Vaginal Drug Delivery of P-glycoprotein Substrates

描述（由申请人提供）：我们研究的长期目标是改善口服生物利用度有限的药物的全身递送，这是由于女性通过特定部位的阴道给药进行广泛的首次通过消除而导致的。本研究的中心假设是黄体酮调节阴道粘膜p -糖蛋白（P-gp）的功能表达，导致可变的药物外排。因此，P-gp底物的最大阴道生物利用度限制在月经周期的一个确定的时间窗口内。MDR1基因产物P-gp是膜外排系统的原型，该系统限制药物分子在战略性上皮和内皮膜屏障（包括胃肠道粘膜）的跨细胞转运，具有广泛的特异性。因此，本文提出了三个具体目的：(1)在体内和体外验证黄体酮调节人阴道粘膜P-gp表达的假设。(2)确定体外人阴道上皮细胞P-gp外排活性是否受黄体酮调节。(3)验证黄体酮在转录水平调控P-gp表达的预测。Northern blot分析将对月经周期采集的人阴道粘膜活检组织和黄体酮处理的人阴道上皮细胞中的P-gp mRNA进行定量分析。蛋白表达对周期性激素变化的反应将通过免疫印迹分析来确定。P-gp的功能活性将在体外通过细胞内P-gp底物地高辛的积累来评估。将设计实验来确定黄体酮诱导的地高辛积累变化是否符合p- gp介导外排的既定标准，包括方向性、对能量和底物浓度的依赖性以及对选择性抑制的敏感性。最后，将准备启动子/报告子构建体，以确定MDR1基因上游区域促进黄体酮信号传导的DNA序列。本研究的意义在于为进一步阐明女性生殖激素在阴道P-gp调节中的相互作用机制奠定了基础。从给药角度来看，该结果可能适用于确定月经周期中P-gp底物阴道有效给药的最佳时间窗口，以最大限度地提高全身生物利用度。

DESCRIPTION (provided by applicant): The long-term goal of our research is to improve systemic delivery of drugs with limited oral bioavailability due to extensive first-pass elimination by means of site-specific vaginal administration in the female. The central hypothesis of the present proposal is that progesterone regulates functional expression of P-glycoprotein (P-gp) in the vaginal mucosa leading to variable drug efflux. As a consequence, maximum vaginal bioavailability of P-gp substrates is restricted to a defined time window during the menstrual cycle. The MDR1 gene product P-gp is a prototype of membrane efflux systems that limit transcellular transport of drug molecules with broad specificity at strategic epithelial and endothelial membrane barriers, including the gastrointestinal mucosa. Thus, there are three specific aims proposed: (l) To test the hypothesis that progesterone regulates P-gp expression in human vaginal mucosa in vivo and in vitro. (2) To determine whether P-gp efflux activity in human vaginal epithelial cells in vitro is regulated by progesterone. (3) To test the prediction that progesterone regulates P-gp expression at the transcriptional level. Northern blot analysis will be conducted to quantify mRNA of P-gp in biopsies of human vaginal mucosa collected during the menstrual cycle as well as progesterone-treated immortalized human vaginal epithelial cells. Protein expression in response to cyclic hormonal changes will be determined by immunoblot analysis. Functional activity of P-gp will be assessed in vitro using intracellular accumulation of the P-gp substrate digoxin. Experiments will be designed to determine whether progesterone-induced changes in digoxin accumulation satisfy established criteria for P-gp-mediated efflux, including directionality, dependency on energy and substrate concentration, and sensitivity to selective inhibition. Finally, promoter/reporter constructs will be prepared to determine DNA sequences in the upstream region of the MDR1 gene that facilitate progesterone signaling. The significance of this research is that it will set the stage for future mechanistic studies to elucidate the interplay of female reproductive hormones in the regulation of vaginal P-gp. From the drug delivery point of view, the results may be applicable to determine an optimum time window during the menstrual cycle for effective vaginal delivery of P-gp substrates in order to maximize systemic bioavailability.