Li A P, Lu C, Brent J A, Pham C, Fackett A, Ruegg C E, Silber P M
In Vitro Technologies Inc., University of Maryland Technology C., Baltimore, 21044, USA.
Chem Biol Interact. 1999 Jun 1;121(1):17-35. doi: 10.1016/s0009-2797(99)00088-5.
Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.
我们实验室对冻存的人肝细胞进行了广泛表征。通过染料排斥法测得的解冻后活力，对于来自17名供体的冻存肝细胞，范围在55%至83%之间。在长达120天的最长评估储存期内，解冻后活力和产量（每瓶活细胞数）保持稳定。冻存肝细胞（十位供体的平均值）的药物代谢酶活性相对于新鲜分离细胞的百分比为：细胞色素P450同工酶（CYP）1A2为97%，CYP2A6为78%，CYP2C9为96%，CYP2C19为86%，CYP2D6为90%，CYP3A4为164%，尿苷二磷酸葡萄糖醛酸转移酶为76%，伞形酮磺基转移酶为88%。人、大鼠、兔、狗和猴的冻存肝细胞再现了7-乙氧基香豆素（7-EC）代谢中已知的种属差异，这表明多种动物种属的冻存肝细胞在评估药物代谢种属差异方面具有实用性。利用冻存的人肝细胞开发了用于肝毒性、代谢稳定性和抑制性药物-药物相互作用的高通量筛选（HTS）测定法。以MTT代谢为终点测量，观察到已知的肝毒性化学物质他莫昔芬、氯氮平、氯化镉、双氯芬酸、胺碘酮、反苯环丙胺、早熟素II存在剂量依赖性细胞毒性，但2-硫尿嘧啶没有。在用于代谢稳定性的HTS测定法中观察到了7-EC和右美沙芬的细胞密度和时间依赖性代谢。在用于抑制性药物-药物相互作用的HTS测定法中评估了已知的CYP同工酶特异性抑制剂。发现呋拉茶碱、磺胺苯吡唑、奎尼丁和酮康唑分别是CYP1A2、CYP2C9、CYP2D6和CYP3A4的特异性抑制剂。发现反苯环丙胺和二乙基二硫代氨基甲酸盐特异性较低，对几种CYP同工酶包括CYP2A6、CYP2C9、CYP2C19和CYP2E1有抑制作用。这些结果表明，冻存的人肝细胞是评估药物代谢、毒性和抑制性药物-药物相互作用潜力的有用实验工具。
