Rougée Luc R A, Mohutsky Michael A, Bedwell David W, Ruterbories Kenneth J, Hall Stephen D
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana.
Drug Metab Dispos. 2017 Sep;45(9):1008-1018. doi: 10.1124/dmd.117.076331. Epub 2017 Jul 5.
Surrogate assays for drug metabolism and inhibition are traditionally performed in buffer systems at pH 7.4, despite evidence that hepatocyte intracellular pH is 7.0. This pH gradient can result in a pK-dependent change in intracellular/extracellular concentrations for ionizable drugs that could affect predictions of clearance and P450 inhibition. The effect of microsomal incubation pH on in vitro enzyme kinetic parameters for CYP2C9 (diclofenac, (S)-warfarin) and CYP3A4 (midazolam, dextromethorphan, testosterone) substrates, enzyme specific reversible inhibitors (amiodarone, desethylamiodarone, clozapine, nicardipine, fluconazole, fluvoxamine, itraconazole) and a mechanism-based inhibitor (amiodarone) was investigated. Intrinsic clearance through CYP2C9 significantly increased (25% and 50% for diclofenac and (S)-warfarin respectively) at intracellular pH 7.0 compared with traditional pH 7.4. The CYP3A4 substrate dextromethorphan intrinsic clearance was decreased by 320% at pH 7.0, while midazolam and testosterone remained unchanged. Reversible inhibition of CYP2C9 was less potent at pH 7.0 compared with 7.4, while CYP3A4 inhibition potency was variably affected. Maximum enzyme inactivation rate of amiodarone toward CYP2C9 and CYP3A4 decreased at pH 7.0, while the irreversible inhibition constant remained unchanged for CYP2C9, but decreased for CYP3A4 at pH 7.0. Predictions of clearance and drug-drug interactions made through physiologically based pharmacokinetic models were improved with the inclusion of predicted intracellular concentrations based at pH 7.0 and in vitro parameters determined at pH 7.0. No general conclusion on the impact of pH could be made and therefore a recommendation to change buffer pH to 7.0 cannot be made at this time. It is recommended that the appropriate hepatocyte intracellular pH 7.0 be used for in vitro determinations when in vivo predictions are made.
尽管有证据表明肝细胞内pH值为7.0，但传统上药物代谢和抑制的替代试验是在pH值为7.4的缓冲系统中进行的。这种pH梯度可能导致可电离药物的细胞内/细胞外浓度发生pK依赖性变化，从而影响清除率预测和P450抑制作用。研究了微粒体孵育pH值对CYP2C9（双氯芬酸、（S）-华法林）和CYP3A4（咪达唑仑、右美沙芬、睾酮）底物、酶特异性可逆抑制剂（胺碘酮、去乙基胺碘酮、氯氮平、尼卡地平、氟康唑、氟伏沙明、伊曲康唑）和基于机制的抑制剂（胺碘酮）的体外酶动力学参数的影响。与传统的pH值7.4相比，在细胞内pH值7.0时，通过CYP2C9的内在清除率显著增加（双氯芬酸和（S）-华法林分别增加25%和50%）。在pH值7.0时，CYP3A4底物右美沙芬的内在清除率降低了320%，而咪达唑仑和睾酮保持不变。与pH值7.4相比，在pH值7.0时CYP2C9的可逆抑制作用较弱，而CYP3A4的抑制作用强度受到不同程度的影响。在pH值7.0时，胺碘酮对CYP2C9和CYP3A4的最大酶失活率降低，而对于CYP2C9，不可逆抑制常数保持不变，但在pH值7.0时CYP3A4的不可逆抑制常数降低。通过基于生理的药代动力学模型进行的清除率预测和药物-药物相互作用预测，在纳入基于pH值7.0的预测细胞内浓度和在pH值7.0时测定的体外参数后得到了改善。目前无法就pH值的影响得出一般性结论，因此此时不能建议将缓冲液pH值改为7.0。建议在进行体内预测时，使用适当的肝细胞内pH值7.0进行体外测定。
