Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2).2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16) > BCRP (9) > OCT1 (8) > OATP1B1 (5) > MRP3 (2).3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2.4. Structure–activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

1. 肝脏代谢或转运的调控可能会导致药物清除率升高，进而削弱药物的药效或安全性。本研究采用冻存人肝细胞，通过实时定量聚合酶链反应（qPCR）技术检测309种化合物对细胞色素P450 1A2（CYP1A2）、细胞色素P450 2B6（CYP2B6）及细胞色素P450 3A4（CYP3A4）的活性与mRNA表达量的影响，同时检测6种肝脏转运蛋白的mRNA表达量，分别为OATP1B1（SCLO1B1）、OCT1（SLC22A1）、MDR1（ABCB1）、MRP2（ABCC2）、MRP3（ABCC3）及BCRP（ABCG2）。
2. 研究结果显示，6%的化合物可诱导CYP1A2活性升高（增幅达1.5倍）；30%的化合物可诱导CYP2B6，23%的化合物可诱导CYP3A4。通过qPCR数据分别鉴定出16%、33%及32%的CYP1A2、CYP2B6及CYP3A4诱导剂。受27种化合物诱导的转运蛋白为MRP2，其次依次为MDR1（16种）、BCRP（9种）、OCT1（8种）、OATP1B1（5种）及MRP3（2种）。
3. 共有53种化合物可使CYP3A4的mRNA表达量下调（降幅≥2倍），下调CYP2B6的化合物有10种，下调OCT1的有6种，下调BCRP的有4种，下调CYP1A2与OATP1B1的各有2种，下调MDR1与MRP2的各有1种。
4. 构效关系（Structure-activity relationship）分析显示，CYP2B6与CYP3A4诱导剂均为大体积亲脂性分子，其重原子数量更多、氢键供体数量更少。最后，本研究提出了一种在药物发现阶段检测CYP诱导剂的策略。