Zhang J George, Ho Thuy, Callendrello Alanna L, Clark Robert J, Santone Elizabeth A, Kinsman Sarah, Xiao Deqing, Fox Lisa G, Einolf Heidi J, Stresser David M
Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.)
Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, Massachusetts (J.G.Z., T.H., A.L.C., R.J.C., E.A.S., S.K., D.X., L.G.F., D.M.S.); and Novartis Institutes for Biomedical Research, East Hanover, New Jersey (H.J.E.).
Drug Metab Dispos. 2014 Sep;42(9):1379-91. doi: 10.1124/dmd.114.058602. Epub 2014 Jun 12.
Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, Cmax/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6β-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R(2) and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6β-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model.
细胞色素P450（P450）诱导作用在药物研发中通常被视为一项不利因素。我们采用基于校准曲线的方法，评估了诱导参数R3（一个表示肝脏中P450诱导量的术语，以0到1之间的比率表示）、相对诱导评分、Cmax/EC50以及曲线下面积（AUC）/F2（使剂量反应曲线基线浓度增加2倍的浓度），这些参数源自体外CYP3A4 mRNA浓度反应曲线和酶活性数据，以此作为体内CYP3A4诱导潜力的预测指标。用来自三名供体的冻存人肝细胞铺板，并用20种受试化合物进行处理，其中包括几种CYP3A4的临床诱导剂和非诱导剂。经过2天的处理后，分别通过实时逆转录聚合酶链反应和液相色谱 - 串联质谱分析来测定CYP3A4 mRNA水平和睾酮6β - 羟化酶活性。我们的结果表明，人体内咪达唑仑AUC变化程度与从CYP3A4 mRNA和酶活性计算出的各种参数之间存在强烈且具有预测性的关系。总体而言，与非咪达唑仑体内探针呈现的关系并不理想。一般来说，当使用未结合而非总血浆Cmax来计算诱导参数时，模型拟合效果更好，这表现为更高的R(2)、更低的均方根误差（RMSE）和几何平均倍数误差。对于咪达唑仑，美国食品药品监督管理局指南建议的R3截止值0.9能够有效区分强诱导剂，但在对中等强度或弱诱导剂进行分类时效果较差。本研究支持使用从体外mRNA诱导反应曲线生成的校准曲线来预测人体中CYP3A4的诱导潜力。需要注意的是，此处评估的大多数化合物并非酶活性的强抑制剂，在该检测模型中，睾酮6β - 羟化酶活性也被证明是CYP3A4诱导潜力的有力预测指标。
