Halladay Jason S, Wong Susan, Khojasteh S Cyrus, Grepper Susan
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
J Pharmacol Toxicol Methods. 2012 Nov-Dec;66(3):270-5. doi: 10.1016/j.vascn.2012.07.004. Epub 2012 Jul 15.
The traditional in vitro approach for assessing potential CYP induction has been to simply compare changes in CYP activities using known CYP-specific probe substrates following exposure to the test compound to that of vehicle and/or positive controls in primary cultured human hepatocytes. The objective of these current studies was to develop and implement a highly efficient 96-well CYP induction assay in which mRNA levels, protein levels, and the conventional enzyme activities of CYP1A2, CYP2B6, and CYP3A4/5 are all measured in the same well after 48 h. Cytotoxicity is also assessed in the same well after 24 and 48 h of incubation. Since enzymatic activity data alone often 'misses' CYP induction due to compounding factors, such as CYP mechanism-based inactivation, this 'all-inclusive' approach efficiently maximizes the generation of additional useful and comprehensive data. This data can more readily identify potential CYP induction liabilities in the drug discovery process and, therefore, avoid potential drug-drug interactions in the clinic.
One 96-well plate with cryopreserved human hepatocytes accommodated up to nine test compounds at three clinically relevant concentrations, positive and negative controls for CYP1A2, CYP2B6, and CYP3A4/5, and a vehicle control (0.1% DMSO) in three different lots of cryopreserved human hepatocytes. Ritonavir, a positive control for CYP3A inactivation/induction, and staurosporine, a positive control for cytotoxicity, were included. The compounds 3-methylcholanthrene (a CYP1A2 inducer), phenobarbital (a CYP2B6 inducer), and rifampicin (a CYP3A4/5 inducer) served as positive controls.
Data showed a strong correlation between the fold-increases in CYP activity, mRNA level, and protein level after incubation of the CYP isoforms with positive controls compared to the vehicle control. Ritonavir resulted in a decrease in CYP3A/5 activity, yet a concomitant increase in mRNA and protein levels of CYP3A4. Cytotoxicity was positive for staurosporine but negative for the other compounds.
An 'all-inclusive' 96-well method for identifying potential drug-drug interactions in vitro was successfully developed and implemented. This is timely, as the recent FDA draft guidance on such studies now recommends using mRNA levels as an important endpoint.
传统的体外评估潜在CYP诱导作用的方法是，在原代培养的人肝细胞中，将测试化合物作用后的已知CYP特异性探针底物的CYP活性变化，与溶剂和/或阳性对照的变化进行简单比较。当前这些研究的目的是开发并实施一种高效的96孔CYP诱导测定法，在48小时后，在同一孔中同时测量CYP1A2、CYP2B6和CYP3A4/5的mRNA水平、蛋白质水平和传统酶活性。在孵育24小时和48小时后，也在同一孔中评估细胞毒性。由于诸如基于CYP机制的失活等复合因素，仅酶活性数据常常会“遗漏”CYP诱导作用，这种“全包式”方法有效地最大化了额外有用且全面数据的产生。这些数据能够在药物发现过程中更轻松地识别潜在的CYP诱导风险，从而避免临床中潜在的药物相互作用。
一块装有冻存人肝细胞的96孔板，可容纳三种临床相关浓度的多达九种测试化合物、CYP1A2、CYP2B6和CYP3A4/5的阳性和阴性对照，以及三种不同批次冻存人肝细胞中的溶剂对照（0.1% DMSO）。包含CYP3A失活/诱导的阳性对照利托那韦和细胞毒性的阳性对照星形孢菌素。化合物3 - 甲基胆蒽（一种CYP1A2诱导剂）、苯巴比妥（一种CYP2B6诱导剂）和利福平（一种CYP3A4/5诱导剂）用作阳性对照。
数据显示，与溶剂对照相比，CYP同工型与阳性对照孵育后，CYP活性、mRNA水平和蛋白质水平的增加倍数之间存在强相关性。利托那韦导致CYP3A/5活性降低，但CYP3A4的mRNA和蛋白质水平同时升高。星形孢菌素的细胞毒性为阳性，而其他化合物为阴性。
成功开发并实施了一种用于体外识别潜在药物相互作用的“全包式”96孔方法。这很及时，因为最近FDA关于此类研究的草案指南现在建议将mRNA水平用作重要终点。
