Substrate Report for: Clopidogrel

The carboxylesterase drug hydrolysis pathway has been used extensively to improve the oral availability of drugs under the assumption that the high capacity and low substrate specificity of hydrolytic enzymes would ensure rapid, complete, and consistent conversion of prodrugs to their active metabolite. However, a growing body of literature indicates that drug hydrolysis is usually catalyzed by one primary enzyme, either carboxylesterase-1 or carboxlylesterase-2, and that there is wide variability in enzyme activity affecting the metabolism of prodrugs to their active metabolites. This review identifies carboxylesterase substrates and describes our current understanding of the influence of genetic polymorphisms on substrate disposition and clinical effects. Several polymorphisms are described in the literature and included in the personalized medicine database PharmGKB, but there are no carboxylesterase genotypes referenced in Food and Drug Administration approved drug labeling. The limited validation of metabolic pathways for drugs undergoing hydrolysis, and the small number of studies evaluating genotype-drug interactions confirm that this is an emerging field of drug metabolism research. The dependence of prodrugs, many with low therapeutic indexes, on carboxylesterase-mediated hydrolysis indicate that genetic variation plays an important role in prodrug activation, and that carboxylesterase genotyping will become an important component of personalized medicine.

1. Clopidogrel is widely prescribed in patients with cardiovascular disease. Most research has focused on the role of hepatic CYP450 metabolism as the primary source of response variability despite 85-90% of clopidogrel being hydrolyzed by human carboxylesterase-1 (CES1). 2. The purpose of this study is to determine the effects of the known CES1 inhibitor alcohol on clopidogrel metabolism: (1) in vitro in human recombinant CES1 and human liver S9 fractions (HLS9), and (2) in a plasma carboxylesterase deficient mouse (Es1(e) ) strain administered 25 mg/kg oral clopidogrel alone and with 3 g/kg alcohol. 3. Alcohol significantly inhibited the hydrolysis of clopidogrel (IC50 161 mM) and 2-oxo-clopidogrel (IC50 6 mM). In HLS9, alcohol treatment formed ethylated metabolites via transesterification and an increased formation of the H4 active metabolite. These results were replicated in Es1(e) mice as alcohol increased clopidogrel (91%) and H4 (22%) AUC and reduced formation of the clopidogrel (48%) and 2-oxo-clopidogrel (42%) carboxylate metabolites. 4. Clopidogrel metabolism is highly sensitive to alterations in CES1 activity. The Es1(e) mouse may represent a suitable model of human CES1 drug metabolism that can be used to rapidly assess how alterations in CES1 function impact the disposition of the substrate drugs.

Background The aim of this study was to evaluate the association of the carriage of the rs2244613 polymorphism of the CES1 gene with clopidogrel resistance as well as to evaluate the effectiveness of antiplatelet therapy in the carriers of this marker who have had acute coronary syndrome (ACS). This study also analyzes the procedure of percutaneous coronary intervention and compares the rs2244613 carrier rate between patients with ACS and healthy participants. Methods The study involved 81 patients diagnosed with ACS and 136 conditionally healthy participants. The optical detection of platelet agglutination by VerifyNow was employed to measure residual platelet reactivity in patients with ACS. The rs2244613 polymorphism was determined using real-time polymerase chain reaction. Results According to the results, the AA genotype of the rs2244613 polymorphism of the CES1 gene was detected in 37 patients (45.6%), the CA genotype in 42 patients (51.8%) and the CC genotype in 2 patients (2.6%). The level of residual platelet reactivity in rs2244613 carriers was higher compared with patients who did not have this allelic variant: 183.23 PRU +/- 37.24 vs. 154.3 PRU +/- 60.36 (p = 0.01). The frequencies of the minor allele C were 28.4% and 28.3% in patients with ACS and healthy participants, respectively. The results of the linear statistical model PRU due to CES1 genotype were as follows: df = 1, F = 6.96, p = 0.01). The standardized beta was 0.285 (p = 0.01) and R2 was 0.081. However, we also added CYP2C19*2 and *17 into the linear regression model. The results of the model were as follows: df = 3, F = 5.1, p = 0.003) and R2 was 0.166. Conclusions We identified a statistically significant correlation between the carriage of the rs2244613 polymorphism of the CES1 gene and the level of residual platelet aggregation among patients with ACS and the procedure of percutaneous coronary intervention.

The carboxylesterase drug hydrolysis pathway has been used extensively to improve the oral availability of drugs under the assumption that the high capacity and low substrate specificity of hydrolytic enzymes would ensure rapid, complete, and consistent conversion of prodrugs to their active metabolite. However, a growing body of literature indicates that drug hydrolysis is usually catalyzed by one primary enzyme, either carboxylesterase-1 or carboxlylesterase-2, and that there is wide variability in enzyme activity affecting the metabolism of prodrugs to their active metabolites. This review identifies carboxylesterase substrates and describes our current understanding of the influence of genetic polymorphisms on substrate disposition and clinical effects. Several polymorphisms are described in the literature and included in the personalized medicine database PharmGKB, but there are no carboxylesterase genotypes referenced in Food and Drug Administration approved drug labeling. The limited validation of metabolic pathways for drugs undergoing hydrolysis, and the small number of studies evaluating genotype-drug interactions confirm that this is an emerging field of drug metabolism research. The dependence of prodrugs, many with low therapeutic indexes, on carboxylesterase-mediated hydrolysis indicate that genetic variation plays an important role in prodrug activation, and that carboxylesterase genotyping will become an important component of personalized medicine.

1. Clopidogrel is widely prescribed in patients with cardiovascular disease. Most research has focused on the role of hepatic CYP450 metabolism as the primary source of response variability despite 85-90% of clopidogrel being hydrolyzed by human carboxylesterase-1 (CES1). 2. The purpose of this study is to determine the effects of the known CES1 inhibitor alcohol on clopidogrel metabolism: (1) in vitro in human recombinant CES1 and human liver S9 fractions (HLS9), and (2) in a plasma carboxylesterase deficient mouse (Es1(e) ) strain administered 25 mg/kg oral clopidogrel alone and with 3 g/kg alcohol. 3. Alcohol significantly inhibited the hydrolysis of clopidogrel (IC50 161 mM) and 2-oxo-clopidogrel (IC50 6 mM). In HLS9, alcohol treatment formed ethylated metabolites via transesterification and an increased formation of the H4 active metabolite. These results were replicated in Es1(e) mice as alcohol increased clopidogrel (91%) and H4 (22%) AUC and reduced formation of the clopidogrel (48%) and 2-oxo-clopidogrel (42%) carboxylate metabolites. 4. Clopidogrel metabolism is highly sensitive to alterations in CES1 activity. The Es1(e) mouse may represent a suitable model of human CES1 drug metabolism that can be used to rapidly assess how alterations in CES1 function impact the disposition of the substrate drugs.

Background The aim of this study was to evaluate the association of the carriage of the rs2244613 polymorphism of the CES1 gene with clopidogrel resistance as well as to evaluate the effectiveness of antiplatelet therapy in the carriers of this marker who have had acute coronary syndrome (ACS). This study also analyzes the procedure of percutaneous coronary intervention and compares the rs2244613 carrier rate between patients with ACS and healthy participants. Methods The study involved 81 patients diagnosed with ACS and 136 conditionally healthy participants. The optical detection of platelet agglutination by VerifyNow was employed to measure residual platelet reactivity in patients with ACS. The rs2244613 polymorphism was determined using real-time polymerase chain reaction. Results According to the results, the AA genotype of the rs2244613 polymorphism of the CES1 gene was detected in 37 patients (45.6%), the CA genotype in 42 patients (51.8%) and the CC genotype in 2 patients (2.6%). The level of residual platelet reactivity in rs2244613 carriers was higher compared with patients who did not have this allelic variant: 183.23 PRU +/- 37.24 vs. 154.3 PRU +/- 60.36 (p = 0.01). The frequencies of the minor allele C were 28.4% and 28.3% in patients with ACS and healthy participants, respectively. The results of the linear statistical model PRU due to CES1 genotype were as follows: df = 1, F = 6.96, p = 0.01). The standardized beta was 0.285 (p = 0.01) and R2 was 0.081. However, we also added CYP2C19*2 and *17 into the linear regression model. The results of the model were as follows: df = 3, F = 5.1, p = 0.003) and R2 was 0.166. Conclusions We identified a statistically significant correlation between the carriage of the rs2244613 polymorphism of the CES1 gene and the level of residual platelet aggregation among patients with ACS and the procedure of percutaneous coronary intervention.

Clopidogrel, a clinically used antiplatelet agent, can be readily hydrolyzed by human carboxylesterase 1A (CES1A) to release an inactive metabolite clopidogrel carboxylic acid (CCA). In this study, clopidogrel was used as a tool substrate to investigate the interspecies variation of clopidogrel hydrolysis in hepatic microsomes from various mammals including human and six laboratory animals (such as mouse, rat, rabbit, beagle dog, minipig and cynomolgus monkey). The results demonstrated that clopidogrel could be hydrolyzed into CCA by all tested hepatic microsomes from human or other mammals, but the hydrolytic rates greatly varied among species. Inhibition assays demonstrated that BNPP (an inactivator of mammalian CES) strongly inactivated clopidogrel hydrolytic activity in all tested hepatic microsomes, suggested that mammalian CES were major contributor(s) responsible for clopidogrel hydrolysis in hepatic preparations from all above-mentioned species. By contrast, the response of a reversible inhibitor of human CES1A on clopidogrel hydrolysis in these liver preparations varied significantly among different species. Moreover, the enzymatic kinetics and the apparent kinetic parameters of clopidogrel hydrolysis in hepatic microsomes from various animal species were evaluated and compared to each other. These findings provide crucial information for deeply understanding the differences in catalytic behaviors of mammalian CES, which will be very helpful for choosing suitable laboratory animal(s) for whole tests of CES1A substrate-drugs.

Several single nucleotide variations (SNVs) affect carboxylesterase 1 (CES1) activity, but the effects of genetic variants on CES1 gene expression have not been systematically investigated. Therefore, our aim was to investigate effects of genetic variants on CES1 gene expression in two independent whole blood sample cohorts of 192 (discovery) and 88 (replication) healthy volunteers and in a liver sample cohort of 177 patients. Furthermore, we investigated possible effects of the found variants on clopidogrel pharmacokinetics (n = 106) and pharmacodynamics (n = 46) in healthy volunteers, who had ingested a single 300 mg or 600 mg dose of clopidogrel. Using massively parallel sequencing, we discovered two CES1 SNVs, rs12443580 and rs8192935, to be strongly and independently associated with a 39% (p = 4.0 x 10(-13) ) and 31% (p = 2.5 x 10(-8) ) reduction in CES1 whole blood expression per copy of the minor allele. These findings were replicated in the replication cohort. However, these SNVs did not affect CES1 liver expression, or clopidogrel pharmacokinetics or pharmacodynamics. Conversely, the CES1 c.428G>A missense SNV (rs71647871) impaired the hydrolysis of clopidogrel, increased exposure to clopidogrel active metabolite and enhanced its antiplatelet effects. In conclusion, the rs12443580 and rs8192935 variants reduce CES1 expression in whole blood but not in the liver. These tissue-specific effects may result in substrate-dependent effects of the two SNVs on CES1-mediated drug metabolism.

Carboxylesterase 1 (CES1) is the predominant human hepatic hydrolase responsible for the metabolism of many clinically important medications. CES1 expression and activity vary markedly among individuals; and genetic variation is a major contributing factor to CES1 interindividual variability. In this study, we comprehensively examined the functions of CES1 nonsynonymous single nucleotide polymorphisms (nsSNPs) and haplotypes using transfected cell lines and individual human liver tissues. The 20 candidate variants include CES1 nsSNPs with a minor allele frequency >0.5% in a given population or located in close proximity to the CES1 active site. Five nsSNPs, including L40Ter (rs151291296), G142E (rs121912777), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), were loss-of-function variants for metabolizing the CES1 substrates clopidogrel, enalapril, and sacubitril. In addition, A158V (rs202121317), R199H (rs2307243), E220G (rs200707504), and T290M (rs202001817) decreased CES1 activity to a lesser extent in a substrate-dependent manner. Several nsSNPs, includingL40Ter (rs151291296), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), significantly reduced CES1 protein and/or mRNA expression levels in the transfected cells. Functions of the common nonsynonymous haplotypes D203E-A269S and S75N-D203E-A269S were evaluated using cells stably expressing the haplotypes and a large set of the human liver. Neither CES1 expression nor activity was affected by the two haplotypes. In summary, this study revealed several functional nsSNPs with impaired activity on the metabolism of CES1 substrate drugs. Clinical investigations are warranted to determine whether these nsSNPs can serve as biomarkers for the prediction of therapeutic outcomes of drugs metabolized by CES1.

Carboxylesterase 1 (CES1) hydrolyzes the prodrug clopidogrel to an inactive carboxylic acid metabolite. The effects of CES1 S75N (rs2307240,C>T) on clopidogrel response among 851 acute coronary syndrome patients who came from the north, central and south of China were studied. The occurrence ratios of each endpoint in the CC group were significantly higher than in the CT + TT group for cerebrovascular events (14% vs 4.8%, p < 0.001, OR = 0.31), acute myocardial infarction (15.1% vs 6.1%, p < 0.001, OR = 0.37) and unstable angina (62.8% vs 37.7%, p < 0.001, OR = 0.36). The results showed that there was a significant association between CES1 S75N (rs2307240) and the outcome of clopidogrel therapy. Moreover, the frequency of the T allele of rs2307240 in acute coronary syndrome patients (MAF = 0.22) was more than four times higher than that in the general public (MAF = 0.05).

The relationship between liver function and clopidogrel response has not been studied so far. We therefore sought to investigate the associations between 3 parameters of liver synthesis and on-treatment platelet reactivity to adenosine diphosphate in patients on dual antiplatelet therapy. On-treatment platelet reactivity was determined by the VerifyNow P2Y12 assay in 316 patients undergoing angioplasty with stent implantation for cardiovascular disease. Cholinesterase, serum albumin, and total cholesterol levels were measured by internationally standardized assays in the hospital's central laboratory. On-treatment platelet reactivity by the VerifyNow P2Y12 assay correlated inversely with cholinesterase, serum albumin, and total cholesterol levels (all P <= .02). The inverse associations of cholinesterase and serum albumin levels with platelet reactivity remained significant after adjustment for cytochrome P450 loss-of-function polymorphisms and other factors that were previously associated with high on-treatment residual platelet reactivity (HRPR; both P < .05). Patients without HRPR by the VerifyNow P2Y12 assay had significantly higher levels of serum albumin and total cholesterol than patients with HRPR (both P = .008). In conclusion, liver function is associated with response to clopidogrel therapy in patients undergoing angioplasty and stenting for cardiovascular disease.

1. In this study, we report that gambogic acid (GA), a promising anticancer agent, potentiates clopidogrel-induced apoptosis and attenuates CPT-11-induced apoptosis by down-regulating human carboxylesterase (CES) 1 and -2 via ERK and p38 MAPK pathway activation, which provides a molecular explanation linking the effect of drug combination directly to the decreased capacity of hydrolytic biotransformation. 2. The expression levels of CES1 and CES2 decreased significantly in a concentration- and time-dependent manner in response to GA in Huh7 and HepG2 cells; hydrolytic activity was also reduced. 3. The results showed that pretreatment with GA potentiated clopidogrel-induced apoptosis by down-regulating CES1. Moreover, the GA-mediated repression of CES2 attenuated CPT-11-induced apoptosis. 4. Furthermore, the ERK and p38 MAPK pathways were involved in the GA-mediated down-regulation of CES1 and CES2. 5. Taken together, our data suggest that GA is a potent repressor of CES1 and CES2 and that combination with GA will affect the metabolism of drugs containing ester bonds.

OBJECTIVE: CES1 encodes carboxylesterase-1, an important drug-metabolizing enzyme with high expression in the liver. Previous studies have reported a genomic translocation of the 5' region from the poorly expressed pseudogene CES1P1, to CES1, yielding the structural variant CES1VAR. The aim of this study was to characterize this translocation and its effect on CES1 expression in the human liver. MATERIALS AND
METHODS: Experiments were conducted in human liver tissues and cell culture (HepG2). The promoter and exon 1 of CES1 were sequenced by Sanger and Ion Torrent sequencing to identify gene translocations. The effects of CES1 5'UTRs on mRNA and protein expression were assessed by quantitative real-time PCR, allelic ratio mRNA analysis by primer extension (SNaPshot), quantitative targeted proteomics, and luciferase reporter gene assays.
RESULTS: Sequencing of CES1 identified two translocations: first, CES1VAR (17% minor allele frequency) comprising the 5'UTR, exon 1, and part of intron 1. A second shorter translocation, CES1SVAR, was observed excluding exon 1 and intron 1 regions (<0.01% minor allele frequency). CES1VAR is associated with 2.6-fold decreased CES1 mRNA and approximately 1.35-fold lower allelic mRNA. Luciferase reporter constructs showed that CES1VAR decreases luciferase activity 1.5-fold, whereas CES1SVAR slightly increases activity. CES1VAR was not associated with CES1 protein expression or metabolism of the CES1 substrates enalapril, clopidogrel, or methylphenidate in the liver. CONCLUSION: The frequent translocation variant CES1VAR reduces mRNA expression of CES1 in the liver by approximately 30%, but protein expression and metabolizing activity in the liver were not detectably altered - possibly because of variable CES1 expression masking small allelic effects. Whether drug therapies are affected by CES1VAR will require further in-vivo studies.

AIMS: Clopidogrel and angiotensin converting enzyme (ACE) inhibitors are commonly co-prescribed drugs. Clopidogrel inhibits carboxylesterase 1 (CES1), the enzyme responsible for converting prodrug ACE inhibitors (such as ramipril and perindopril) to their active metabolites. The clinical implications of this potential drug interaction are unknown. The clinical consequences of the potential drug interaction between clopidogrel and prodrug ACE inhibitors were examined.
METHODS: We conducted a nested case-control study of Ontarians aged 66 years and older treated with clopidogrel between September 1 2003 and March 31 2013 following acute myocardial infarction. Cases were subjects who died or were hospitalized for reinfarction or heart failure in the subsequent year, and each was matched with up to four controls. The primary outcome was a composite of reinfarction, heart failure or death. The primary analysis examined whether use of the prodrug ACE inhibitors ramipril or perindopril was more common among cases than use of lisinopril, an active ACE inhibitor.
RESULTS: Among 45 918 patients treated with clopidogrel following myocardial infarction, we identified 4203 cases and 14 964 controls. After adjustment, we found no association between the composite outcome and use of perindopril (adjusted odds ratio (aOR) 0.94, 95% confidence interval (CI) 0.76, 1.16) or ramipril (aOR 0.97, 95% CI 0.80, 1.18), relative to lisinopril. Secondary analyses of each element of the composite outcome yielded similar findings.
CONCLUSIONS: Following myocardial infarction, use of clopidogrel with ACE inhibitors activated by CES1 is not associated with an increased risk of adverse cardiovascular outcomes relative to lisinopril. These findings suggest that the recently described drug interaction between clopidogrel and prodrug ACE inhibitors is of little clinical relevance.

Human arylacetamide deacetylase (AADAC) is an esterase responsible for the hydrolysis of some drugs, including flutamide, indiplon, phenacetin, and rifamycins. AADAC is highly expressed in the human liver, where carboxylesterase (CES) enzymes, namely, CES1 and CES2, are also expressed. It is generally recognized that CES1 prefers compounds with a large acyl moiety and a small alcohol or amine moiety as substrates, whereas CES2 prefers compounds with a small acyl moiety and a large alcohol or amine moiety. In a comparison of the chemical structures of known AADAC substrates, AADAC most likely prefers compounds with the same characteristics as does CES2. However, the substrate specificity of human AADAC has not been fully clarified. To expand the knowledge of substrates of human AADAC, we measured its hydrolase activities toward 13 compounds, including known human CES1 and CES2 substrates, using recombinant enzymes expressed in Sf21 cells. Recombinant AADAC catalyzed the hydrolysis of fluorescein diacetate, N-monoacetyldapsone, and propanil, which possess notably small acyl moieties, and these substrates were also hydrolyzed by CES2. However, AADAC could not hydrolyze another CES2 substrate, procaine, which possesses a moderately small acyl moiety. In addition, AADAC did not hydrolyze several known CES1 substrates, including clopidogrel and oseltamivir, which have large acyl moieties and small alcohol moieties. Collectively, these results suggest that AADAC prefers compounds with smaller acyl moieties than does CES2. The role of AADAC in the hydrolysis of drugs has been clarified. For this reason, AADAC should receive attention in ADMET studies during drug development.

Carboxylesterase 1 (CES1) hydrolyzes the prodrug clopidogrel to an inactive carboxylic acid metabolite. We studied the pharmacokinetics and pharmacodynamics of 600 mg oral clopidogrel in healthy white volunteers, including 10 carriers and 12 noncarriers of CES1 c.428G>A (p.Gly143Glu, rs71647871) single nucleotide variation (SNV). Clopidogrel carboxylic acid to clopidogrel area under the plasma concentration-time curve from 0 hours to infinity (AUC0-infinity ) ratio was 53% less in CES1 c.428G>A carriers than in noncarriers (P = 0.009), indicating impaired hydrolysis of clopidogrel. Consequently, the AUC0-infinity of clopidogrel and its active metabolite were 123% (P = 0.004) and 67% (P = 0.009) larger in the c.428G>A carriers than in noncarriers. Consistent with these findings, the average inhibition of P2Y12 -mediated platelet aggregation 0-12 hours after clopidogrel intake was 19 percentage points higher in the c.428G>A carriers than in noncarriers (P = 0.036). In conclusion, the CES1 c.428G>A SNV increases clopidogrel active metabolite concentrations and antiplatelet effects by reducing clopidogrel hydrolysis to inactive metabolites.

BACKGROUND: Coprescribing of clopidogrel and other drugs is common. Available reviews have addressed the drug-drug interactions (DDIs) when clopidogrel is as an object drug, or focused on combination use of clopidogrel and a special class of drugs. Clinicians may still be ignorant of those DDIs when clopidogrel is a precipitant drug, the factors determining the degree of DDIs, and corresponding risk management.
METHODS: A literature search was performed using PubMed, MEDLINE, Web of Science, and the Cochrane Library to analyze the pharmacokinetic DDIs of clopidogrel and new P2Y12 receptor inhibitors.
RESULTS: Clopidogrel affects the pharmacokinetics of cerivastatin, repaglinide, ferulic acid, sibutramine, efavirenz, and omeprazole. Low efficacy of clopidogrel is anticipated in the presence of omeprazole, esomeprazole, morphine, grapefruit juice, scutellarin, fluoxetine, azole antifungals, calcium channel blockers, sulfonylureas, and ritonavir. Augmented antiplatelet effects are anticipated when clopidogrel is coprescribed with aspirin, curcumin, cyclosporin, St John's wort, rifampicin, and angiotensin-converting enzyme inhibitors. The factors determining the degree of DDIs with clopidogrel include genetic status (eg, cytochrome P540 [CYP]2B6*6, CYP2C19 polymorphism, CYP3A5*3, CYP3A4*1G, and CYP1A2-163C.A), species differences, and dose strength. The DDI risk does not exhibit a class effect, eg, the effects of clopidogrel on cerivastatin versus other statins, the effects of proton pump inhibitors on clopidogrel (omeprazole, esomeprazole versus pantoprazole, rabeprazole), the effects of rifampicin on clopidogrel versus ticagrelor and prasugrel, and the effects of calcium channel blockers on clopidogrel (amlodipine versus P-glycoprotein-inhibiting calcium channel blockers). The mechanism of the DDIs with clopidogrel involves modulating CYP enzymes (eg, CYP2B6, CYP2C8, CYP2C19, and CYP3A4), paraoxonase-1, hepatic carboxylesterase 1, P-glycoprotein, and organic anion transporter family member 1B1. CONCLUSION: Effective and safe clopidogrel combination therapy can be achieved by increasing the awareness of potential changes in efficacy and toxicity, rationally selecting alternatives, tailoring drug therapy based on genotype, checking the appropriateness of physician orders, and performing therapeutic monitoring.

Patients with coronary artery disease often receive concurrent treatment with clopidogrel and a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor medication. Accordingly, potential drug-drug interactions associated with the concomitant use of these agents present an area of concern. Both CYP enzymes and carboxylesterase 1 (CES1) are involved in the metabolism of clopidogrel, while CES1 is believed to be the enzyme responsible for the activation of simvastatin. Some in vitro studies have suggested that simvastatin could attenuate clopidogrel activation via inhibiting CYP3A activity. However, these findings have not found support in several recently published clinical investigations. The present study addresses these inconsistencies by exploring the potential role of CES1 in the metabolism of clopidogrel and simvastatin. Our in vitro human liver s9 fraction incubation study demonstrated that simvastatin significantly enhanced the formation of the intermediate metabolite 2-oxo-clopidogrel, and inhibited the CES1-mediated hydrolysis of clopidogrel, 2-oxo-clopidogrel, and the active metabolite. However, the production of the active metabolite remained unchanged. Conversely, clopidogrel was not found to influence the CES1 mediated hydrolysis (activation) of simvastatin. Moreover, we provided evidence that CES1 is not an efficient enzyme for catalyzing simvastatin activation. In summary, the inhibitory effect of simvastatin on the hydrolysis of clopidogrel and its principal metabolites may have offset the influence of simvastatin-mediated inhibition of CYP3A, and permitted the unaltered formation of the clopidogrel active metabolite. These data help explain the conflicting accounts in previous reports regarding clopidogrel and simvastatin interactions by taking into consideration CES1; they suggest that the interactions are unlikely to significantly influence clinical outcomes.

Ticlopidine is a first-generation thienopyridine antiplatelet drug that prevents adenosine 5'-diphosphate (ADP)-induced platelet aggregation. We identified the enzymes responsible for the two-step metabolic bioactivation of ticlopidine in human liver microsomes and plasma. Formation of 2-oxo-ticlopidine, an intermediate metabolite, was NADPH dependent and cytochrome P450 (CYP) 1A2, 2B6, 2C19, and 2D6 were involved in this reaction. Conversion of 2-oxo-ticlopidine to thiol metabolites was observed in both microsomes (M1 and M2) and plasma (M1). These two metabolites were considered as isomers, and mass spectral analysis suggested that M2 was a thiol metabolite bearing an exocyclic double bond, whereas M1 was an isomer in which the double bond was migrated to an endocyclic position in the piperidine ring. The conversion of 2-oxo-ticlopidine to M1 in plasma was significantly increased by the addition of 1 mM CaCl2. In contrast, the activity in microsomes was not changed in the presence of CaCl2. M1 formation in plasma was inhibited by EDTA but not by other esterase inhibitors, whereas this activity in microsomes was substantially inhibited by carboxylesterase (CES) inhibitors such as bis-(p-nitrophenyl)phosphate (BNPP), diisopropylphosphorofluoride (DFP), and clopidogrel. The conversion of 2-oxo-ticlopidine to M1 was further confirmed with recombinant paraoxonase 1 (PON1) and CES1. However, M2 was detected only in NADPH-dependent microsomal incubation, and multiple CYP isoforms were involved in M2 formation with highest contribution of CYP2B6. In vitro platelet aggregation assay demonstrated that M2 was pharmacologically active. These results collectively indicated that the formation of M2 was mediated by CYP isoforms whereas M1, an isomer of M2, was generated either by human PON1 in plasma or by CES1 in the human liver.

Clopidogrel is an oral antiplatelet prodrug, the majority of which is hydrolyzed to an inactive metabolite by hepatic carboxylesterase 1 (CES1). Most angiotensin-converting enzyme inhibitors (ACEIs) are also metabolized by this enzyme. We examined the effects of ACEIs on clopidogrel bioactivation in vitro and linked the results with a pharmacoepidemiological study. In vitro, ACEIs inhibited CES1-mediated hydrolysis of a model substrate, and trandolapril and enalapril increased formation of clopidogrel active metabolite. In 70,934 patients with myocardial infarction, hazard ratios for clinically significant bleeding in ACEI-treated patients cotreated with or without clopidogrel were 1.10 (95% confidence interval (CI): 0.97-1.25, P = 0.124) and 0.90 (95% CI: 0.81-0.99, P = 0.025), respectively, as compared with patients who did not receive ACEIs. This difference was statistically significant (P = 0.002). We conclude that cotreatment with selected ACEIs and clopidogrel may increase the risk of bleeding. Combination of in vitro and pharmacoepidemiological studies may be a useful paradigm for assessment of drug-drug interactions.

Abstract Human carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are serine esterases responsible for the hydrolysis of ester and amide bonds present in a number of pharmaceutical products. Several common genetic variants of the CES1 and CES2 genes have been shown to influence drug metabolism and clinical outcomes. Polymorphisms of the CES1 gene have been reported to affect the metabolism of dabigatran etexilate, methylphenidate, oseltamivir, imidapril, and clopidogrel, whereas variants of the CES2 gene have been found to affect aspirin and irinotecan. Although the findings of these studies may be preliminary, they demonstrate the potential clinical utility of CES polymorphisms; however, more research is required, especially with respect to CES2. In this review, we outline the functional, molecular, and genetic properties of CES1 and CES2, and highlight recent studies that have shown relations between CES1 and CES2 variants and contemporary pharmacotherapy.

OBJECTIVE: Carboxylesterase 1 hydrolyzes the majority of clopidogrel to the inactive metabolite. The aim of this study was to assess the effects of the CES1A2 A(-816)C polymorphism and other genetic and clinical factors on clopidogrel response variability. An additional aim was to investigate the relationship between genetic variations and development of stent thrombosis (ST).
METHODS: We recruited 162 coronary heart disease patients treated with aspirin and clopidogrel, and we genotyped them for the CES1A2 A(-816)C, CYP2C19 *2/*3, PON1 Q192R, and ABCB1 C3435T polymorphisms. Platelet reactivity was analyzed using the VASP-PRI assay. We also carried out a case-control study in which 22 patients undergoing stent implantation who had ST were matched with 86 ST-free controls.
RESULTS: The VASP-PRI values were significantly higher in the carriers of the CES1A2 -816C allele (P=0.014) and CYP2C19 loss of function (LOF) alleles (P=0.004). Furthermore, the patients with CYP2C19 LOF alleles showed an increased risk of ST (ORadj=4.28, P=0.033). However, there was no significant association between the CES1A2 -816C allele and the development of ST. The CYP2C19 and CES1A2 genotypes alone could explain 6.1 and 3.7% of the interindividual variability in the VASP-PRI results, respectively. The value increased to 12.5% when clinical factors (e.g. BMI and triglycerides) were also considered. The PON1 Q192R and ABCB1 C3435T genetic variations produced no significant impact. CONCLUSION: The CES1A2 -816C and the CYP2C19 LOF alleles were associated with attenuated platelet reactivity to clopidogrel. CYP2C19 LOF was also predictive of ST; however, the association between the CES1A2 -816C allele and development of ST requires further study.

INTRODUCTION: Carboxylesterase 1 (CES1) is the primary enzyme responsible for converting clopidogrel into biologically inactive carboxylic acid metabolites.
METHODS: We genotyped a functional variant in CES1, G143E, in participants of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) study (n=566) and in 350 patients with coronary heart disease treated with clopidogrel, and carried out an association analysis of bioactive metabolite levels, on-clopidogrel ADP-stimulated platelet aggregation, and cardiovascular outcomes.
RESULTS: The levels of clopidogrel active metabolite were significantly greater in CES1 143E-allele carriers (P=0.001). Consistent with these findings, individuals who carried the CES1 143E-allele showed a better clopidogrel response as measured by ADP-stimulated platelet aggregation in both participants of the PAPI study (P=0.003) and clopidogrel-treated coronary heart disease patients (P=0.03). No association was found between this single nucleotide polymorphism and baseline measures of platelet aggregation in either cohort. CONCLUSION: Taken together, these findings suggest, for the first time, that genetic variation in CES1 may be an important determinant of the efficacy of clopidogrel.

Clopidogrel pharmacotherapy is associated with substantial interindividual variability in clinical response, which can translate into an increased risk of adverse outcomes. Clopidogrel, a recognized substrate of hepatic carboxylesterase 1 (CES1), undergoes extensive hydrolytic metabolism in the liver. Significant interindividual variability in the expression and activity of CES1 exists, which is attributed to both genetic and environmental factors. We determined whether CES1 inhibition and CES1 genetic polymorphisms would significantly influence the biotransformation of clopidogrel and alter the formation of the active metabolite. Coincubation of clopidogrel with the CES1 inhibitor bis(4-nitrophenyl) phosphate in human liver s9 fractions significantly increased the concentrations of clopidogrel, 2-oxo-clopidogrel, and clopidogrel active metabolite, while the concentrations of all formed carboxylate metabolites were significantly decreased. As anticipated, clopidogrel and 2-oxo-clopidogrel were efficiently hydrolyzed by the cell s9 fractions prepared from wild-type CES1 transfected cells. The enzymatic activity of the CES1 variants G143E and D260fs were completely impaired in terms of catalyzing the hydrolysis of clopidogrel and 2-oxo-clopidogrel. However, the natural variants G18V, S82L, and A269S failed to produce any significant effect on CES1-mediated hydrolysis of clopidogrel or 2-oxo-clopidogrel. In summary, deficient CES1 catalytic activity resulting from CES1 inhibition or CES1 genetic variation may be associated with higher plasma concentrations of clopidogrel-active metabolite, and hence may enhance antiplatelet activity. Additionally, CES1 genetic variants have the potential to serve as a biomarker to predict clopidogrel response and individualize clopidogrel dosing regimens in clinical practice.

BACKGROUND AND PURPOSE: Clopidogrel is a prodrug bioactivated by cytochrome P450s (CYPs). More recently, paraoxonase-1 (PON1) has been proposed as a major contributor to clopidogrel metabolism. The purpose of this study was to assess the relative contribution of CYPs and PON1 to clopidogrel metabolism in vitro. EXPERIMENTAL APPROACH: Clopidogrel metabolism was studied in human serum, recombinant PON1 enzyme (rePON1), pooled human liver microsomes (HLMs), HLMs with the CYP2C19*1/*1 genotype and HLMs with the CYP2C19*2/*2 genotype. Inhibition studies were also performed using specific CYP inhibitors and antibodies. Clopidogrel and its metabolites were measured using LC/MS/MS method. KEY
RESULTS: PON1 activity was highest in the human serum and there was no difference in PON1 activity between any of the HLM groups. The production of clopidogrel's active metabolite (clopidogrel-AM) from 2-oxo-clopidogrel in pooled HLMs was approximately 500 times that in serum. When 2-oxo-clopidogrel was incubated with rePON1, clopidogrel-AM was not detected. Clopidogrel-AM production from 2-oxo-clopidogrel was lower in CYP2C19*2/*2 HLMs compared with CYP2C19*1/*1 HLMs, while PON1 activity in HLMs with both genotypes was similar. Moreover, incubation with inhibitors of CYP3A, CYP2B6 and CYP2C19 significantly reduced clopidogrel bioactivation while a PON1 inhibitor, EDTA, had only a weak inhibitory effect. CONCLUSION AND IMPLICATIONS: This in vitro study shows that the contribution of PON1 to clopidogrel metabolism is limited at clinically relevant concentrations. Moreover, CYP2C19, CYP2B6 and CYP3A play important roles in the bioactivation of clopidogrel.

BACKGROUND: A poor biological response to clopidogrel is associated with an increased risk of major cardiovascular ischemic events (MACE). Paraoxonase 1 (PON1) enzyme activity is modulated by the PON1-Q192R variant (rs662) and was recently suggested to be strongly involved in clopidogrel bioactivation, but the influence of the PON1-Q192R variant on the risk of MACE in clopidogrel-treated patients is controversial. OBJECTIVES: To determine whether the PON1-Q192R variant influences clopidogrel biological responsiveness and the risk of MACE in patients treated with clopidogrel.
METHODS: Systematic review and meta-analysis of studies of the association between the PON1-Q192R polymorphism and the biological response to clopidogrel and/or the risk of MACE during clopidogrel administration.
RESULTS: Seventeen studies were included. In the 12 studies of the biological response to clopidogrel (n = 5302 patients), there was no significant difference between 192QQ and 192QR + 192RR subjects, whatever the laboratory method used (global mean standardized difference = 0.10 [-0.06; 0.25], P = 0.22). Eleven studies assessed the risk of MACE, four using a case-control design (n = 2739 patients) and seven a prospective design (n = 5353 patients). Overall, MACE occurred in 19% of patients in case-control studies and in 6% of patients in prospective cohort studies, with no significant difference between 192QQ and 192QR + 192RR patients (OR = 1.28 [0.97; 1.68], P = 0.08). Similar results were obtained when study design was taken into account. Heterogeneity was mainly driven by one publication.
CONCLUSIONS: This meta-analysis suggests that the PON1-Q192R polymorphism has no major impact on the risk of MACE and does not alter the biological response to clopidogrel in clopidogrel-treated patients.

The aim of this study was to conclusively determine the enzyme responsible for the hydrolysis of oxybutynin in human liver. Hydrolysis in human liver microsomes (HLMs) and human liver cytosol (HLC) followed Michaelis-Menten kinetics with similar K(m) values. In recombinant human carboxylesterase (CES)-expressing microsomes, CES1 was much more efficient than CES2 and yielded a K(m) value more comparable with that found in HLMs or HLC than did CES2. A correlation analysis using a set of individual HLMs, in which both CESs acted independently showed that the hydrolysis rate of oxybutynin, correlated significantly with a CES1 marker reaction, clopidogrel hydrolysis, but not with a CES2 marker reaction, irinotecan (CPT-11) hydrolysis. Chemical inhibition studies using bis-(p-nitrophenyl) phosphate, clopidogrel, nordihydroguaiaretic acid, procainamide, physostigmine, and loperamide revealed that the effects of these compounds in HLMs, HLC, and recombinant CES1-expressing microsomes were similar, whereas those in CES2-expressing microsomes were clearly different. These results strongly suggest that CES1, rather than CES2, is the principal enzyme responsible for the hydrolysis of oxybutynin in human liver..

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.

BACKGROUND: The paraoxonase activity of the enzyme paraoxonase-1 (PON-1) associated with high-density lipoprotein (HDL) may significantly influence clopidogrel's antiplatelet and clinical efficacy as a result of its involvement in the clopidogrel biotransformation to the pharmacologically active thiol metabolite. We evaluated the possible relationships of HDL levels as well as PON-1 activities and the Q192R genotype with clopidogrel's antiplatelet efficacy in acute coronary syndrome (ACS) patients. METHODS AND
RESULTS: The platelet aggregation, P-selectin expression and platelet/leukocyte conjugates as well as the clopidogrel response variability (evaluated by the VASP phosphorylation test and expressed as platelet reactivity index, PRI) were assessed in 74 ACS patients undergoing percutaneous coronary intervention (PCI) in relation to the PON-1 Q192R genotype and to serum HDL-cholesterol levels, and PON-1 (paraoxonase and arylesterase) activities. Patients were loaded with 600 mg of clopidogrel followed by 75 mg per day. HDL-cholesterol levels and PON-1 activities at baseline (before clopidogrel loading) were not altered at 5- and 30-day post-clopidogrel loading, whereas baseline platelet activation parameters were significantly attenuated. At 5 days, 17 patients were clopidogrel non-responders (PRI: 64.2 +/- 11.1%). HDL-cholesterol was inversely associated with platelet activation parameters independently on platelet response variability to clopidogrel whereas a negative association between platelet activation parameters and paraoxonase activity was observed in patients adequately responding to clopidogrel but not in clopidogrel non-responders. Similarly, the platelet activation markers were significantly higher in PON-1 Q192Q genotype carriers compared with those having one or two R alleles only in patients adequately responding to clopidogrel.
CONCLUSIONS: PON-1 is an important determinant of clopidogrel antiplatelet efficacy only in patients adequately responding to clopidogrel. These findings may be clinically important in ACS patients receiving clopidogrel therapy, especially the first days after the episode.

Ticlopidine, clopidogrel, and prasugrel are thienopyridine prodrugs that inhibit adenosine-5'-diphosphate (ADP)-mediated platelet aggregation in vivo. These compounds are converted to thiol-containing active metabolites through a corresponding thiolactone. The 3 compounds differ in their metabolic pathways to their active metabolites in humans. Whereas ticlopidine and clopidogrel are metabolized to their thiolactones in the liver by cytochromes P450, prasugrel proceeds to its thiolactone following hydrolysis by carboxylesterase 2 during absorption, and a portion of prasugrel's active metabolite is also formed by intestinal CYP3A. Both ticlopidine and clopidogrel are subject to major competing metabolic pathways to inactive metabolites. Thus, varying efficiencies in the formation of active metabolites affect observed effects on the onset of action and extent of inhibition of platelet aggregation (IPA). Knowledge of the CYP-dependent formation of ticlopidine and clopidogrel thiolactones helps explain some of the observed drug-drug interactions with these molecules and, more important, the role of CYP2C19 genetic polymorphism on the pharmacokinetics of and pharmacodynamic response to clopidogrel. The lack of drug interaction potential and the absence of CYP2C19 genetic effect result in a predictable response to thienopyridine antiplatelet therapy with prasugrel. Current literature shows that greater ADP-mediated IPA is associated with significantly better clinical outcomes for patients with acute coronary syndrome.

IMPORTANCE OF THE FIELD: The administration of dual antiplatelet therapy with aspirin and a thienopyridine for the prevention of thrombosis in patients with acute coronary syndrome undergoing percutaneous coronary intervention is proven to reduce mortality. The original thienopyridine, ticlopidine, has largely been displaced by clopidogrel, which has a superior adverse effect profile. Prasugrel is a new thienopyridine that has been purported to have a faster onset of activity, a lower rate of nonresponders and a greater potency than clopidogrel. AREAS COVERED IN THIS REVIEW: This review compares the metabolism of clopidogrel and prasugrel by carboxylesterases and the CYP system with emphasis on the formation of their respective active metabolites. WHAT THE READER WILL GAIN: The reader will gain an understanding of the pharmacokinetics of prasugrel and clopidogrel and how the differences in their respective metabolic pathways explain the difference in their therapeutic activity. TAKE HOME MESSAGE: The superior therapeutic profile of prasugrel is explained by the different roles of carboxylesterases in their metabolic pathways. Though prasugrel is superior to clopidogrel as a prodrug of the active P(2)Y(12) inhibitor, caution is advised because limited information is available on genetic polymorphisms or drug interactions affecting carboxylesterase metabolism.

The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing active metabolites through corresponding thiolactone metabolites. The formation rate of clopidogrel active metabolite was much lower and more variable [0.164 + or - 0.196 microl/min/mg protein, coefficient of variation (CV) = 120%] compared with the formation of prasugrel active metabolite (8.68 + or - 6.64 microl/min/mg protein, CV = 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone metabolite (2.24 + or - 1.00 microl/min/mg protein, CV = 45%) compared with the formation of prasugrel thiolactone metabolite (55.2 + or - 15.4 microl/min/mg protein, CV = 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inactive acid metabolite (approximately 90% of total metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone metabolite formed was converted to an inactive thiolactone acid metabolite. The oxidation of clopidogrel to its thiolactone metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the active metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone metabolite to inactive acid metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone metabolite, which may contribute to the poorer and more variable active metabolite formation for clopidogrel than prasugrel.

Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wild-type enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90% when the equal concentration was assayed. Given the fact that hydrolysis of oseltamivir is required for its therapeutic activity, concurrent use of both drugs would inhibit the activation of oseltamivir, thus making this antiviral agent therapeutically inactive. This is epidemiologically of significance because people who receive oseltamivir and clopidogrel simultaneously may maintain susceptibility to influenza infection or a source of spreading influenza virus if already infected.