C19*17 gain-offunction variant. We do not expect the presence of this variant in some subjects to be a major source of confounding in the current analysis since stratified and adjusted analyses in a larger sample from the Amish population have previously shown that the CYP2C19*17 variant has a minimal effect on clopidogrel related traits in this population. 19 Additionally, a functional variant in CES1 is associated with better clopidogrel response in this and other populations. Only one IM was a carrier for the CES1 variant in the current investigation, thus we were not able to fully evaluate the effect of this variant. Fifth, our study was restricted to Caucasians, although we know of no data to support that the CYP2C19*2 enzyme functions differently by race. Finally, our primary outcome in this study of healthy participants was platelet aggregation and not a clinical measure. Although platelet aggregation may be of limited utility for prospective doseadjustment and in medically managed populations, 31,32 it is a well-established way to assess the effects of antiplatelet therapy, and it is correlated with clinical outcomes in patients undergoing PCI.,21 Whether using quadruple daily doses of clopidogrel in PMs will result in better outcomes than the standard dose therapy will require a very large, prospective clinical trial. It is also unclear whether chronically higher exposures to the parent drug would present other non-bleeding safety concerns. In conclusion, consistent with other studies, we show that IMs and PMs administered clopidogrel have higher levels of platelet aggregation and lower concentrations of the clopidogrel active metabolite than do EMs. We observed increasing active metabolite concentrations as the clopidogrel dose was increased from 75 to 1 to in each of the three genotype groups, with a similar relationship observed for reductions in ex vivo platelet aggregation, although, the magnitude of the changes were less than proportional to the dose. A dose of daily appears to be required to normalize active metabolite concentrations and antiplatelet responses in PMs. Thus based our findings and those of Mega et al, 23 the dose that is being studied in ongoing, genotype-guided outcomes trials, 17 may not be adequate to overcome reduced CYP2C19 metabolism, particularly in PMs. Declaration of Conflicting Interests None. Funding This study was funded by the Bench to Bedside Program of the National Institutes of Health (128475) and the National Institutes of Health (U01GM and U01HL5198). References 1. Gurbel PA, Bliden KP, Hiatt BL, O Connor CM. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation. 03;7 (23): Bouman HJ, Schomig E, van Werkum JW, et al. Paraoxonase-1 is a major determinant of clopidogrel efficacy. Nat Med. 11;17 (1): Abell LM, Liu EC-K. Dissecting the activation of thienopyridines by cytochromes P4 using a pharmacodynamic assay in vitro. J Pharmacol Exp Ther. 11;339(2): Hulot JS, Collet JP, Silvain J, et al. Cardiovascular risk in clopidogrel-treated patients according to cytochrome P4 2C19*2 loss-of-function allele or proton pump inhibitor coadministration: a systematic meta-analysis. J Am Coll Cardiol. ;56(2): Scott SA, Sangkuhl K, Stein CM, et al. 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