An improved method for specific and quantitative determination of the clopidogrel active metabolite isomers in human plasma
An improved method for specific and quantitative determination of the clopidogrel active metabolite isomers in human plasma
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DOI：
10.1160/th10-09-0582
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发表时间：
2011-04-01
影响因子：
6.7
通讯作者：
Sultan, Eric
中科院分区：
文献类型：
--
作者：
Tuffal, Gilles;Roy, Sebastien;Sultan, Eric
氯吡格雷的药代动力学分析因存在多种活性代谢物异构体（H1至H4）及其在血液中的不稳定性而受到阻碍。我们试图在体外重新测试四种活性代谢物异构体的药效学活性，最终目的是确定体内氯吡格雷活性的异构体。通过测定[P-33]-2-甲硫基-ADP与表达P2 Y（12）的中国仓鼠卵巢（CHO）细胞和富血小板血浆（PRP）中人血小板的结合，评价体外活性。开发了一种使用反相超高效液相色谱（UHPLC）和串联质谱（MS）的立体选择性方法来测量H1-H4的稳定的3 '-甲氧基苯乙酮（MP）衍生物的个体浓度。新方法用于分析来自参加I期临床试验的氯吡格雷治疗受试者的血浆样品，体外结合试验证实了先前观察到的H4的生物活性（IC 50：CHO-P2 Y（12）：0.12 μ M; PRP：0.97 μ M）和H3的无活性，并证明H1也无活性。此外，与H4相比，H2在体外表现出约一半的生物活性。优化UHPLC条件和MS碰撞参数，可分离和检测4种衍生化活性代谢物异构体（MP-H1至MP-H4）。对立体选择性测定进行了广泛验证，在0.5-250 ng/ml浓度范围内具有准确度和精密度。在真实临床样本中，仅MP-H3和MP-H4可定量。根据体外药效学数据和实测浓度，活性代谢产物异构体H4是唯一具有临床意义的非对映异构体，可用于记录氯吡格雷活性代谢产物的药代动力学特征。
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 [P-33]-2-methyl-thio-ADP on P2Y(12)-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 trialin vitro binding assays confirmed the previously observed biological activity of H4 (IC50: CHO-P2Y(12): 0.12 mu M; PRP: 0.97 mu M) 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.