Cellular Efflux and Metabolism of Protease Inhibitors

蛋白酶抑制剂的细胞流出和代谢

• 批准号: 6496490
• 负责人: Ashim K. Mitra
• 金额: $ 20.3万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6496490
• 关键词: P glycoprotein; antiAIDS agent; biological transport; chemical structure function; cytochrome P450; drug classification; drug metabolism; drug screening /evaluation; gastrointestinal drug absorption; intermolecular interaction; mathematical model; model design /development; multidrug resistance; oral administration; physical model; polymerase chain reaction; protease inhibitor; tissue /cell culture; western blottings

DESCRIPTION (Provided by the applicant): Cytochrome P450 3A4 (CYP3A4), most abundantly presently both in the liver and upper intestinal enterocytes, limits the systemic bioavailability of anti-HIV agents. P-glycoprotein (P-gp), the MDR-1 gene product is also known to reduce the oral bioavailability of protease inhibitors. Moreover the existence of "sanctuary" sites of HIV-1 may potentially endanger the efficacy of highly active antiretroviral therapy (HAART). High cellular expression of P-gp in brain capillary endothelial cells, P-gp and CYP3A4 in mature enterocytes and alveolar cells and their similar substrate specificity suggest that the function of these proteins may be complementary and may form a coordinated intestinal, blood-brain and pulmonary barrier. The purpose of this study is to classify all anti-AIDS drugs, currently used clinically, under a new proposed Absorption-Metabolism Classification System (AMCS). Under AMCS, four classes of drugs, A, B, C and D, will be proposed based on their specificity towards P-gp and/or CYP3A4. We propose to investigate the simultaneous P-gp mediated efflux and CYP3A4 mediated metabolism of the above mentioned 14 anti-AIDS compounds across l alpha, 2,5-di-OH vitamin D3 treated Caco-2 cell monolayers expressing high levels of P-gp and CYP3A4. Moreover, in some experiments, isolated hepatocytes containing liver CYP3A, representing first pass liver metabolism, wil1 also be included. Under AMCS we hypothesize that apparent order of oral bioavailability will be A > BEC > D. Our proposed classification system may prove to be an important tool in screening anti-AIDS compounds for their oral absorption potential. According to this hypothesis Class A compounds should not interact with any other class. There should be no interaction between compounds of class B and C, whereas Class B and C compounds are expected to interact with Class D compounds. Also, maximum interaction is anticipated within Class D compounds compared to interaction with Class B and Class C compound. Our proposed theoretical model may predict, a priori, the extent of drug absorption and more importantly drug interactions among four WCS classes. Therefore, the specific aims of this project are: 1) to develop an in vitro Caco-2 cell culture model expressing high levels of P-gp and CYP3A4 to a priori predict oral absorption potential and drug interactions among the four AMCS class drugs, to utilize the model in predicting first pass metabolism using human hepatocytes. Specific inhibitors of P-gp and CYP3A4 will be used to demonstrate the involvement of each of these proteins. In addition, the expression of these proteins will be determined by Western Blot and RT PCR. 2) to develop a quantitative model for oral drug absorption and interactions based on mathematical expression of passive influx, saturable efflux, and saturable CYP3A4 metabolism and to correlate model predicted effluxes with experimentally determined efflux values. This theoretical model will also predict the fluxes of each of the A, B, C, and D classes of drugs. The theoretical model will be first validated using model compound in each class: Tolbutamide - Class-A, Rhodamine 123 - Class-B, Carbamazepine - Class-C, and Cortisol - Class-D. 3) to study the transport and metabolism across two other in vitro cell culture models, bovine brain microvessels endothelial cell (BBMEC) and Calu-3 monolayers representing blood-brain and alveolar cells respectively. The brain parenchymal and alveolar lining - two sanctuary sites that are 'protected' from optimal antiretroviral drug access. 4) to study the effect of protein binding on the influx and efflux of model compounds, and anti-HIV drugs.

描述（申请人提供）：细胞色素P450 3A4（CYP3A4），大多数 目前在肝脏和上肠肠上皮细胞中大量限制 抗HIV药物的全身生物利用度。 P-糖蛋白（P-GP）， 还已知MDR-1基因产物可降低蛋白酶的口服生物利用度 抑制剂。此外，HIV-1的“庇护所”五月的存在 潜在地危害高度活性抗逆转录病毒疗法的功效 （Haart）。 P-gp在脑毛细管内皮细胞中的高细胞表达， 成熟的肠细胞和牙槽细胞中的P-gp和CYP3A4及其相似 底物特异性表明这些蛋白质的功能可能是 互补，可能形成协调的肠道，血脑和肺部 障碍。这项研究的目的是对所有抗AIDS药物进行分类， 目前在临床上，在新提出的吸收代谢下使用 分类系统（AMC）。在AMC下，四类药物A，B，C和D， 将根据其对P-gp和/或CYP3A4的特异性提出。我们 建议研究同时p-gp介导的外排和CYP3A4 上述介导的代谢，遍布L Alpha，2,5-Di-OH维生素D3处理过的Caco-2细胞单层，表达高 P-gp和CYP3A4的水平。此外，在某些实验中，孤立的肝细胞 含有肝CYP3A，代表第一通过肝代谢，Wil1也是 包括。在AMC下，我们假设口服生物利用度的明显顺序 将是一个> bec>D。我们提出的分类系统可能被证明是 筛选抗AID的重要工具，用于口服吸收 潜在的。根据这个假设类别A的化合物不应相互作用 与任何其他班级。班级化合物之间不应相互作用 B和C，而B类和C级化合物有望与D类相互作用 化合物。另外，预计在D类化合物中将有最大的相互作用 与与B类和C类化合物的相互作用相比。我们提出的 理论模型可以预测，先验，药物吸收程度和更多 重要的是四个WCS类之间的药物相互作用。因此，具体 该项目的目的是： 1）开发一种体外CACO-2细胞培养模型，表达高水平的 P-gp和CYP3A4先验预测口服吸收潜力和药物 四种AMCS类药物之间的相互作用，以利用该模型 使用人肝细胞预测首次通过代谢。特定抑制剂 P-gp和CYP3A4将用于证明每一种 蛋白质。另外，这些蛋白质的表达将由 蛋白质印迹和RT PCR。 2）开发一种用于口服药物吸收和相互作用的定量模型 基于被动流入，饱和流出和的数学表达 可饱和的CYP3A4代谢，并将模型相关联预测外排与 实验确定的外排值。这个理论模型也将 预测A，B，C和D类药物的每一种的通量。这 理论模型将首先使用每个类中的模型化合物验证： TOLBUTAMIDE- A级，Rhodamine 123 -B类，Carbamazepine -C级C，和Class -C和 皮质醇-D类。 3）研究其他两个体外细胞培养的运输和代谢 模型，牛脑微血管内皮细胞（BBMEC）和CALU-3 分别代表血脑和牙槽细胞的单层。大脑 实质和肺泡衬里 - 两个“保护”的保护区 最佳抗逆转录病毒药物进入。 4）研究蛋白质结合对模型涌入和外流的影响 化合物和抗HIV药物。