AIDS THERAPEUTICS & BLOOD-BRAIN BARRIER AZT DRUG EFFLUX

艾滋病治疗

• 批准号: 6392722
• 负责人: WILLIAM M PARDRIDGE
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6392722
• 关键词: AIDS /HIV neuropathy; AIDS therapy; Xenopus oocyte; blood brain barrier; brain circulation; capillary; complementary DNA; drug design /synthesis /production; drug vehicle; electron microscopy; expression cloning; genetic library; immunocytochemistry; laboratory rabbit; membrane transport proteins; messenger RNA; northern blottings; nucleic acid sequence; reverse transcriptase inhibitors; transport inhibitor; vascular endothelium permeability; zidovudine

The goal of this research is to develop new brain drug delivery strategies for azidothymidine (AZT) and other nucleoside reverse transcriptase inhibitors (NRTI) so that the clinical potential of these drugs is enhanced for the treatment of the cerebral component of acquired immune deficiency syndrome (AIDS). Nearly all current drugs in clinical practice for the treatment of AIDS (NRTI, non-NRTI, and HIV protease inhibitors) are not transported from blood in brain in pharmacologically significant amounts owing to the active efflux of these drugs across the blood-brain barrier (BBB). The clinical potential of these drugs could be enhanced for the treatment of cerebral AIDS by the administration of co-drugs, wherein the co-drugs are inhibitors of the BBB active efflux systems. The development of co-drugs is straightforward for HIV protease inhibitors, because these drugs are substrates for a known efflux transporter, p-glycoprotein. However, the development of co-drugs for the NRTI's cannot be achieved presently because the putative active efflux systems for AZT and the other NRTI's has not been defined at the molecular level. The purpose of the proposed studies is to clone, sequence, and express a full length cDNA encoding the AZT active efflux transporter protein localized at the BBB. The availability of the cDNA will allow for the expression of the AZT active efflux transporter in a defined format and this will allow for the subsequent identification of drugs that are inhibitors of the BBB AZT efflux transporter. The following specific aims are proposed: (1) expression cloning of the AZT efflux cDNA using the frog oocyte expression cloning system and RNA derived from a bovine brain capillary cDNA library; (2) DNA sequencing of the cloned cDNA and DNA sequence analysis; (3) expression of the cDNA in COS cells followed by measurements of the Michaelis-Menten Kinetics of radiolabeled AZT transport into these cells; (4) functional analysis of the BBB AZT efflux transporter mRNA and protein using Northern blotting, immunocytochemistry, and electron microscopy of brain and isolated brain capillaries.

这项研究的目标是开发叠氮胸苷（AZT）和其他核苷逆转录酶抑制剂（NRTI）的新脑部药物递送策略，从而增强这些药物治疗获得性免疫缺陷综合征（AIDS）脑部部分的临床潜力。 目前临床实践中用于治疗艾滋病的几乎所有药物（NRTI、非 NRTI 和 HIV 蛋白酶抑制剂）都不会以药理学上显着的量从脑部血液中转运，因为这些药物会主动穿过血脑屏障 (BBB)。 通过施用联合药物可以增强这些药物治疗脑艾滋病的临床潜力，其中联合药物是BBB主动流出系统的抑制剂。 对于 HIV 蛋白酶抑制剂来说，联合药物的开发非常简单，因为这些药物是已知的外排转运蛋白 p-糖蛋白的底物。然而，目前无法实现 NRTI 联合药物的开发，因为 AZT 和其他 NRTI 的推定活性外排系统尚未在分子水平上定义。 拟议研究的目的是克隆、测序和表达编码位于 BBB 的 AZT 活性外排转运蛋白的全长 cDNA。 cDNA 的可用性将允许 AZT 活性外排转运蛋白以定义的形式表达，这将允许随后鉴定作为 BBB AZT 外排转运蛋白抑制剂的药物。 提出以下具体目标：（1）利用青蛙卵母细胞表达克隆系统和源自牛脑毛细血管cDNA文库的RNA表达AZT外排cDNA； (2) 对克隆的cDNA进行DNA测序和DNA序列分析； (3) COS 细胞中 cDNA 的表达，然后测量放射性标记的 AZT 转运到这些细胞中的 Michaelis-Menten 动力学； (4)使用Northern印迹、免疫细胞化学以及脑和离体脑毛细血管的电子显微镜对BBB AZT外排转运蛋白mRNA和蛋白质进行功能分析。