Development of a method to predict in vivo drug metabolism and excretion from in vitro data with human hepatic tissues and/or recombinant proteins

本研究的内容可以分为两个部分。每个部分分别描述：1.用人微粒体/P-450表达系统从体外数据定量预测体内代谢通过对以前报道的数据进行动力学分析，我们确定了人微粒体体外测定的代谢活性和人体内测定的代谢活性。我们发现这两个参数之间有很好的1：1相关性，表明可以从体外数据推断体内处置。为了证实这一结论，我们还用CYP 3A 4酶代谢的YM 796进行了实验。用微粒体在体外测定动力学参数（Km和Vmax）。这些参数值进一步用于预测口服给药后的体内药物分布。在该预测中，使用弥散模型，其中考虑了非线性代谢。通过分析YM 796口服后的处置，我们发现， ...更多信息 这种药物的体内分布可以从体外数据中推断出来。此外，我们还用转染CYP 3A 4 cDNA的白血病细胞微粒体研究了YM 796的代谢，结果表明，用重组酶测定的动力学参数，经校正人微粒体中同工酶的量后，可以预测人微粒体的代谢数据。这些结果表明，可以从重组酶测定的数据预测人体中的处置。用转染了转运蛋白cDNA的哺乳动物细胞测定转运活性通过比较在分离的肝细胞中测定的转运活性沿着在转染了克隆的转运蛋白cDNA的细胞中测定的转运活性，我们确定了每种转运蛋白对肝摄取配体的贡献。此外，我们进行了attransporter（小管多特异性有机阴离子转运蛋白; cMOAT）的遗传分析，负责有机阴离子排泄到胆汁中。我们还通过制备稳定的转染子来检测克隆的cMOAT cDNA的功能。通过转染大鼠cMOAT cDNA，刺激cMOAT的非典型底物2，4-二硝基苯基-S-谷胱甘肽进入NIH/3 T3细胞分离的膜囊的ATP依赖性摄取。这些结果表明，本研究中采用的方法可能是有用的，从克隆的cDNA产物测定的活性定量预测运输。少

The content of the present study can be classified into two parts. Each part is described separately :1. Quantitative prediction of in vivo metabolism from in vitro data with human microsomes/P-450 expressing systemBy kinetically analyzing the previously reported data, we determined the metabolic activity determined with human microsomes in vitro and that determined in humans in vivo. We found a nice 1 : 1 correlation between the two parameters, suggesting that in vivo disposition can be extrapolated from in vitro data. In order to comfirm this conclusion, we also performed experiments with YM796, which is metabolized by CYP3A4 enzyme. Kinetic parameters (Km and Vmax) were determined in vitro with microsomes. These parameter values were further used in predicting the drug disposition in vivo after oral administration. In this prediction, dispersion model was used, in which the nonlinear metabolism was considered. By analyzing the disposition of YM796 after oral administration, we found … More that the in vivo disposition of this drug can be extrapolated from in vitro data. Moreover, we examined the metabolism of YM796 by using the microsomes from leukoblastoma transfected with CYP3A4 cDNA.It was indicated that the metabolic data with human microsomes can be predicted from the kineticparameters determined with the recombinant enzymes after correcting the amount of the isozymes in the human microsomes. These results suggest that the disposition in humans can be predicted from the data determined with the recombinant enzymes.2. Determination of transport activity using the mammalian cells transfected with cDNA for transportersBy comparing the transport activity determined in isolated hepatocytes along with that determined in the cells transfected with the cloned cDNA for transporters, we determined the contribution of each transporter to the hepatic uptake of ligands. Moreover, we performed the genetic analysis of atransporter (canalicular multispecific organic anion transporter ; cMOAT) responsible for the excretion of organic anions into the bile. We had also examined the function of cloned cMOAT cDNA by preparing the stable transfectant. ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione, atypical substrate for cMOAT,into membrane vesicles isolated from NIH/3T3 cells was stimulated by transfection of rat cMOAT cDNA These results suggest that the methodology employed in the present study may be useful in the quantitative prediction of transport from the activity determined with the cloned cDNA products. Less