Vignati Luisella, Turlizzi Elisa, Monaci Sonia, Grossi Pietro, Kanter Ruben de, Monshouwer Mario
Department of Pre-Clinical Development, Nerviano Medical Sciences S.r.l., V.le Pasteur, 10, 20014, Nerviano, MI, Italy.
Toxicology. 2005 Dec 15;216(2-3):154-67. doi: 10.1016/j.tox.2005.08.003. Epub 2005 Sep 19.
Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a "detoxification system" using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.
许多药物不良反应是由细胞色素P450（CYP）介导的药物活化成反应性代谢产物所致。为了减少因代谢介导的毒性导致的药物研发失败，并提高候选药物的安全性，我们通过将激活系统（人CYP3A4）与靶细胞（HepG2细胞）相结合，开发了两种基于细胞的体外试验：在第一种方法中，我们将含有cDNA表达的CYP3A4的微粒体与HepG2细胞一起孵育；在第二种方法中，HepG2细胞被瞬时转染CYP3A4。在这两种试验系统中，发现CYP3A4催化的代谢与肝细胞中报道的高水平相当。这两种试验系统都用于研究10种已知有潜力形成比母体化合物毒性更高的代谢产物的CYP3A4底物。评估了几个毒性终点，并选择测量MTT还原和细胞内ATP水平来评估细胞活力。结果表明，与各自的对照系统相比，这两种试验系统都能够代谢测试化合物，导致毒性增加。与CYP3A4抑制剂酮康唑共同孵育证实，反应性代谢产物的形成依赖于CYP3A4。为了进一步验证这两种试验系统的功能，它们还被用作“解毒系统”，使用可被CYP3A4代谢为毒性低于其母体化合物的代谢产物的选定化合物。这些结果表明，这两种试验系统都可用于筛选代谢活化或失活，这对于预测CYP3A4代谢介导的毒性可能是一种快速且相对廉价的体外试验。
