AIDS Neurotherapeutics and BBB Drug Efflux

描述（由申请人提供）：高活性抗逆转录病毒疗法（HAART）对艾滋病相关痴呆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）的渗透能力差。尽管HAART药物可通过脉络膜丛或血-脑脊液屏障进入脑脊液（CSF），但由于药物通过脑毛细血管内皮壁（在体内形成血-脑屏障（BBB））的运输有限，因此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药物应该穿过血脑屏障。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-糖蛋白血脑屏障主动外排转运体（AET）[在核苷类逆转录酶抑制剂（NRTIs）的情况下]从脑外排回血液。虽然制药公司正致力于开发“联合药物”，即p糖蛋白抑制剂，但由于这些转运体的分子特性尚不清楚，因此尚未对血脑屏障处的非p糖蛋白aet进行研究。本基金的目的是继续利用青蛙卵母细胞表达克隆系统和pSPORT载体中大鼠或兔cDNA文库体外转录获得的克隆RNA，进行转运nrti的BBB aet的分子克隆工作。利用这种方法，我们克隆了大鼠血脑屏障腺苷转运体CNT2，并发现二脱氧肌苷（DDI）由BBBCNT2转运。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。由于药物在血脑屏障主动外排转运中存在物种差异，因此我们将从大鼠和家兔的脑毛细血管来源的多a +RNA中制备血脑屏障cdna文库。一旦克隆和测序全长cdna， AET系统的氨基酸序列推断。从这个序列中，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在脑血脑屏障的特异性表达，(b)将转运蛋白表达位点定位于管腔或管腔脑内皮膜。本研究为未来发现AET阻滞剂提供了药物基础，可作为增加HAART药物中枢神经系统渗透的联合药物。

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.