Drug Transport at the Blood-Testis Barrier

血睾屏障的药物转运

• 批准号: 8278026
• 负责人: Nathan J Cherrington
• 金额: $ 37.5万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-17 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8278026
• 关键词: Acquired Immunodeficiency Syndrome; Activities of Daily Living; Anti-Retroviral Agents; Basement membrane; Biological; Blood - brain barrier anatomy; Blood-Testis Barrier; Carrier Proteins; Cessation of life; Characteristics; Chemicals; Development; Didanosine; Diffusion; Disease; Drug Delivery Systems; Drug Exposure; Drug Transport; Ensure; Exclusion; Germ Cells; HIV; Highly Active Antiretroviral Therapy; Human; Immunohistochemistry; Indinavir; Lopinavir; Measures; Methods; Molecular; Movement; Mus; Organ; P-Glycoprotein; Patients; Penetration; Permeability; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Property; Protease Inhibitor; Regulation; Relative (related person); Reproduction; Resistance; Rest; Risk; Role; Seminal fluid; Seminiferous tubule structure; Site; Specificity; Surface; System; Tenofovir; Testing; Testis; Therapeutic; Tight Junctions; United States; Viral Load result; Virus; Wild Type Mouse; Zidovudine; abacavir; antiretroviral therapy; base; basolateral membrane; carrier mediated transport; design; emtricitabine; inhibitor/antagonist; luminal membrane; men; non-nucleoside reverse transcriptase inhibitors; novel; nucleoside analog; public health relevance; research study; response; sertoli cell; small hairpin RNA; transmission process; uptake

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. PUBLIC HEALTH RELEVANCE: Even when effectively managed by antiretroviral therapy, patients can transmit HIV virus because the blood-testis barrier provides a sanctuary where the virus can hide from the drugs. This project seeks to determine the molecular mechanisms whereby specific nucleoside analog drugs can cross the blood-testis barrier and the possibility of utilizing endogenous transporter systems as a means of circumventing the blood-testis barrier. This mechanism could be utilized in the development of new drugs to reduce the risk of transmission by infected patients.

描述（由申请人提供）：自 1996 年推出以来，高效抗逆转录病毒疗法 (HAART) 被认为显着且持续地减少了美国与艾滋病相关的死亡和疾病。与任何药物治疗剂一样，绝对需要将药物递送至作用部位才能引起有利的反应。从患者体内彻底根除艾滋病毒的一个重大障碍是生物屏障的存在，这些生物屏障为病毒提供了避难所，使病毒免受药物暴露。存在两个这样的屏障，确保病毒的生存并可能发展出耐药表型：血脑屏障（BBB）和血睾屏障（BTB）。虽然目前还没有完全重现 BBB 来研究药物转运的方法，但我们开发了一种研究完整 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学影响的影响。虽然 BTB 的这种正常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 病毒提供庇护所。这个艾滋病毒庇护所具有特别重要的意义，因为感染男性的精液中存在艾滋病毒病毒，这是传播给未感染者的主要途径。因此，即使患者正在接受抗逆转录病毒药物混合物的有效治疗，在 BTB 处选择性排除药物也会增加精液中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逆转录酶抑制剂在 BTB 处被主动排除，并且在睾丸中未达到治疗浓度。另外，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 NSA 能够在接受治疗的男性精液中积累。作为血睾屏障的主要组成部分，支持细胞通过在发育中的生殖细胞下方形成紧密连接，将曲细精管 (ST) 的腔内室与身体的其他部分分开。为了到达腔内空间，药物必须首先避开屏障功能转运蛋白，然后再通过被动扩散或载体介导的转运穿过支持细胞。面向内的转运蛋白位于基底外侧膜和腔膜处，允许无法通过被动扩散通过的特定化合物通过跨上皮分泌穿过 BTB。因此，化合物跨支持细胞的选择性运动构成了 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外侧和腔膜上，允许特定的 NSA 药物穿透 BTB。设计了以下目标来检验这一假设： 1. 确定主要药物转运蛋白在血睾屏障处的组成型表达和细胞定位，以及 NSAs 和 PI 渗透到睾丸中。 2. 确定选定的内向转运蛋白（特别是 Ent1、Ent2、Mate1 和 Mate2）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 NSAs（去达诺辛、叠氮胸苷、阿巴卡韦、恩曲他滨、替诺福韦）和 PI（洛匹那韦和茚地那韦）的功能能力和分子特异性。当前提案的主要重点是关注机制通过这种方法，治疗可以成功地利用内源性跨上皮转运蛋白在腔内积聚，甚至在功能性生理屏障的背景下也是如此。 公共卫生相关性：即使通过抗逆转录病毒治疗有效控制，患者也可能传播 HIV 病毒，因为血睾屏障提供了病毒可以躲避药物的避难所。该项目旨在确定特定核苷类似物药物穿过血睾屏障的分子机制，以及利用内源性转运系统作为绕过血睾屏障的手段的可能性。这种机制可用于开发新药，以降低受感染患者传播的风险。