Vaginal Drug Delivery of P-glycoprotein Substrates

P-糖蛋白底物的阴道给药

• 批准号: 6595690
• 负责人: GIOVANNI M PAULETTI
• 金额: $ 14.9万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-10 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6595690
• 关键词: P glycoprotein; biological signal transduction; biopsy; clinical research; digoxin; drug administration routes; drug metabolism; epithelium; female; human subject; menstrual cycle; messenger RNA; mucosa; multidrug resistance; northern blottings; nucleic acid sequence; pharmacokinetics; progesterone; protein structure function; technology /technique development; vagina; western blottings; young adult human (21-34)

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.

描述(由申请人提供)：我们研究的长期目标是改善药物的全身给药，这些药物由于在女性体内通过特定部位的阴道给药进行广泛的首过消除而具有有限的口服生物利用度。本研究的中心假设是孕酮调节P-糖蛋白(P-gp)在阴道粘膜中的功能性表达，导致不同的药物外排。因此，在月经周期期间，P-gp底物的最大阴道生物利用度被限制在规定的时间窗口内。Mdr1基因产物P-gp是膜外排系统的原型，它限制药物分子的跨细胞转运，具有广泛的特异性，具有战略上皮和内皮细胞膜屏障，包括胃肠道粘膜。因此，提出了三个具体的目标：(L)在体内和体外验证孕酮调节人阴道黏膜P-gp表达的假说。(2)探讨孕酮对体外培养的人阴道上皮细胞P-gp外流活性的调节作用。(3)验证孕酮在转录水平调节P-gp表达的预测。Northern印迹分析将对月经周期期间收集的人阴道粘膜活检组织以及经孕酮处理的永生化的人阴道上皮细胞中P-gp的mRNA进行定量。免疫印迹分析将确定对激素周期变化作出反应的蛋白质表达。P-gp的功能活性将通过细胞内P-gp底物地高辛的积累在体外进行评估。实验将被设计来确定孕酮引起的地高辛积累的变化是否满足P-gp介导的外流的既定标准，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/报告结构来确定mdr1基因上游区域促进孕激素信号转导的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调节中的相互作用奠定了基础。从药物传递的角度来看，这一结果可能适用于确定月经周期内有效经阴道传递P-gp底物的最佳时间窗口，以使全身生物利用度最大化。