AIDS THERAPEUTICS & BLOOD-BRAIN BARRIER AZT DRUG EFFLUX

艾滋病治疗

• 批准号: 6145728
• 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/6145728
• 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.

本研究的目的是为叠氮胸苷（AZT）和其他核苷类逆转录酶抑制剂（NRTI）开发新的脑药物递送策略，从而提高这些药物治疗获得性免疫缺陷综合征（AIDS）脑部分的临床潜力。几乎所有目前用于治疗艾滋病的临床药物（NRTI、非NRTI和HIV蛋白酶抑制剂），由于这些药物通过血脑屏障（BBB）的主动外排，在药理学上都不能从大脑血液中大量转运。这些药物的临床潜力可以通过联合用药来增强，其中联合用药是血脑屏障主动外排系统的抑制剂。开发HIV蛋白酶抑制剂的联合药物很简单，因为这些药物是已知外排转运蛋白p-糖蛋白的底物。然而，由于AZT和其他NRTI的推定主动外排系统尚未在分子水平上定义，因此目前无法实现NRTI的联合药物开发。本研究的目的是克隆、测序并表达位于血脑屏障的AZT活性外排转运蛋白全长cDNA。cDNA的可用性将允许以确定的格式表达AZT主动外排转运蛋白，这将允许随后识别血脑屏障AZT外排转运蛋白抑制剂的药物。提出了以下具体目标：(1)利用青蛙卵母细胞表达克隆系统和牛脑毛细管cDNA文库提取的RNA进行AZT外排cDNA的表达克隆；(2)克隆cDNA的DNA测序和DNA序列分析；(3)在COS细胞中表达cDNA，并测量放射性标记AZT转运到这些细胞的Michaelis-Menten动力学；(4)利用Northern blotting、免疫细胞化学和电镜对脑和离体脑毛细血管血脑屏障AZT外排转运蛋白mRNA和蛋白进行功能分析。