Evaluation and Optimization of a Microcavity Plate-Based Human Hepatocyte Spheroid Model for Predicting Clearance of Slowly Metabolized Drug Candidates.

IF 4.4 3区 医学 Q1 PHARMACOLOGY & PHARMACY
David A Kukla, David G Belair, David M Stresser
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引用次数: 0

Abstract

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days, achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning Elplasia microcavity 96-well microplates enable the culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared with microsomes, hepatocyte suspensions, two-dimensional-plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted intrinsic clearance (CLint) and blood clearance (CLb) within threefold for 53% [9/17; average absolute fold error (AAFE), 3.9] and 82% (14/17; AAFE, 2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE, 4.0) and CLb (14/17; AAFE, 2.1) without the need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 μM itraconazole was within 25% of observed values for 6 of 8 compounds, with 5 of 8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. SIGNIFICANCE STATEMENT: Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance, which is improved with the consideration of mass transport within the spheroid. Incubations with 3 μM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.

评估和优化基于微腔板的人肝细胞球体模型,以预测缓慢代谢候选药物的清除率。
体外清除率测定是药物发现中的常规方法,用于预测体内清除率,但低代谢周转化合物通常难以评估。肝细胞球体可以培养数天,以实现更高的药物周转率,但由于每孔细胞数量的限制而受到阻碍。Corning® Elplasia® 微腔 96 孔微孔板每孔可培养 79 个肝细胞球。本研究评估并优化了微腔球形细胞的特性(大小、肝细胞功能、寿命、培养技术),以进行清除率测定,然后将其与微粒体、肝细胞悬液、二维电镀肝细胞和每孔培养一个的大孔球形细胞进行比较。微腔球体中的酶活性较高,肝细胞浓度也较高,因此所有 17 种测试化合物的周转率均可测量,而其他模型的药物周转率较低。使用线性回归校正模型,微腔球体还能分别预测 53% (9/17;AAFE=3.9)和 82% (14/17;AAFE=2.6)化合物的 CLint 和 CLb 在 3 倍以内。另一种方法结合了机理建模,考虑了球体内的质量传输(渗透性和扩散性),无需经验缩放因子即可提高对 CLint(12/17;AAFE=4.0)和 CLb(14/17;AAFE=2.1)的预测能力。使用 3 μM 伊曲康唑估计细胞色素 P450 3A4 代谢的药物比例(fm,CYP3A4),6/8 种化合物在观察值的 25% 以内,5/8 种化合物在 10% 以内。总之,微腔板中的球形培养物能够测试和预测低代谢率化合物的清除率和 fm,CYP3A4,是对传统体外清除率测定的重要补充。意义说明 在超低附着微腔中培养多个球形体,可以在底物耗竭测定中准确定量代谢稳定的化合物,克服了单个培养球形体的局限性。反过来,这也允许对内在清除率进行可靠的估计,而考虑到球体内的质量传输,这种估计会得到改善。用 3 µM 的伊曲康唑进行培养,可以评估 CYP3A4 参与肝脏清除的情况。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.50
自引率
12.80%
发文量
128
审稿时长
3 months
期刊介绍: An important reference for all pharmacology and toxicology departments, DMD is also a valuable resource for medicinal chemists involved in drug design and biochemists with an interest in drug metabolism, expression of drug metabolizing enzymes, and regulation of drug metabolizing enzyme gene expression. Articles provide experimental results from in vitro and in vivo systems that bring you significant and original information on metabolism and disposition of endogenous and exogenous compounds, including pharmacologic agents and environmental chemicals.
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