Enhancing the Delivery of Amphotericin B Across the Blood Brain Barrier for Treatment of Cryptococcal Meningoencephalitis

研究背景和意义。新生隐球菌和加蒂氏杆菌是被包裹的酵母菌，它能引起 免疫功能正常和免疫抑制的人的慢性感染，并导致超过 每年有62.5万人死亡。两性霉素B(Amb)一直是治疗隐球菌脑膜炎的主要药物。 脑炎(CME)。然而，在这些接受有效和有效治疗的患者中，显著的死亡率(~20%)。 真菌制剂表明需要更好的治疗方法。AMB是一个相对较大的分子，不会影响- 有效地穿过血脑屏障(BBB)。我们的目标是开发AMB的配方，使其在 能够有效清除脑组织中真菌生物的浓度，用于治疗CME。 假设。血脑屏障内皮细胞高水平表达转铁蛋白受体(TFR)以转运体内的铁 血液进入大脑。已发现一种多肽(CRTIGPSVC)，它通过与转铁蛋白和铁蛋白结合来模拟铁 可能被用来针对大脑的药物。我们已经产生了一种链霉亲和素突变体(SAM7)，它与 脑靶向肽(BTP)CRTIGPSVC。该融合蛋白(BTP-SAm7)与TFR结合。此外，BTP- SAm7与生物素结合的Amb*(BTP-SAm7-[Amb*])在体外对新生葡萄球菌有较强的抑制作用。 在体外跨越血脑屏障，并减少小鼠慢性粒细胞白血病的脑CFU。我们假设一种融合蛋白包含 生物素结合蛋白(例如链霉亲和素)和BTP将把AmB*运送到大脑中。我们进一步假设- 脑靶向AMB的大小将对CME有更大的疗效。最后，我们假设以Amb为目标 对大脑的使用将允许使用较低的总剂量，从而减少对AMB全身毒性的担忧。 明确的目标。1)优化AMB跨血脑屏障的运输和递送；2)确定抗 新型Amb类似物的隐球菌活性，3)确定脑靶向Amb对小鼠CME的疗效， 4)检测脑靶向AMB的毒性。 建议的工作。对于目标1，运输机BTP-SAM的各个部件将进行并行优化。 我们将研究其他多肽，如BTP和SA的其他单体变体，它们对生物素有不同的亲和力。我们 将在hCMEC/D3细胞单层上系统地测试这些第二代分子，该细胞单层已经 建立了血脑屏障的体外模型，并与CRTIGPSVC-SAm7和无关肽进行比较 控制。我们将确定BTP-SAm7是否能有效地将Amb*运送到小鼠的脑实质中。 在目标2中，我们将确定Amb和Amb的类似物是否与具有协同作用的抗真菌多肽相连。 AMB具有较强的抗新生葡萄球菌活性和/或较低的毒性。对于目标3，我们将确定疗效 BTP-SAM-[AMB*]在CME小鼠体内的作用。对于目标4，我们将评估BTP-SAM-[AMB*]的毒性与 原代培养的神经细胞和未感染的小鼠的AMB。

Background and Significance. Cryptococcus neoformans and C. gattii are encapsulated yeasts that cause chronic infections in both immunocompetent and immunosuppressed individuals and are responsible for over 625,000 deaths each year. Amphotericin B (AmB) has been the mainstay of treatment for cryptococcal menin- goencephalitis (CME). However, the significant mortality rate (~20%) in these patients treated with available an- tifungal agents indicates the need for better therapies. AmB is a relatively large molecule and does not effec- tively cross the blood brain barrier (BBB). Our goal is to develop formulations of AmB that will cross the BBB in concentrations capable of effectively eliminating fungal organisms from the brain tissue for treating CME. Hypotheses. BBB endothelial cells express transferrin receptor (TfR) at high levels to transport iron from the blood into the brain. A peptide (CRTIGPSVC) has been identified that mimics iron by binding to transferrin and could be used to target drugs to the brain. We have produced a mutant of streptavidin (SAm7) fused to the brain targeting peptide (BTP) CRTIGPSVC. This fusion protein (BTP-SAm7) binds to TfR. In addition, BTP- SAm7 complexed with biotinylated AmB* (BTP-SAm7-[AmB*]) is effective against C. neoformans in vitro, crosses BBB in vitro, and reduces brain CFUs in murine CME. We hypothesize that a fusion protein consisting of a biotin binding protein (e.g., streptavidin) and a BTP will transport AmB* into the brain. We further hypothe- size that brain-targeted AmB will have greater efficacy against CME. Finally, we hypothesize that targeting AmB to the brain will allow use of lower total doses and thus reduce concerns of AmB systemic toxicity. Specific Aims. 1) to optimize the transport and delivery of AmB across the BBB, 2) to determine the anti- cryptococcal activity of novel AmB analogs, 3) to determine the efficacy of brain-targeted AmB in murine CME, and 4) to examine the toxicity of brain-targeted AmB. Work Proposed. For Aim 1, Individual components of the transporter BTP-SAm will be optimized in parallel. We will study other peptides as BTP and other monomeric variants of SA that have varying affinity for biotin. We will systematically test these second-generation molecules in vitro on hCMEC/D3 cell monolayers, which has been established as an in vitro model of the BBB, in comparison to CRTIGPSVC-SAm7 and irrelevant peptide controls. We will determine whether BTP-SAm7 can effectively deliver AmB* into the brain parenchyma in mice. In Aim 2, we will determine whether analogs of AmB and AmB linked to antifungal peptides that are synergistic with AmB have greater activity against C. neoformans and/or less toxicity. For Aim 3, we will determine efficacy of BTP-SAm-[AmB*] in mice with CME. For Aim 4, we will evaluate toxicity of BTP-SAm-[AmB*] compared to AmB in primary neuronal cell cultures and uninfected mice.