Wong Susan, Doshi Utkarsh, Vuong Peter, Liu Ning, Tay Suzanne, Le Hoa, Kosaka Mika, Kenny Jane R, Li Albert P, Yan Zhengyin
Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA, 94080, United States.
In vitro ADMET Laboratories, Columbia, MD 21045, United States.
Drug Metab Lett. 2018;12(1):3-13. doi: 10.2174/1872312812666171213114422.
A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes.
It was found that, for CYP3A4/5 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a known CES2 substrate, the measured apparent clearance was higher in hepatocytes, but formation of 4-aminobenzoic acid, a CE2-specific metabolite, was more pronounced in enterocytes, suggesting that CE2 is more active in enterocytes. Salbutamol, a SULT1A3 substrate, showed little turnover in both enterocytes and hepatocytes, and more abundant sulfate conjugate was detected in enterocytes, indicating higher SULT activity in enterocytes than hepatocytes. As expected, ketoconazole inhibited CYP3A4/5-mediated metabolite formation in enterocytes for midazolam, amprenavir and loperamide, suggesting that cryopreserved enterocytes may be useful in determining intestinal CYP3A inhibition parameters. Interestingly, elacridar, a P-gp inhibitor, suppressed metabolite formation in enterocytes for loperamide, a substrate of CYP3A4 and P-gp, suggesting that enterocytes in suspension do not have active P-gp efflux functions, and the suppression of metabolism in enterocytes is probably caused by inhibition of CYP3A4/5 by elacridar.
Our results suggest that pooled cryopreserved human enterocytes, specifically the MetMax™ cryopreserved human enterocytes, represent a valuable in vitro model for assessing first-pass clearance and potential drug interactions in human intestine.
分离和冷冻保存技术的最新进展已使市售的原代人肠上皮细胞得以应用，这些细胞表达多种药物代谢酶（DMEs）和转运蛋白。本研究的主要目的是进一步评估混合冷冻保存的肠上皮细胞，特别是MetMax™冷冻保存的人肠上皮细胞（体外ADMET实验室）作为评估肠道清除率的体外模型相对于肝细胞的实用性。
研究发现，对于咪达唑仑、安普那韦和洛哌丁胺等CYP3A4/5底物，与肝细胞相比，肠上皮细胞中的体外代谢清除率通常较低，这与该酶在肠道和肝脏中的相对丰度一致。相比之下，雷洛昔芬（一种替代UGT活性底物）在肠上皮细胞中的周转率比肝细胞高3倍,这可能归因于人类肝脏和肠道中各个UGT的差异表达。对于已知的CES2底物普鲁卡因，肝细胞中测得的表观清除率较高，但在肠上皮细胞中，CE2特异性代谢物4-氨基苯甲酸的形成更为明显，这表明CE2在肠上皮细胞中更具活性。沙丁胺醇是一种SULT1A3底物，在肠上皮细胞和肝细胞中的周转率都很低，并且在肠上皮细胞中检测到更丰富的硫酸酯缀合物，表明肠上皮细胞中的SULT活性高于肝细胞。正如预期的那样,酮康唑抑制了咪达唑仑、安普那韦和洛哌丁胺在肠上皮细胞中CYP3A4/5介导的代谢物形成，这表明冷冻保存的肠上皮细胞可能有助于确定肠道CYP3A抑制参数,有趣的是，P-糖蛋白抑制剂艾拉司群抑制了洛哌丁胺（一种CYP3A4和P-糖蛋白的底物）在肠上皮细胞中的代谢物形成，这表明悬浮的肠上皮细胞不具有活跃的P-糖蛋白外排功能，并且肠上皮细胞中代谢抑制可能是由艾拉司群对CYP3A4/5 的抑制引起的。
我们的结果表明，混合冷冻保存的人肠上皮细胞，特别是MetMax™冷冻保存的人肠上皮细胞,是评估人肠道首过清除率和潜在药物相互作用的有价值的体外模型。
