Clopidogrel metabolites from both actions were only produced at clinically relevant concentrations in HLMs. important functions in the bioactivation of clopidogrel. loss of function alleles or *are often resistant to clopidogrel treatment (Hulot and showed that CYP1A2, 2B6 and 2C19 are involved in the first step of clopidogrel metabolism, whereas CYP3A4, 2C9, 2C19 and 2B6 are principally involved in the second step (Kazui studies did not confirm these findings, as polymorphisms responsible for loss of PON1 activity were not correlated with increased frequency of cardiovascular events in cohorts of cardiovascular patients treated with clopidogrel. Therefore, Rabbit Polyclonal to CLIC3 the involvement of PON1 in clopidogrel metabolism remains unclear (Fontana or genotype, respectively) were purchased from Tebu Bio (Offenbach, Germany). Determination of kinetic parameters of S-mephenytoin in genotyped CYP2C19 microsomes S-Mephenytoin was used as probe for the determination of CYP2C19 activity. We incubated 0.5 mg proteinmL?1HLMs or CYP2C19*HLMs with different concentrations of S-mephenytoin (0, 10, 20, 30, 50, 100, 150 and 250 M) in 0.1 M potassium phosphate buffer at pH 7.4. Mixtures were pre-incubated for 3 min at 37C before the reaction was initiated by the addition of the NADPH-generating system (1 mM NADP, 5 mM isocitrate, 5 mM MgCl2 and 1 UImL?1 isocitrate dehydrogenase in reaction buffer). After 40 min incubation at 37C, the reaction was halted with acetonitrile that contained 100 ngmL?1 OH-mephenytoin-D3 as an internal standard. After centrifugation at 10 000for 3 min at room heat, the supernatants were diluted 1:5 in the mobile phase before injection of 10 L Smilagenin diluted sample into the LC/MS/MS. Determination of kinetic parameters of clopidogrel in HLMs with and genotypes The kinetic parameters for the production of both 2-oxo-clopidogrel from clopidogrel and the clopidogrel-AM from 2-oxo-clopidogrel were evaluated in HLMs and HLMs. Clopidogrel or 2-oxo-clopidogrel at increasing concentrations (0, 0.5, 1, 5, 10, 50 and 100 M) in incubation buffer (0.1 M potassium phosphate, pH 7.4) was added to samples containing 0.5 mg proteinmL?1 microsomes with 5 mM NaF to inhibit esterases activity and 5 mM glutathione for 3 min at 37C. Then, we added the NADPH-generating system and incubated them for 30 min at 37C. The reaction was stopped and the clopidogrel-AM was stabilized by adding 30 mM BMAP in acetonitrile. After centrifugation at 10 000for 3 min, supernatants were diluted 1:5 in the mobile phase before injection of 10 L sample into the LC/MS/MS system. Inhibition of clopidogrel metabolism by PON1 and CYP isoform-specific inhibitors The metabolism of Smilagenin clopidogrel was investigated at 37C under linear conditions. We prepared 0.5 mg microsomal proteinmL?1 CYP2C19-genotyped HLMs suspensions in reaction buffer (0.1 M phosphate potassium at pH 7.4). Samples were pre-incubated for 3 min at 37C with 10 M clopidogrel or 2-oxo-clopidogrel, 5 mM NaF, 5 mM glutathione, reaction buffer and CYP-specific inhibitors. The specific CYP inhibitors (Dierks and isolated as explained previously (Deakin values 0.05 were considered statistically significant. Graphic representations of data were created using GraphPad Prism version 4.0 software (GraphPad Software Inc., La Jolla, Smilagenin CA, USA). Results Paraoxonase activity in supersomes, pooled HLMs, HLMs and HLMs PON1 activity (imply SD) was 295 28.5 UmL?1, 2.01 0.1 Umg?1, 1.99 0.04 Umg?1 and 2.0 0.04 Umg?1 in human serum, pooled HLMs, HLMs and HLMs, respectively. No PON1 activity was detectable in any of the supersomes. PON1 activity was high in serum and there was no difference in PON1 between any of the HLM groups. CYP2C19 activity assessment in and HLMs OH-mephenytoin from S-mephenytoin in HLMs was 20 occasions that in HLMs (Vmax = 1212 31.3 pmolmin?1mg?1 protein; CI95: 1147C1277 vs. Vmax = 52.7 3.85 pmolmin?1mg?1 protein; CI95: 44.7C60.7) as shown in Physique 2. These results confirm that there is a good correlation between genotype and CYP2C19 activity when S-mephenytoin is used as a probe drug. Open in a separate window Physique 2 Kinetic analysis of OH-mephenytoin formation from mephenytoin in CYP2C19-genotyped human liver microsomes. Data are mean SD of three impartial data points. Effect of the polymorphism on clopidogrel metabolism Kinetic parameters of clopidogrel or 2-oxo-clopidogrel biotransformation.