Rysz Marta, Schäfer Anima M, Paloumpis Nikolaos, Kinzi Jonny, Brecht Karin, Seibert Isabell, Schmidlin Seraina, In-Albon Katja, Ricklin Daniel, Meyer Zu Schwabedissen Henriette E
Biopharmacy, Department of Pharmaceutical Sciences (M.R., A.M.S., N.P., J.K., K.B., I.S., S.S., K.I.-A., H.E.M.Z.S.) and Molecular Pharmacy, Department of Pharmaceutical Sciences (D.R.), University of Basel, Basel, Switzerland.
Biopharmacy, Department of Pharmaceutical Sciences (M.R., A.M.S., N.P., J.K., K.B., I.S., S.S., K.I.-A., H.E.M.Z.S.) and Molecular Pharmacy, Department of Pharmaceutical Sciences (D.R.), University of Basel, Basel, Switzerland
J Pharmacol Exp Ther. 2024 Mar 15;389(1):87-95. doi: 10.1124/jpet.123.001884.
The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 ()] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of knockin and knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used and rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing and rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.
有机阴离子转运多肽（OATP）2B1[基因：溶质载体有机阴离子转运体家族成员2B1（）]是一种摄取转运体，可促进其底物在细胞内的积累。敲入和敲除大鼠的比较显示，人源化动物中rCYP3A1的表达更高。我们假设OATP2B1的人源化不仅影响细胞摄取，还影响代谢活性。为了进一步研究这一假设，我们使用了和大鼠以及OATP2B1和rCYP3A1的底物厄洛替尼（其代谢为OSI-420）进行体内和体外实验。单次给药厄洛替尼1小时后，敲入大鼠的厄洛替尼血清水平显著降低，但代谢物浓度或OSI-420/厄洛替尼比值未观察到变化。比较和大鼠的肝脏组织水平也得到了类似的结果。使用睾酮对从厄洛替尼处理的动物中分离的肝微粒体进行体外rCYP3A活性表征，结果显示敲入大鼠的活性更高。当评估从未经处理的动物中分离的微粒体将厄洛替尼代谢为OSI-420的情况时，观察到相反的结果。后者与通过蛋白质印迹分析在大鼠肝脏裂解物和从未经处理的大鼠中分离的肝微粒体中观察到的较高rCYP3A1蛋白量形成对比。总之，OATP2B1人源化的大鼠肝脏中rCYP3A1表达更高，厄洛替尼血清水平降低，但尽管分离的肝微粒体中OSI-420形成减少，OSI-420/厄洛替尼比值未发生变化。有必要对CYP3A特异性底物进行研究，以评估人源化是否不仅影响rCYP3A1表达，还影响体内代谢活性。重要声明：大鼠的有机阴离子转运多肽（OATP）2B1人源化增加了肝脏中rCYP3A1的表达和活性。使用OATP2B1/CYP3A底物厄洛替尼评估所产生的表型，我们观察到厄洛替尼血清和肝脏浓度较低，但对肝脏/血清比值没有影响。此外，比较人源化大鼠和敲除大鼠时，OSI-420/厄洛替尼比值没有差异，这表明OSI-420不适用于监测rCYP3A1表达的差异，大鼠肝脏微粒体的体外实验数据也支持这一点。
