AIDS THERAPEUTICS & BLOOD-BRAIN BARRIER AZT DRUG EFFLUX

本研究的目的是开发新的脑给药策略叠氮胸苷（AZT）和其他核苷类逆转录酶抑制剂（NRTI），使这些药物的临床潜力增强，用于治疗获得性免疫缺陷综合征（AIDS）的脑成分。 几乎所有目前在临床实践中用于治疗AIDS的药物（NRTI、非NRTI和HIV蛋白酶抑制剂）由于这些药物的主动外排穿过血脑屏障（BBB）而不以非常显著的量从脑中的血液转运。 这些药物用于治疗脑AIDS的临床潜力可以通过给予联合药物来增强，其中联合药物是BBB主动外排系统的抑制剂。 HIV蛋白酶抑制剂的联合药物的开发是简单的，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，目前无法实现NRTI的联合药物的开发，因为AZT和其他NRTI的推定主动外排系统尚未在分子水平上定义。 本研究的目的是克隆、测序和表达位于血脑屏障的AZT主动外排转运蛋白的全长cDNA。 cDNA的可用性将允许AZT主动外排转运蛋白以确定的形式表达，这将允许随后鉴定作为BBB AZT外排转运蛋白抑制剂的药物。 提出了以下具体目标：（1）利用蛙卵母细胞表达克隆系统和牛脑毛细血管cDNA文库中的RNA表达克隆AZT外排cDNA;（2）克隆cDNA的DNA测序和DNA序列分析;（3）在COS细胞中表达cDNA，然后测量放射性标记AZT转运到这些细胞中的Michaelis-Menten动力学;（4）采用北方印迹、免疫细胞化学和脑和分离的脑毛细血管的电子显微镜分析BBB AZT外排转运体mRNA和蛋白的功能。

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.