In vitro assays confirmed the biological activity of the H4 isomer (IC50 0.12 μM in CHO-P2Y12 cells) and inactivity of H1 and H3, establishing H4 as the only clinically relevant diastereoisomer.
The active metabolite isomer H4 is the only clinically relevant diastereoisomer for documenting the pharmacokinetic profile of clopidogrel's active metabolite.
Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of ³³P-2-methylthio-ADP on P2Y₁₂-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC₅₀: CHO-P2Y₁₂: 0.12 μM; PRP: 0.97 μM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.
Roy et al. (Sat,) conducted a other in Clopidogrel-treated subjects. Clopidogrel active metabolite isomers (H1 to H4) was evaluated on In vitro biological activity and quantifiable concentrations in clinical samples. In vitro assays confirmed the biological activity of the H4 isomer (IC50 0.12 μM in CHO-P2Y12 cells) and inactivity of H1 and H3, establishing H4 as the only clinically relevant diastereoisomer.