Jones Hannah M, Houston J Brian
Centre for Applied Pharmaceutical Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, UK.
Drug Metab Dispos. 2004 Sep;32(9):973-82. doi: 10.1124/dmd.104.000125.
The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL(int)). However, the incubation conditions used in these studies can vary, the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam, and triazolam. Subsequent predictions of in vivo CL(int) (ranging from 3 to 200 ml/min) and hepatic clearance (CL(H)) (ranging from 0.3 to 15 ml/min) demonstrated that the general predictive ability of this approach was similar to that of the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL(int) estimates indicated that the concentration of enzyme used is of particular importance. The metabolism of triazolam, clonazepam, and diazepam was monoexponential at all cell densities using hepatocytes; however, with microsomes, biphasic depletion was apparent, particularly at higher microsomal protein concentrations (2-5 mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8 to 65% activity after a 1-h incubation) compared with the hepatocyte system (approximately 100% activity after a 1-h incubation). For clonazepam (a low clearance substrate), these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL(int) and CL(H) when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.
底物消耗法是一种用于测量体外内在清除率（CL(int)）的常用方法。然而，这些研究中使用的孵育条件可能会有所不同，但其后果尚未得到系统的探索。使用大鼠微粒体和肝细胞对8种苯二氮䓬类药物进行了初始底物消耗孵育：阿普唑仑、氯巴占、氯硝西泮、氯氮卓、地西泮、氟硝西泮、咪达唑仑和三唑仑。随后对体内CL(int)（范围为3至200 ml/min）和肝清除率（CL(H)）（范围为0.3至15 ml/min）的预测表明，该方法的总体预测能力与传统的代谢物形成法相似。对底物消耗曲线和CL(int)估计值的更详细研究表明，所用酶的浓度尤为重要。使用肝细胞时，在所有细胞密度下，三唑仑、氯硝西泮和地西泮的代谢均为单指数型；然而，对于微粒体，双相消耗很明显，特别是在较高的微粒体蛋白浓度（2 - 5 mg/ml）下。酶活性研究表明，与肝细胞系统（孵育1小时后活性约为100%）相比，微粒体系统中的酶损失更为明显（孵育1小时后活性范围为8%至65%）。对于氯硝西泮（一种低清除率底物），这些双相曲线可以用酶活性的损失来解释。为了在使用底物消耗法时确保准确预测体内CL(int)和CL(H)，基于这类药物获得的结果，建议尽可能使用低酶浓度和短孵育时间。
