Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes
Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes
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DOI：
10.1124/dmd.115.067173
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发表时间：
2016-02-01
影响因子：
3.9
通讯作者：
Hariparsad, Niresh
中科院分区：
文献类型：
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作者：
Dixit, Vaishali;Moore, Amanda;Hariparsad, Niresh
肝细胞的长期共培养模型是研究药物转运、清除和肝毒性的有前途的工具。在这份报告中，我们比较了药物处置基因的基础表达和原型诱导剂（利福平，苯巴比妥，苯妥英钠）在肝细胞二维单一培养和长期共培养模型（HepatoPac）的诱导反应。研究中使用的所有诱导剂均增加了HepatoPac培养物中CYP 3A 4、CYP 2B 6和CYP 2C酶的表达和活性。与单一培养物相比，共培养物模型显示出一致且更高的CYP 2C酶诱导。HepatoPac中利福平对CYP 3A 4和CYP 2C 9的EC 50比单一培养物低10倍。根据临床药物相互作用研究计算的利福平EC 50与HepatoPac培养物中观察到的EC 50相关性良好。由于HepatoPac培养物的长期稳定性，我们能够使用mRNA和功能活性的消耗动力学直接测量CYP 3A 4和CYP 2B 6的半衰期（t（1/2））。CYP 3A 4 mRNA的t（1/2）为26 h，功能蛋白的t（1/2）为49 h。CYP 2B 6的t（1/2）为38小时（mRNA）和68小时（活性），比CYP 3A 4长，显示了这两种蛋白质的差异转换。据我们所知，这是第一项报告人肝细胞中CYP 2B 6转换率的研究。本文提供的数据表明，HepatoPac培养物有潜力用于长期培养，以模拟复杂的临床情况。
Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t(1/2)) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t(1/2) for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t(1/2) of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios.