AIDS THERAPEUTICS & BLOOD-BRAIN BARRIER AZT DRUG EFFLUX

本研究的目的是为叠氮胸苷(AZT)和其他核苷逆转录酶抑制剂(NRTI)开发新的脑部给药策略，从而增强这些药物在治疗获得性免疫缺陷综合征(AIDS)脑成分方面的临床潜力。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)在药理学上都不是从血液中转运的，这是因为这些药物主动地通过血脑屏障(BBB)外排。这些药物的临床潜力可以通过给予辅助药物来增强，其中辅助药物是血脑屏障主动外排系统的抑制剂。对于HIV蛋白酶抑制剂，联合药物的开发是直接的，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，由于AZT和其他NRTI的主动外排系统还没有在分子水平上定义，目前还不能实现针对NRTI的辅药的开发。本研究的目的是克隆、测序和表达定位于血脑屏障的AZT活性外排转运蛋白的全长c DNA。该基因的问世将允许AZT主动外排转运体以定义的形式表达，这将允许随后识别作为BBB AZT外排转运体抑制剂的药物。建议的具体目标如下：(1)利用青蛙卵母细胞表达克隆系统和来自牛脑毛细血管cDNA文库的RNA，表达克隆AZT外排基因；(2)对克隆的cDNA进行DNA测序和DNA序列分析；(3)在COS细胞中表达该cDNA，然后测量放射性标记的AZT转运到这些细胞的Michaelis-Menten动力学；(4)利用Northern印迹、免疫细胞化学和脑及分离的脑毛细血管的电子显微镜等方法分析BBB AZT外排转运体的mRNA和蛋白功能。

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.