Hormonal Regulation of Human CYP3A

描述（由申请人提供）：小肠中CYP3A4活性的个体间差异导致许多CYP3A底物药物的口服生物利用度低且多变。这种可变性似乎是由于十二指肠肠细胞中CYP3A4特异性含量的巨大差异。从治疗的角度来看，首次通过肠道提取效率的巨大差异可能导致不同的全身药物暴露和不同的药理作用，口服剂量适合“普通”患者，增加治疗失败和不良毒性的风险。肠道CYP3A表达变化的原因在很大程度上是未知的，但被认为涉及遗传和环境因素。重要的是，我们之前已经证明了维生素D最具生物活性的形式，1,25-二羟基维生素D3 (1,25(OH)2D3)，以vdr依赖的方式增强CYP3A4基因的转录，而CYP3A4反过来可以催化1,25(OH)2D3的代谢清除。本研究的总体目标是确定肠道CYP3A4在体内是否受以下顺序过程的调节：25(OH)D3-葡萄糖醛酸缀合物的形成和胆汁排泄，近端肠腔中缀合物水解为25(OH)D3，并被初级肠细胞吸收，在那里它被CYP27B1转化为1,25(OH)2D3。在肠细胞中以这种方式产生的活性激素可以调节CYP3A4和其他VDR靶基因的表达，包括钙转运蛋白TRPV6和钙结合蛋白D9k。我们将通过鉴定和表征参与人类维生素D偶联物胆汁排泄的肝脏转运体，并测试这些偶联物是否会影响培养的人类肠细胞中VDR靶基因的表达和功能，来验证这一假设。由于CYP3A4可以催化1,25(OH)2D3的氧化代谢，我们还提出，已知受体激动剂在小肠中激活hPXR可以增强肠道1,25(OH)2D3的清除，从而导致钙转运蛋白形成减少，并可能改变钙稳态的全身指标。我们将利用培养的人肝细胞、肠细胞和原代小管上皮细胞、一种新型微流体、人肠粘膜三维模型以及在健康志愿者中进行CYP3A4相互作用的体内研究来验证这一假设。阐明CYP3A依赖性药物代谢个体间差异的分子基础，可以通过更清楚地了解其他药物、环境和疾病状态如何影响肠道CYP3A底物的新候选药物的处置，从而增强制药行业开发安全有效药物的能力。此外，如果我们关于CYP3A4参与小肠内1,25(OH)2D3基因组效应负反馈控制的假设被证明是正确的，那么它可以指出相对简单的方法（例如，饮用葡萄柚汁）来预防强效hPXR激动剂对“高危”患者骨骼健康的不利影响。

DESCRIPTION (provided by applicant): Inter-individual differences in the activity of CYP3A4 in the small intestine contribute to the low and variable oral bioavailability observed for many drugs that are CYP3A substrates. This variability appears to be the result of large differences in the specific content of CYP3A4 in duodenal enterocytes. From a therapeutic perspective, large differences in first-pass intestinal extraction efficiency can lead to variable systemic drug exposure and variable pharmacological effects following oral administration of doses that are appropriate for the "average" patient, increasing the risk of therapeutic failure and adverse toxicity. The cause of variable intestinal CYP3A expression is largely unknown, but thought to involve both genetic and environmental factors. Importantly, we have shown previously that the most biologically active form of vitamin D, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), enhances transcription of the CYP3A4 gene in a VDR-dependent manner and that CYP3A4 in turn can catalyze the metabolic clearance of 1,25(OH)2D3. The overall objectives of this grant proposal are to determine whether or not intestinal CYP3A4 is regulated in vivo by the following sequential process: formation and biliary excretion of a 25(OH)D3-glucuronide conjugate, hydrolysis of the conjugate to 25(OH)D3 in the proximal intestinal lumen and its absorption into the primary enterocytes, where it is converted to 1,25(OH)2D3 by CYP27B1. Active hormone produced in the enterocyte in this manner can regulate the expression of CYP3A4 and other VDR target genes, including the calcium transport proteins TRPV6 and calbindin D9k. We will test this hypothesis by identifying and characterizing the hepatic transporters involved in the biliary excretion of vitamin D conjugates in humans and testing whether or not these conjugates can affect the expression and function of VDR target genes in cultured human enterocytes. Because CYP3A4 can catalyze the oxidative metabolism of 1,25(OH)2D3, we also propose that activation of hPXR in the small intestine by known receptor agonists enhances intestinal 1,25(OH)2D3 clearance, resulting in a decrease in the formation of calcium transporters, and a potential change in systemic indices of calcium homeostasis. We will test this hypothesis with the use of cultured human hepatocytes, enterocytes and primary tubular epithelial cells, a novel microfluidic, 3-dimensional model of the human intestinal mucosa and the conduct of an in vivo CYP3A4 interaction study in healthy volunteers. Elucidating the molecular basis of inter-individual differences in CYP3A-dependent drug metabolism could enhance the ability of the drug industry to develop safe and efficacious drugs through a clearer understanding of how other medications, the environment, and disease states might impinge on the disposition of new drug candidates that are intestinal CYP3A substrates. In addition, if our hypothesis about the participation of CYP3A4 in negative feedback control of 1,25(OH)2D3 genomic effects within the small intestine proves correct, it could point to relatively simple ways (e.g., grapefruit juice consumption) to prevent the adverse effects of potent hPXR agonists on bone health in "at-risk" patients.