AIDS Neurotherapeutics and BBB Drug Efflux

描述(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治疗对不涉及大脑的艾滋病相关疾病的影响，这归因于HAART药物对中枢神经系统(CNS)的渗透性较差。虽然HAART药物可以通过脉络丛或血脑脊液屏障进入脑脊液(CSF)，但由于在体内形成血脑屏障(BBB)的脑毛细血管内皮壁转运有限，药物对脑实质的实际渗透受到限制。由于脂质介导的小分子转运，许多HAART药物应该通过血脑屏障。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-糖蛋白的BBB活性外排转运体(AET)[在核苷逆转录酶抑制剂(NRTI)的情况下]主动地从大脑流出到血液中。虽然制药公司正在致力于开发p-糖蛋白抑制剂的联合药物，但还没有对BBB的非p-糖蛋白AET进行研究，因为这些转运蛋白的分子身份尚不清楚。本资助的目的是继续利用青蛙卵母细胞表达克隆系统克隆运送NRTI的BBB AETs，并通过在pSPORT载体中体外转录大鼠或兔的cDNA文库获得克隆的RNA。利用这种方法，我们克隆了大鼠血脑屏障腺苷转运蛋白CNT2，并证明了二脱氧肌苷(DDI)是由BBBCNT2转运的。作为两栖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已缩减为实践。由于血脑屏障主动外排药物转运存在物种差异，大鼠和兔脑毛细血管来源的PolA+RNA均可制备血脑屏障cDNA文库。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了。根据这一序列，我们将制备抗肽抗血清，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AET在脑内血脑屏障的特异性表达，以及(B)将转运蛋白的表达定位于管腔或腔隙的脑内皮膜。本工作为AET受体阻滞剂的药物开发奠定了基础，可作为辅助药物提高HAART药物的中枢神经系统渗透率。

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.