Drug Transport at the Blood-Testis Barrier

描述（由申请人提供）：自1996年引入以来，高效抗逆转录病毒疗法（HAART）已被认为在美国显著和持续地减少了艾滋病相关的死亡和疾病。与任何药物制剂一样，药物递送到作用部位是绝对需要的，以引起有利的反应。从病人体内彻底根除艾滋病毒的一个重大障碍是存在生物屏障，造成病毒不受药物影响的庇护所。存在两个这样的屏障，确保病毒的存活和可能的耐药表型的发展：血脑屏障（BBB）和血睾丸屏障（BTB）。虽然目前还没有完全概括血脑屏障的方法来研究药物的转运，但我们已经开发出一种研究完整BTB的新方法。BTB保护发育中的生殖细胞免受潜在破坏性化学相互作用以及免疫影响的影响。虽然BTB的这种正常的保护功能在生理上有利于生殖，但它也可以在抗逆转录病毒治疗期间为HIV病毒提供避难所。这个艾滋病毒庇护所特别重要，因为感染男性射精中存在的艾滋病毒是传播给未感染者的主要途径。因此，即使患者正在有效地接受抗逆转录病毒药物的鸡尾酒治疗，在BTB选择性排除药物也会增加射精的病毒载量和传播的可能性。事实上，蛋白酶抑制剂和非核苷类逆转录酶抑制剂在BTB中被积极排除，在睾丸中达不到治疗浓度。或者，核苷类似物药物（NSAs）提供了一个独特的机会来检查BTB的特征，以及利用内源性转运蛋白作为规避BTB的手段的可能性，因为许多NSAs能够在接受治疗的男性精液中积累。支持细胞作为血-睾丸屏障的主要组成部分，通过在发育中的生殖细胞下方形成紧密连接，将生精小管（ST）的近腔室与身体的其他部分分开。为了到达近腔空间，药物必须首先避开屏障功能转运蛋白，然后通过被动扩散或载体介导的转运通过支持细胞。位于基底膜和腔膜的向内转运蛋白允许不能通过被动扩散的特定化合物通过跨上皮分泌穿过BTB。因此，化合物在支持细胞中的选择性移动构成了BTB的生理或功能方面。因此，我们假设特定的转运蛋白存在于支持细胞的基底膜和管腔膜上，允许特定的NSA药物穿透BTB。以下目标已被设计来测试这一假设：1。确定主要药物转运蛋白在血-睾丸屏障上的组成型表达和细胞定位，以及NSA和PI向睾丸的渗透。2.确定选定的内向转运蛋白（特别是Ent 1、Ent 2、Mate 1和Mate 2）支持NSA和PI跨上皮分泌的能力。3.确定分离的支持细胞和完整ST转运NSA的功能能力和分子特异性（去羟肌苷、叠氮胸苷、阿巴卡韦、恩曲他滨、替诺福韦）和PI当前提议的主要重点是关注治疗剂可以成功地利用内源性跨上皮转运蛋白在近腔室中积累的机制，甚至在功能性生理屏障的情况下。

DESCRIPTION (provided by applicant): Since its introduction in 1996, highly active antiretroviral therapy (HAART) has been credited with a marked and sustained reduction in AIDS-related death and disease in the United States. As with any pharmacotherapeutic agent, drug delivery to the site of action is absolutely required to elicit a favorable response. A significant obstacle to complete eradication of the HIV virus from the patient's body is the existence of biological barriers creating sanctuaries where the virus remains free from drug exposure. Two such barriers exist, ensuring the virus' survival and possible development of a resistant phenotype: the blood- brain barrier (BBB) and the blood-testis barrier (BTB). While there are no current methods that fully recapitulate the BBB to study the transport of drugs, we have developed a novel means of studying the intact BTB. The BTB protects the developing germ cells from the effects of potentially disruptive chemical interactions as well as immunological influences. Although this normal, protective function of the BTB is physiologically beneficial for reproduction, it can also provide a sanctuary for the HIV virus during antiretroviral therapy. This sanctuary site for HIV is particularly significant because the presence of HIV virus in the ejaculate of infected men is the major means of transmission to uninfected persons. Therefore, even while a patient is being effectively treated with a cocktail of antiretroviral drugs, the selective exclusion of the drugs at the BTB increases the viral load of the ejaculate and potential for transmission. Indeed, protease inhibitors and non-nucleoside reverse transcriptase inhibitors are actively excluded at the BTB and do not reach therapeutic concentrations in the testis. Alternatively, nucleoside analog drugs (NSAs) offer a unique opportunity to examine the characteristics of the BTB as well as the possibility of utilizing endogenous transporters as a means of circumventing the BTB since a number of NSAs are capable of accumulating in the semen of treated men. As the major component of the blood-testis barrier, Sertoli cells separate the adluminal compartment of the seminiferous tubules (STs) from the rest of the body by the formation of tight junctions below developing germ cells. In order to reach the adluminal space, drugs must first avoid barrier-function transporters before subsequently passing through Sertoli cells either by passive diffusion or carrier-mediated transport. Inward-facing transporter proteins, located at the basolateral and luminal membranes, allow specific compounds that cannot pass by passive diffusion to cross the BTB via transepithelial secretion. Selective movement of compounds across Sertoli cells therefore comprises the physiologic or functional aspect of the BTB. Therefore, we hypothesize that specific transporters are present at the basolateral and luminal membranes of Sertoli cells that allow specific NSA drugs to penetrate the BTB. The following aims have been designed to test this hypothesis: 1. Determine the constitutive expression and cellular localization of the major drug transporter proteins at the blood-testis barrier, as well as penetration of NSAs and PIs into the testis. 2. Determine the ability of selected inward-facing transporters (particularly Ent1, Ent2, Mate1 and Mate2) to support transepithelial secretion of NSAs and PIs. 3. Determine the functional capacity and molecular specificity of isolated Sertoli cells and intact STs to transport NSAs (didanosine, azidothymidine, abacavir, emtricitabine, tenofovir) and PIs (lopinavir and indinavir).The major emphasis of the current proposal is to focus on mechanisms by which therapeutics can successfully utilize endogenous transepithelial transporters to accumulate in the adluminal compartment, even within the context of the functional physiologic barrier.