Thiol Metabolite Research Articles

The pharmacokinetic and pharmacodynamic interaction of clopidogrel and cilostazol in relation to CYP2C19 and CYP3A5 genotypes.

The primary objective of the present study was to evaluate the pharmacokinetic and pharmacodynamic interactions between clopidogrel and cilostazol in relation to the CYP2C19 and CYP3A5 genotypes. In a randomized, three-way crossover study, 27 healthy subjects were administered clopidogrel (300mg), cilostazol (100mg) or clopidogrel+cilostazol orally. Plasma concentrations of clopidogrel, cilostazol and their active metabolites (clopidogrel thiol metabolite, 3,4-dehydrocilostazol and 4″-trans-hydroxycilostazol), and adenosine diphosphate-induced platelet aggregation were measured for pharmacokinetic and pharmacodynamic assessment. The area under the plasma concentration-time curve (AUC) of the active thiol metabolite of clopidogrel was highest in the CYP2C19 extensive metabolizers (EM) and lowest in the poor metabolizers (PM). Cilostazol decreased the thiol metabolite AUC by 29% in the CYP3A5*1/*3 genotype [geometric mean ratio (GMR) 0.71; 90% confidence interval (CI) 0.58, 0.86; P=0.020] but not in the CYP3A5*3/*3 genotype (GMR 0.93; 90% CI 0.80, 1.10; P=0.446). Known effects of the CYP2C19 and CYP3A5 genotypes on the exposure of cilostazol and its metabolites were observed but there was no significant difference in the AUC of cilostazol and 3,4-dehydrocilostazol between cilostazol and clopidogrel+cilostazol. The inhibition of platelet aggregation from 4h to 24h (IPA4-24 ) following the administration of clopidogrel alone was highest in the CYP2C19 EM genotype and lowest in the CYP2C19 PM genotype (59.05±18.95 vs. 36.74±13.26, P=0.023). However, the IPA of the CYP2C19 PM following co-administration of clopidogrel and cilostazol was comparable with that of the CYP2C19 EM and intermediate metabolizers (IM) only in CYP3A5*3/*3 subjects. The additive antiplatelet effect of cilostazol plus clopidogrel is maximized in subjects with both the CYP2C19 PM and CYP3A5*3/*3 genotypes because of a lack of change of clopidogrel thiol metabolite exposure in CYP3A5*3/*3 as well as the highest cilostazol IPA in CYP2C19 PM and CYP3A5*3/*3 subjects.

Differential neurogenic effects of casein-derived opioid peptides on neuronal stem cells: implications for redox-based epigenetic changes

Food-derived peptides, such as β-casomorphin BCM7, have potential to cross the gastrointestinal tract and blood–brain barrier and are associated with neurological disorders and neurodevelopmental disorders. We previously established a novel mechanism through which BCM7 affects the antioxidant levels in neuronal cells leading to inflammatory consequences. In the current study, we elucidated the effects of casein-derived peptides on neuronal development by using the neurogenesis of neural stem cells (NSCs) as an experimental model. First, the transient changes in intracellular thiol metabolites during NSC differentiation (neurogenesis) were investigated. Next, the neurogenic effects of food-derived opioid peptides were measured, along with changes in intracellular thiol metabolites, redox status and global DNA methylation levels. We observed that the neurogenesis of NSCs was promoted by human BCM7 to a greater extent, followed by A2-derived BCM9 in contrast to bovine BCM7, which induced increased astrocyte formation. The effect was most apparent when human BCM7 was administered for 1day starting on 3days postplating, consistent with immunocytochemistry. Furthermore, neurogenic changes regulated by bovine BCM7 and morphine were associated with an increase in the glutathione/glutathione disulfide ratio and a decrease in the S-adenosylmethionine/S-adenosylhomocysteine ratio, indicative of changes in the redox and the methylation states. Finally, bovine BCM7 and morphine decreased DNA methylation in differentiating NSCs. In conclusion, these results suggest that food-derived opioid peptides and morphine regulated neurogenesis and differentiation of NSCs through changes in the redox state and epigenetic regulation.

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel.

Clopidogrel, a thienopyridine antiplatelet prodrug, is metabolized by oxidation to 2-oxo-clopidogrel, followed by conversion to its pharmacologically active thiol metabolite. After oral administration of clopidogrel to humans, two thiol isomers (H3 and H4) are observed in plasma, with similar concentrations, and only H4 is active in humans. In this work, the mechanism of stereoselectivity in the formation and S-methylation of H3 and H4 was investigated in vitro. The two diastereomers of 2-oxo-clopidogrel were epimerized rapidly at physiologic pH. The intrinsic clearance (CLint) for H3 formation from 2-oxo-clopidogrel in human liver microsomes (HLMs) was 3.1-fold higher than that for H4 formation, indicating stereoselective metabolism. Kinetic studies using expressed enzymes demonstrated that the contributions of CYP2B6, CYP2C19, and CYP3A4 to the formation of H4 from 2-oxo-clopidogrel were 18.5%, 26.1%, and 53.5%, respectively. The CLint ratios of H3 formation to H4 formation from 2-oxo-clopidogrel by CYP2B6, CYP2C19, and CYP3A4 were 2.2, 1.0, and 1.7, respectively. In HLMs, H3 and H4 were further S-methylated, and the S-methylation was inhibited by 2,3-dichloromethyl benzylamine, indicating the involvement of thiol S-methyltransferase. The CLint value for the S-methylation of H3 in HLMs was 98.1-fold higher than that for H4. The stereoselective formation of H3 from 2-oxo-clopidogrel and the stereoselective S-methylation of H3 may lead to the similar exposure levels of H3 and H4 previously reported in humans. The epimerization of 2-oxo-clopidogrel and the variations of thiol S-methyltransferase may affect the exposure to H4 in humans.

Bioactivation of clopidogrel and prasugrel: factors determining the stereochemistry of the thiol metabolite double bond.

The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a further oxidation with the formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and trans thiols were formed upon the metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH), whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.

A quantitative metabolomics peek into planarian regeneration.

The fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate a whole animal from a small piece of tissue. It is one of the best model systems to address the basic mechanisms essential for regeneration. Here, we are interested in studying the roles of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated the differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. The levels of thiols such as cysteine and GSH are 651 and 1107 ng mg(-1) in planarians. Among the nucleotides, the level of cGMP is the lowest (0.03 ng mg(-1)) and the level of AMP is the highest (187 ng mg(-1)) in both of the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, the thiol (cysteine and GSH) levels are well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in intact planarians, the level of cGMP is also very low in regenerating planarians. Surprisingly, the levels of amines and thiols in head regenerating blastemas are ∼3 times higher compared to those for tail regenerating blastemas. Thus our results strongly indicate the potential roles of amines, thiols and nucleotides in planarian regeneration.

Quantitation of thiol metabolites from mammalian cells using fluorous tagging and HILIC-MS

Oxidative stress diminishes reduced thiols by decreasing their production or by forming disulfide bonds.

Impact of common ABCB1 polymorphism on pharmacokinetics and pharmacodynamics of clopidogrel and its metabolites.

The reasons of clopidogrel (CLP) resistance are still unclear. The response to CLP may be influenced by both genetic and non-genetic factors. Among genetic factors, common polymorphisms in the gene coding glycoprotein-P (P-gp, MDR1 and ABCB1) are considered as potential determinants of the efficacy of CLP treatment. The aim of this study was to evaluate the influence of ABCB1 3435C>T genetic polymorphism on the pharmacokinetics and pharmacodynamics of CLP and its metabolites: diastereoisomers of thiol metabolite (the inactive H3 and the active H4) and inactive carboxylic derivative. The study group included 42 patients undergoing elective coronary angiography and percutaneous coronary intervention. The plasma concentrations of CLP and its metabolites were measured by a validated HPLC-MS/MS method. Whole-blood aggregation was determined with Multiplate analyzer. For evaluation of ABCB1 3435C>T polymorphism, PCR-RFLP method was applied. It was found that Exposition to the unchanged CLP, measured by AUC0-t of the drug, was significantly lower (P=0·012) in TT homozygotes comparing to that observed in CC and CT genotypes, although no correlation was found between platelet aggregation and ABCB1 genetic polymorphism. Our findings show that the presence of 3435C>T allele has an impact on CLP pharmacokinetics but not on the drug pharmacodynamics. Therefore, the 3435C>T genotype may not be the primary determinant influencing the pharmacokinetics of the active H4 metabolite and antiplatelet effect of the drug.

Abstract 17314: Impact of Mild Therapeutic Hypothermia on Platelet Inhibition in Acute Coronary Syndrome

Background: Mild therapeutic hypothermia (MTH) improves outcome for patients with acute coronary syndrome (ACS) after cardiac arrest (CA). Previous data point to an interaction between hypothermia and drug metabolism, thus potentially impacting on platelet function under antiplatelet therapy. Purpose: The aim of the study is to determine clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with mild hypothermia (33°C, n=12) compared with ACS patients (troponin positive) with normal body temperature (n=14). Methods: Platelet function was measured by light transmittance aggregometry (LTA), multiple electrode platelet aggregometry (MEA) and VASP phosphorylation analysis before, 2h, 4h and 24h after administration of a 600 mg clopidogrel loading dose. Furthermore, plasma concentrations of clopidogrel, the active thiol metabolite and the inactive carboxyl metabolite were determined. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. Mild hypothermia was carried out according to current guidelines for 24 hours at a target temperature of 33°C. Results: Plasma concentration of clopidogrel and metabolites were lower in the MTH group after 2h and 4h, respectively (all p<0.005). All platelet function tests showed an attenuated response to clopidogrel with respect to baseline platelet activity in the MTH group. This was significant for VASP analysis and LTA (p<0.05). Moreover, there was no difference in genotype and platelet function determined ex vivo with 33°C and 37°C, respectively. Conclusion: Inhibition of platelet function is decreased under MTH, presumably due to a diminished clopidogrel absorption and metabolization. Thus, these patients might have a higher risk for cardiovascular events despite antiplatelet therapy.

Effects of cytochrome P450 2C19 and paraoxonase 1 polymorphisms on antiplatelet response to clopidogrel therapy in patients with coronary artery disease.

Clopidogrel is an antiplatelet prodrug that is recommended to reduce the risk of recurrent thrombosis in coronary artery disease (CAD) patients. Paraoxonase 1 (PON1) is suggested to be a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to active thiol metabolite with inconsistent results. Here, we sought to determine the associations of CYP2C19 and PON1 gene polymorphisms with clopidogrel response and their role in ADP-induced platelet aggregation. Clopidogrel response and platelet aggregation were determined using Multiplate aggregometer in 211 patients with established CAD who received 75 mg clopidogrel and 75–325 mg aspirin daily for at least 14 days. Polymorphisms in CYP2C19 and PON1 were genotyped and tested for association with clopidogrel resistance. Linkage disequilibrium (LD) and their epistatic interaction effects on ADP-induced platelet aggregation were analysed. The prevalence of clopidogrel resistance in this population was approximately 33.2% (n = 70). The frequencies of CYP2C19*2 and *3 were significantly higher in non-responder than those in responders. After adjusting for established risk factors, CYP2C19*2 and *3 alleles independently increased the risk of clopidogrel resistance with adjusted ORs 2.94 (95%CI, 1.65–5.26; p<0.001) and 11.26 (95%CI, 2.47–51.41; p = 0.002, respectively). Patients with *2 or *3 allele and combined with smoking, diabetes and increased platelet count had markedly increased risk of clopidogrel resistance. No association was observed between PON1 Q192R and clopidogrel resistance (adjusted OR = 1.13, 95%CI, 0.70–1.82; p = 0.622). Significantly higher platelet aggregation values were found in CYP2C19*2 and *3 patients when compared with *1/*1 allele carriers (p = 1.98×10−6). For PON1 Q192R genotypes, aggregation values were similar across all genotype groups (p = 0.359). There was no evidence of gene-gene interaction or LD between CYP2C19 and PON1 polymorphisms on ADP-induced platelet aggregation. Our findings indicated that only CYP2C19*2 and *3 alleles had an influence on clopidogrel resistance. The risk of clopidogrel resistance increased further with smoking, diabetes, and increased platelet count.

The influence of diabetic status on the pharmacokinetics of clopidogrel and its metabolites in patients suffered from cardiovascular diseases

Aim. A significant percentage of individuals treated with an anti-platelet agent clopidogrel do not receive the expected therapeutic effect. Clopidogrel resistance is even more prevalent in patients with type 2 diabetes mellitus (DM). An extensive investigation on pharmacokinetics of clopidogrel and its metabolites in patients with type 2 DM suffering from cardiovascular diseases were performed following an administration of 75 mg of the drug.Material and methods. Plasma concentrations of clopidogrel, its carboxylic metabolite (CLPM) and diastereoisomers of a thiol metabolite (the inactive H3 and the active H4) were determined by a validated HPLC-MS/MS method. The pharmacokinetic parameters of the analytes in diabetic (n = 16) and non-diabetic (n = 28) patients were compared and correlated with platelet aggregation. Results. DM patients exhibited a slightly higher Cmax of clopidogrel (2.34 ± 2.29 ng/mL) compared with non-diabetic group (1.82 ± 1.86 ng/mL), whereas plasma levels of clopidogrel metabolites were lower in DM than in non-DM patients (2339 ± 989 ng/mL vs. 2662 ± 2090 ng/mL, 4.64 ± 4.79 ng/mL vs. 5.42 ± 4.55 ng/mL and 6.42 ± 4.80 ng/mL vs. 7.44 ± 7.18 ng/mL, respectively for CLPM, H3 and H4). A significant correlation was found between platelet aggregation and the Cmax of the active H4 metabolite in non-diabetic patients. Conclusions. Pharmacokinetic parameters of clopidogrel, CLPM, H3 and H4 isomers in patients with DM did not differ significantly from those determined in non-diabetic group. Moreover, the antiplatelet response to clopidogrel therapy measured by ADP-stimulated platelet aggregation was similar in both groups of patients.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry.

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4 ± 11.5 and 10.1 ± 4.0 nmol/mg protein, respectively.

The influence of genetic polymorphism of Cyp2c19 isoenzyme on the pharmacokinetics of clopidogrel and its metabolites in patients with cardiovascular diseases

An extensive investigation on pharmacokinetics of clopidogrel and its metabolites as well as pharmacodynamics of the drug was performed in patients with cardiovascular disease carrying various alleles coding CYP2C19 isoenzyme. The influence of non-genetic factors on the clopidogrel response was also studied. Plasma concentrations of clopidogrel, its carboxylic metabolite, and diastereoisomers of a thiol metabolite (the inactive H3 and the active H4) following an administration of 75 mg of the drug were determined in three groups of patients divided with respect to their CYP2C19 genotype: ultrametabolizers, extensive metabolizers, and intermediate metabolizers. The mean peak plasma concentration of H4 in intermediate metabolizers was 3.1- and 2.8-fold lower than that of ultrametabolizers (P = 0.055) and extensive metabolizers (P = 0.026), respectively. The mean H4 area under the curve (AUC0-24 h ) for intermediate metabolizers were significantly lower than that for ultrametabolizers (P = 0.046). Intermediate metabolizers exhibited a significantly higher platelet aggregation than ultrametabolizers and extensive metabolizers (P = 0.035). A multivariate analysis showed that the effect of CYP2C19*2 allele on an ADP-induced platelet aggregation was better pronounced in the presence of non-genetic risk factors (P = 0.008). We concluded that the CYP2C19*2 genotype is the primary determinant of the antiplatelet response to clopidogrel therapy.

Effect of alcohol on clopidogrel metabolism: Identification of a new alcohol‐dependent metabolic pathway (1141.14)

The majority of orally administrated clopidogrel, a platelet aggregation inhibitor, is hydrolyzed by human carboxyesterase‐1 (CES1) to the inactive clopidogrel acid metabolite with the remainder metabolized by CYP450 enzymes into the inactive intermediate, 2‐oxo‐clopidogrel, that is further oxidized to the active thiol metabolite. The objective of this study was to determine the effect of alcohol, a known CES1 inhibitor, on the metabolism of clopidogrel. Clopidogrel metabolism was studied in human recombinant CES1, human liver S9 fractions (HLS9), and in plasma‐carboxylesterase knockout mice. Liquid chromatography/triple quadrupole mass spectrometry (LC‐MS/MS) was utilized for the quantification of clopidogrel and its metabolites. Alcohol‐derived metabolites were identified by product ion scan using LC‐MS/MS. The addition of alcohol resulted in the formation of the transesterified clopidogrel metabolite, ethyl‐clopidogrel in CES1 and HLS9 incubations. Similar to clopidogrel, ethyl‐clopidogrel was metabolized to ethyl‐2‐oxo‐clopidogrel followed by the subsequent oxidation to thiol metabolites in HLS9. Co‐administration of alcohol (3 g/kg) and clopidogrel (25 mg/kg) to plasma‐carboxylesterase knockout mice resulted in a reduced systemic exposure to clopidogrel acid (control AUC0‐8h 39 µg·h/mL versus alcohol AUC0‐8h 20 µg·h/mL) and the formation of transesterified metabolites. Alcohol significantly inhibits the hydrolysis of clopidogrel to clopidogrel acid, and produces transesterified metabolites of clopidogrel, 2‐oxo‐clopidogrel, and the active metabolite.Grant Funding Source: Supported by National Institutes of Health National Institute of General Medical Sciences

Highlights from recent advances in antiplatelet pharmacogenomics.

Pers. Med. (2014) 11(2), 135–138 ISSN 1741-0541 CYP2C19-guided antiplatelet therapy in coronary artery disease Coronary artery disease (CAD) is the leading cause of morbidity and mortality worldwide [1]. Clinical practice guidelines currently recommend dual antiplatelet therapy with aspirin and a P2Y12 inhibitor in patients experiencing an acute coronary syndrome, with or without a percutaneous coronary intervention (PCI), and in stable CAD patients undergoing an elective PCI [2]. Clopidogrel, a prodrug that requires hepatic biotransformation into its active thiol metabolite via CYP450 isoenzyme CYP2C19, remains the most commonly prescribed P2Y12 inhibitor. Importantly, CYP2C19 is highly polymorphic and includes common loss-offunction alleles (*2 or *3) and a common gain-of-function (GOF) allele (*17) in multiple populations [3]. It is now clear that individuals carrying one or two loss-of-function alleles exhibit a significantly lower capacity to metabolize clopidogrel into its active metabolite and inhibit platelet activation, compared with wild-type individuals, and are, therefore, at significantly higher risk of adverse cardiovascular events following acute coronary syndrome or PCI [4]. These data led the US FDA to place a black-box warning in the clopidogrel prescribing information in March 2010 to alert clinicians of these risks [5]. Moreover, carriers of the GOF *17 allele, which enhances CYP2C19 transcription, have been reported to exhibit enhanced inhibition of platelet function and increased bleeding risk; however, conflicting results in the literature exist regarding the association between the *17 allele and bleeding risk [6]. By contrast, CYP2C19 genotype does not influence the pharmacokinetics, pharmacodynamics or clinical response to the more recently approved P2Y12 inhibitors, prasugrel and ticagrelor, which are more expensive than clopidogrel and associated with higher bleeding risk. The rapid accumulation of these data has generated considerable debate surrounding the following question: should CYP2C19 genotyping be used to guide selection of a P2Y12 inhibitor following PCI? Expert insight into this question, including the Clinical Pharmacogenetics Implementation Consortium [6] and the American College of Cardiology Foundation/American Heart Association [2], has collectively concluded that this approach should be considered in certain circumstances and more data are needed. Three notable gaps in knowledge have served as a barrier to more widespread implementation of CYP2C19-guided antiplatelet therapy in practice. First, it remains unclear whether clinically relevant associations between CYP2C19 genotype and clinical outcomes exist in clopidogrel-treated stable CAD patients undergoing elective PCI. Second, it remains unclear whether a clinically relevant association between the GOF CYP2C19*17 allele, clopidogrel use and bleeding risk truly exists. The third, and most important, gap in knowledge remains to be the lack of prospective, randomized clinical studies evaluating the impact of a CYP2C19-guided dual antiplatelet therapy strategy on clinical outcomes in patients undergoing PCI. The three studies highlighted below collectively offer new insight into each of these gaps in evidence regarding the potential clinical utility of CYP2C19 genotype-directed antiplatelet therapy in patients with CAD, part of Research Highlights

Abstract T MP108: A Concentration Response Relationship Between the Active Metabolite of Clopidogrel and its Antiplatelet Activity

Background: Variability in antiplatelet response to clopidogrel is reported to be due, in part, to the reduced ability to metabolize clopidogrel to its active form. This can be a result of a genetic difference in metabolizer status or to drug interactions which can cause inhibition of either the CYP2C19 or CYP3A4 P450 isoenzymes. Although up to this point it has remained unclear whether there is a concentration dependent relationship between clopidogrel's active thiol metabolite and its antiplatelet effects and whether there is a threshold concentration of the active metabolite (AM) needed to produce an adequate antiplatelet effect, this 3-arm, complete crossover study demonstrates a statistical relationship between AM concentrations and antiplatelet response. Methods: Fourteen healthy male volunteers took clopidogrel doses of 25 mg, 50 mg, and 75 mg. Participants were excluded for tobacco, illicit substance, and alcohol use. AM concentrations were measured using liquid chromatography-mass spectrometry over a 4 hour period and an area under the curve (AUC) was calculated. Results: The median baseline whole blood platelet aggregation value was 8 Ω (IQR=7-8.5 Ω) and responsiveness to clopidogrel was defined as &lt;6 Ω and a ≥60% decrease from baseline. Using a receiver operating characteristic (ROC) curve the optimal AUC 0-4 was found to be 784ng*min/mL. Sensitivity was 92.0% (95% CI: 48.0-100.0%) and specificity was 71.4% (95% CI: 46.4-85.7%). Area under the ROC curve was 84.0% (95% CI: 73.0-95.0%, p=1.867e-5) demonstrating statistical validity of the estimates of the AM threshold concentration needed to inhibit platelet aggregation. A Bayesian breakpoint analysis found the optimal threshold AUC to be in a range from 732-882 ng*min/mL. Conclusions: These findings could make it possible for clinicians to immediately tailor antiplatelet therapy to individual patients by measuring AM levels after the first dose of clopidogrel, conceivably preventing a secondary event at a time when risk is the highest. Additionally, research is ongoing to explore whether disease states commonly seen in stroke patients, such as diabetes, change the amount of the AM produced or whether they may actually result in a shift of the concentration response curve.

Antiplatelet Effect of Clopidogrel Can Be Reduced by Calcium-Channel Blockers

PurposeClopidogrel is metabolized by the hepatic cytochrome P450 (CYP) system into its active thiol metabolite. CYP3A4 is involved in the metabolism of both clopidogrel and dihydropyridine calcium channel blockers (CCBs). A few reports have suggested an inhibitory interaction between CCBs and clopidogrel. Accordingly, the aim of this study was to determine the effect of CCBs on the antiplatelet activity of clopidogrel by serial P2Y12 reaction unit (PRU) measurements.Materials and MethodsWe assessed changes in antiplatelet activity in patients receiving both clopidogrel and CCBs for at least 2 months prior to enrollment in the study. The antiplatelet activity of clopidogrel was measured by VerifyNow P2Y12 assay in the same patient while medicated with CCBs and at 8 weeks after discontinuation of CCBs. After discontinuation of the CCBs, angiotensin receptor blockers were newly administered to the patients or dosed up for control of blood pressure.ResultsThirty patients finished this study. PRU significantly decreased after discontinuation of CCBs (238.1±74.1 vs. 215.0±69.3; p=0.001). Of the 11 patients with high post-treatment platelet reactivity to clopidogrel (PRU≥275), PRU decreased in nine patients, decreasing below the cut-off value in seven of these nine patients after 8 weeks. Decrease in PRU was not related to CYP2C19 genotype.ConclusionCCBs inhibit the antiplatelet activity of clopidogrel.

The effect of CYP2C19 genotype on the time course of platelet aggregation inhibition after clopidogrel administration

We evaluated the effect of CYP2C19 genotype over time on the antiplatelet response of clopidogrel in healthy subjects. Seventy subjects enrolled for a pharmacodynamic study and 22 subjects for a pharmacokinetic and pharmacodynamic study took 300 mg clopidogrel on the first day and 75 mg once daily for six consecutive days. The subjects with CYP2C19 poor metabolizers (PM, N = 22) and intermediate metabolizers (IM, N = 37) had significantly delayed time to inhibition of platelet aggregation (IPA) compared with CYP2C19 extensive metabolizers (EM, N = 33) (12 vs. 9 vs. 2 hours as median Tmax , P < .05) after a 300 mg of clopidogrel. During maintenance doses of clopidogrel, IPA values of only CYP2C19 PM subjects were gradually decreased from 30.0 ± 21.9% on day 2 to 23.7 ± 16.6% on day 8 (P > .05 for time effect; P < .05 for time and genotype interaction effect). CYP2C19 PM had decreased Cmax and AUC of thiol metabolite compared with CYP2C19 EM (0.42- and 0.37-fold on day 1, P < .01; 0.39- and 0.34-fold on day 7, P < .01, respectively). Delayed time to reach maximal IPA as well as decreased IPA may influence the increased risk of the acute cardiac events in CYP2C19 PM and IM.

Multiple Cytochrome P450 Isoforms Are Involved in the Generation of a Pharmacologically Active Thiol Metabolite, whereas Paraoxonase 1 and Carboxylesterase 1 Catalyze the Formation of a Thiol Metabolite Isomer from Ticlopidine

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.

Clinical Pharmacokinetics of Clopidogrel and Its Metabolites in Patients with Cardiovascular Diseases

Background and ObjectiveApproximately 5–40 % of patients treated with clopidogrel do not display an adequate antiplatelet response. Clopidogrel resistance may be caused by insufficient drug absorption or impaired metabolic activation of the drug. The aim of this study was to evaluate the pharmacokinetics of clopidogrel and its metabolites in plasma samples from patients treated with high and low doses of clopidogrel, to obtain a possible explanation for antiplatelet resistance.MethodsThe study included patients receiving either a single 300 mg loading dose of clopidogrel (n = 17) or a 75 mg dose (n = 45) for at least 7 days before sample collection. The concentrations of clopidogrel and its metabolites—the inactive H3 and the pharmacologically active H4 isomers of the thiol metabolite and the inactive carboxylic acid metabolite—in plasma samples (stabilized with 2-bromo-3′-methoxyacetophenone) from three patients after 300 mg and from 41 patients after 75 mg of the drug were determined using a validated high-performance liquid chromatography method with tandem mass spectrometry. The non-stabilized samples from the remaining patients were analysed using a validated capillary electrophoresis method. The calculated concentrations were used to determine the pharmacokinetic parameters of the analytes. The pharmacodynamic response to clopidogrel treatment, expressed as adenosine diphosphate-induced platelet aggregation, was measured using a Multiplate analyser.ResultsThe pharmacokinetic parameter values for the H3 and H4 isomers determined in the studied group of patients treated with clopidogrel 75 mg (maximum plasma concentration [C max] 5.29 ± 5.54 and 7.13 ± 6.32 ng/mL for H3 and H4, respectively; area under the plasma concentration-time curve from time zero to time t [AUCt] 7.37 ± 6.71 and 11.30 ± 9.58 ng·h/mL for H3 and H4, respectively) were lower than those reported in healthy volunteers, according to the literature data. Platelet aggregation measured with a Multiplate analyser ranged between 37 and 747 AU·min. A significant correlation was found between the C max of the active H4 isomer and platelet aggregation (p = 0.025).ConclusionThe C max of the active H4 isomer and platelet aggregation measured by the Multiplate analyser may serve as markers of the patient response to clopidogrel therapy.

Harnessing cerium oxide nanoparticles to protect normal tissue from radiation damage

At the onset of radiation exposure, free radicals are formed through ionizing reactions that are then capable of destroying normal tissues. While cells release a level of protective molecules, such as glutathione and metallothionine, they are not capable of blocking all damage, thus resulting in the death of normal tissues and therefore, we must continue to develop strategies to protect normal tissues from radiation-induced damage. One such strategy is the development of radiation protectors. Several compounds have been described, but Amifostine (Ethyol), whose active free thiol metabolite WR-1065 has been shown to prevent both radiation-induced cell death and mutagenesis while facilitating the repair of normal cells remains the only agent currently in clinical use. Major limitations to the clinical use of Amifostine are its short half-life, daily dosing requirements, toxicity based on route of administration, and its cost. Recent studies have shown the effects of engineered cerium oxide (CeO 2 ) nanoparticles for protection against radiation-induced damage in a variety of tissue types. The role of nanoparticles as radioprotectants is a cutting-edge development in decades of scientific interest regarding the protection of normal cells and tissues from radiation. The chemistry of engineered CeO 2 nanoparticles supports a potential role as a biological free radical scavenger or antioxidant. The work presented in this review article will address the effectiveness of CeO 2 nanoparticles in radioprotection in a variety of cells and in animal models during radiation exposure which will encourage the development of innovative and new approaches to radiation protection, using nanotechnology.