Substrate depletion approach for determining in vitro metabolic clearance: Time dependencies in hepatocyte and microsomal incubations
Substrate depletion approach for determining in vitro metabolic clearance: Time dependencies in hepatocyte and microsomal incubations
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DOI：
10.1124/dmd.104.000125
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发表时间：
2004-09-01
影响因子：
3.9
通讯作者：
Houston, JB
中科院分区：
文献类型：
--
作者：
Jones, HM;Houston, JB
底物耗尽法是一种常用的方法，用于测量体外固有清除率（CLint）。然而，这些研究中使用的孵育条件可能会有所不同，其后果尚未得到系统的探讨。用大鼠微粒体和肝细胞对八种苯二氮卓类药物进行初始底物耗尽培养：阿普唑仑、氯巴唑仑、氯硝西泮、氯二氮平、地西泮、氟硝西泮、咪达唑仑和三唑仑。随后对体内CLint（范围从3到200 ml/min）和肝清除率（CLH）（范围从0.3到15 ml/min）的预测表明，该方法的总体预测能力与传统代谢物形成方法相似。对底物消耗谱和CLint估计的更详细的研究表明，所使用的酶的浓度特别重要。肝细胞在所有细胞密度下，三唑安定、氯硝西泮和地西泮的代谢均呈单指数；然而，对于微粒体，两相耗损是明显的，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（孵育1小时后活性为8%至65%）的酶损失更为明显。对于氯硝西泮（一种低清除率底物），这些双相特征可以通过酶活性的丧失来解释。为了确保在使用底物耗尽法时准确预测体内CLint和CLH，根据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。
The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CLint). 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 CLint (ranging from 3 to 200 ml/min) and hepatic clearance (CLH) (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 CLint 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 CLint and CLH 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.