sanofi-aventis R&D, Drug Disposition, Disposition Safety and Animal Research, Montpellier, France.
Thromb Haemost. 2011 Apr;105(4):696-705. doi: 10.1160/TH10-09-0582. Epub 2011 Feb 8.
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.
氯吡格雷的药代动力学分析受到多种活性代谢物异构体（H1 至 H4）及其在血液中不稳定性的阻碍。我们试图重新测试四种单个活性代谢物异构体在体外的药效学活性，最终目的是确定体内负责氯吡格雷活性的异构体。通过测量在富含血小板的血浆（PRP）中表达 P2Y₁₂ 的中国仓鼠卵巢（CHO）细胞和人血小板上[³³P]-2-甲基硫代-ADP 的结合来评估体外活性。开发了一种立体选择性方法，该方法使用反相超高效液相色谱（UHPLC）和串联质谱（MS）来测量 H1-H4 的稳定 3'-甲氧基苯乙酮（MP）衍生物的单个浓度。该新方法用于分析在 I 期临床试验中接受氯吡格雷治疗的受试者的血浆样本。体外结合测定证实了先前观察到的 H4 的生物学活性（IC₅₀：CHO-P2Y₁₂：0.12 μM；PRP：0.97 μM）和 H3 的无活性，并且表明 H1 也是无活性的。此外，H2 在体外的生物学活性约为 H4 的一半。UHPLC 条件和 MS 碰撞参数的优化允许四种衍生的活性代谢物异构体（MP-H1 至 MP-H4）的分辨率和检测。立体选择性测定法经过广泛验证，在 0.5-250 ng/ml 的浓度范围内具有准确性和精密度。仅可在获得的临床样品中定量测定 MP-H3 和 MP-H4。根据体外药效学数据和发现的浓度，活性代谢物异构体 H4 是唯一具有临床相关性的非对映异构体，用于记录氯吡格雷活性代谢物的药代动力学特征。
