AIDS Neurotherapeutics and BBB Drug Efflux

艾滋病神经治疗和 BBB 药物流出

• 批准号: 6759371
• 负责人: WILLIAM M PARDRIDGE
• 金额: $ 26.85万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6759371
• 关键词: AIDS /HIV neuropathy; AIDS therapy; P glycoprotein; Xenopus; active transport; antiserum; biotechnology; blood brain barrier; confocal scanning microscopy; drug metabolism; expression cloning; genetic library; genetic transcription; immunoelectron microscopy; laboratory rabbit; laboratory rat; lamivudine; membrane transport proteins; molecular cloning; neuropharmacology; nucleic acid sequence; reverse transcriptase inhibitors; transfection /expression vector; transport inhibitor; vascular endothelium permeability; western blottings; zidovudine

DESCRIPTION (provided by applicant): The impact of highly active anti-retroviral therapy (HAART) on AIDS related dementia is less than the effect of drug therapy on AIDS-related illness not involving the brain, and this is attributed to the poor HAART drug penetration into the central nervous system (CNS). Whereas HAART drugs may enter cerebrospinal fluid (CSF) owing to transport across the choroid plexus or blood-CSF barrier, the actual penetration of the drugs into brain parenchyma is restricted owing to limited transport across the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo. Many of the HAART drugs should cross the BBB owing to lipid-mediated transport of small molecules. However, these drugs are actively effluxed from brain back to blood by either p-glycoprotein (in the case of the protease inhibitors) or non-p-glycoprotein BBB active efflux transporters (AET) [in the case of the nucleoside reverse transcriptase inhibitors (NRTIs)]. While drug companies are working on the development of "co-drugs", which are p-glycoprotein inhibitors, no work is being done on the non-p-glycoprotein AETs at the BBB, because the molecular identity of these transporters is not known. The purpose of the present grant is to continue work on the molecular cloning of BBB AETs that transport the NRTIs using the frog oocyte expression cloning system and cloned RNA obtained by in vitro transcription of a rat or rabbit cDNA library in the pSPORT vector. With this methodology, we cloned the rat BBB adenosine transporter, CNT2, and showed that dideoxyinosine (DDI) is transported by BBBCNT2. As an alternative to the amphibian expression system, we will also use a mammalian COS cell expression cloning system, which has been reduced to practice in the past funding period. BBB cDNAlibraries will be prepared from both rat and rabbit brain capillary-derived polyA+RNA, because there are species differences in BBB active efflux transport of drugs. Once the full-length cDNAs are cloned and sequenced, the amino acid sequence of the AET system is deduced. From this sequence, we will prepare anti-peptide antisera, which will be used in confocal microscopy, and immuno-gold electron microscopy. These immunochemical studies will (a) demonstrate the specific expression in brain of the AET at the BBB, and (b) localize the site of transporter expression to either the luminal or abluminal brain endothelial membrane. This work provides the basis for future drug discovery of AET blockers, which can be used as co-drugs to increase CNS penetration of HAART drugs.

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）对艾滋病相关痴呆症的影响小于药物疗法对不涉及大脑的艾滋病相关疾病的影响，这是由于HAART药物渗透到中枢神经系统（CNS）的效果较差。尽管HAART药物可由于穿过脉络丛或血-CSF屏障的运输而进入脑脊液（CSF），但由于穿过脑毛细血管内皮壁（其在体内形成血-脑屏障（BBB））的运输有限，药物向脑实质中的实际渗透受到限制。由于小分子的脂质介导的转运，许多HAART药物都应该穿过BBB。然而，这些药物通过p-糖蛋白（在蛋白酶抑制剂的情况下）或非p-糖蛋白BBB主动外排转运蛋白（AET）[在核苷逆转录酶抑制剂（NRTI）的情况下]从脑主动外排回血液。虽然制药公司正在努力开发“联合药物”，这是p-糖蛋白抑制剂，没有工作正在做的非p-糖蛋白在血脑屏障，因为这些转运蛋白的分子身份是未知的。本资助的目的是继续进行BBB Ablation的分子克隆工作，该Ablation使用青蛙卵母细胞表达克隆系统和通过在pSPORT载体中体外转录大鼠或兔cDNA文库获得的克隆RNA来转运NRTI。用这种方法，我们克隆了大鼠血脑屏障腺苷转运蛋白，CNT 2，并表明，双脱氧肌苷（DDI）的BBBCNT 2运输。作为两栖动物表达系统的替代方案，我们还将使用哺乳动物COS细胞表达克隆系统，该系统在过去的资助期内已减少到实践中。BBB cDNA将从大鼠和兔脑毛细血管来源的polyA+RNA制备，因为药物的BBB主动外排转运存在物种差异。一旦全长cDNA被克隆和测序，AET系统的氨基酸序列就被推导出来。从这个序列，我们将制备抗肽抗血清，这将用于共聚焦显微镜，和免疫金电子显微镜。这些免疫化学研究将（a）证明脑内AET在BB B的特异性表达，和（B）将转运蛋白表达的位点定位于腔或近腔脑内皮膜。这项工作为未来AET阻断剂的药物发现提供了基础，AET阻断剂可用作联合药物以增加HAART药物的CNS渗透。