Centre for Applied Pharmacokinetic Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK.
Drug Metab Dispos. 2012 Aug;40(8):1596-602. doi: 10.1124/dmd.112.045732. Epub 2012 May 16.
Drug depletion-time profiles in isolated hepatocytes, as well as microsomes, have become a standard method of assessing hepatic metabolic clearance in vitro. There is a previously described adaptation of the depletion approach to allow determination of hepatic uptake by transporters in addition to metabolism (Drug Metab Dispos 35:859-865, 2007). Dual incubations are performed where one set of incubations undergo conventional methodology, whereas for the second set, cells and media are separated for determination of drug loss from the media. The utility of this dual incubation approach has been assessed using eight drugs (atorvastatin, clarithromycin, erythromycin, fexofenadine, pitavastatin, repaglinide, rosuvastatin, and saquinavir) with a range of active uptake, passive permeability, cell binding, and metabolic characteristics. Four of these compounds (fexofenadine, rosuvastatin, pitavastatin, and atorvastatin) show a biphasic time profile when assessing drug loss from media indicative of hepatic uptake before elimination within the hepatocyte, which is distinct from the time profile in a conventional incubation, and show higher clearances. The four other compounds (clarithromycin, saquinavir, erythromycin, and repaglinide) show identical depletion-time profiles (and clearances) in both sets of incubations. Whether or not the biphasic nature (and higher clearance) is evident, indicating transporter activity for a particular drug, appears to be dependent on its passive permeability. Using the parameter K(pu) to reflect the relative importance of hepatic transporters versus passive diffusion, a value of 10 was identified as a cutoff for whether the biphasic nature was evident; those compounds in excess of 10 show this characteristic clearly. There appears to be no relationship between the presence of the biphasic nature and any other parameter, including cellular binding, extent of metabolism, or the magnitude of active uptake.
在分离的肝细胞和微粒体中，药物耗尽时间曲线已成为评估体外肝代谢清除率的标准方法。先前已经描述了一种耗尽方法的适应性，除了代谢之外，还可以确定转运蛋白对药物摄取的作用（Drug Metab Dispos 35:859-865, 2007）。进行双孵育，一组孵育采用常规方法，而对于第二组，将细胞和培养基分离，以确定药物从培养基中的损失。使用八种药物（阿托伐他汀、克拉霉素、红霉素、非索非那定、匹伐他汀、瑞格列奈、罗苏伐他汀和沙奎那韦）评估了这种双孵育方法的实用性，这些药物具有不同的主动摄取、被动渗透性、细胞结合和代谢特征。其中四种化合物（非索非那定、罗苏伐他汀、匹伐他汀和阿托伐他汀）在评估从培养基中损失药物时显示出双相时间曲线，表明在肝细胞内消除之前存在肝摄取，这与传统孵育中的时间曲线不同，并且显示出更高的清除率。其他四种化合物（克拉霉素、沙奎那韦、红霉素和瑞格列奈）在两组孵育中显示出相同的耗尽时间曲线（和清除率）。是否存在双相性质（和更高的清除率），表明特定药物的转运体活性，似乎取决于其被动渗透性。使用参数 K(pu) 来反映肝转运体与被动扩散的相对重要性，发现 10 是一个界限值，用于确定双相性质是否明显；那些超过 10 的化合物明显显示出这种特征。双相性质的存在与任何其他参数之间似乎没有关系，包括细胞结合、代谢程度或主动摄取的大小。
