Drug Transport at the CNS Barriers

描述(申请人提供)：有机阳离子(OC)转运体在体内许多内源性和外源性OC的处置和清除中发挥重要作用。与肾脏和肝脏相比，人们对大脑中OC的消除机制知之甚少。这是相当不幸的，因为许多中枢神经系统活性化合物，包括单胺类神经递质(例如多巴胺、5-羟色胺)、中枢神经系统药物(例如金刚烷胺、尼古丁)和神经毒素(例如1-甲基-4-苯基吡啶(MPP+))都是小型亲水性OCS，依赖转运蛋白来调节其大脑水平。本研究以本实验室首次克隆的脑组织OC转运蛋白--质膜单胺转运体(PmAt)为研究对象。虽然在结构上与平衡核苷转运蛋白家族(SLC29)相关，但pmAt具有独特的和令人惊讶的多样性底物专一性，运输结构不同的OCs，如生物胺、临床使用的药物和神经毒素。在人类和啮齿动物中，pmAt在脑中表达最丰富，并在血-脑脊液屏障(即BCSFB或脉络丛)中高度集中。我们假设pmAt是BCSFB的主要OC转运体，负责从脑中清除各种内源性和外源性OCs。提出了三个具体目标(SA)。SA1致力于阐明控制转运蛋白-底物相互作用的分子机制，以解释pmAt独特的和多功能的底物专一性。SA2致力于阐明PMAt的传输机制，并建立BCSFB有机碳通量的细胞模型。最后，在SA3中，我们将构建并验证转基因动物模型，这将使各种机制研究揭示pmAt在体内的作用。我们将应用分子生化技术、计算生物学、电生理学、免疫组织化学和转基因方法等多种实验方法，阐明pmAt在清除脑内神经递质、药物和毒素方面的结构、功能和生物学意义。所提出的研究对于我们理解大脑的正常和病理生理功能具有广泛的意义。对中枢神经系统屏障的OC转运体的详细了解也有助于解释和预测OC药物和毒素在中枢神经系统隔室的药代动力学和药效学，并有助于开发新的药物靶向大脑的策略。

DESCRIPTION (provided by applicant): Organic cation (OC) transporters play important roles in the disposition and clearance of many endogenous and foreign OCs in the body. In contrast to the kidney and liver, little is known about OC elimination mechanisms in the brain. This is rather unfortunate because many CNS active compounds, including monoamine neurotransmitters (e.g. dopamine, serotonin), CNS drugs (e.g. amantadine, nicotine) and neurotoxins (e.g. 1-methyl-4-phenylpyridinium (MPP+)) are small hydrophilic OCs that rely on transporters to regulate their brain levels. This proposal focuses on plasma membrane monoamine transporter (PMAT), a novel brain OC transporter first cloned in our laboratory. While structurally related to the equilibrative nucleoside transporter family (SLC29), PMAT possesses a unique and surprisingly diverse substrate specificity, transporting structurally heterogeneous OCs such as biogenic amines, clinically used drugs and neurotoxins. In humans and rodents, PMAT is most abundantly expressed in the brain and highly concentrated in the blood-cerebrospinal fluid (CSF) barrier (i.e. BCSFB or choroid plexus). We hypothesized that PMAT is the principal OC transporter at the BCSFB and is responsible for removing a variety of endogenous and xenobiotic OCs from the brain. Three Specific Aims (SAs) have been proposed. SA1 is focused on elucidating the molecular mechanisms governing transporter-substrate interactions to explain the unique and versatile substrate specificity of PMAT. SA2 is focused on elucidating the transport mechanism of PMAT and developing a cellular model for OC flux at the BCSFB. Lastly, in SA3, we will construct and validate a transgenic animal model, which will allow a variety of mechanistic studies to reveal the in vivo action of PMAT. We will apply several experimental methods, ranging from molecular biochemical techniques, computational biology, electrophysiology, immunohistochemistry to transgenic approach, to elucidate the structure, function and biological significance of PMAT in clearing neurotransmitters, drugs and toxins in the brain. The proposed studies have broad implications in our understanding of normal and pathophysiological functions of the brain. Detailed knowledge of OC transporters at the CNS barriers can also help to explain and predict the pharmacokinetics and pharmacodynamics of OC drugs and toxins in the CNS compartment and aid in the development of new strategies for drug targeting to the brain.