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

增强两性霉素 B 穿过血脑屏障的递送以治疗隐球菌性脑膜脑炎

• 批准号: 9898292
• 负责人: DAVID Owen BEENHOUWER
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898292
• 关键词: AIDS/HIV problem; Affect; Affinity; Amino Acids; Amphotericin B; Anemia; Antibody Response; Antifungal Agents; Autoimmune Process; Binding; Biodistribution; Biological; Biological Assay; Biotin; Blastomycosis; Blood; Blood - brain barrier anatomy; Blood capillaries; Brain; Cell Culture Techniques; Cells; Central Nervous System Fungal Infections; Cessation of life; Chimeric Proteins; Clinical Trials; Coccidioidomycosis; Complex; Cryptococcosis; Cryptococcus; Cryptococcus gattii; Cryptococcus neoformans; Dose; Drug Targeting; Electrolytes; Encapsulated; Endothelial Cells; Formulation; Generations; Goals; HIV/TB; Hematologic Neoplasms; Hepatotoxicity; Histopathology; Histoplasmosis; Immune response; Immunocompetent; Immunohistochemistry; In Vitro; Individual; Infection; Inflammatory; Iron; Knowledge; Label; Lead; Leukoencephalopathy; Link; Liposomes; Malaria; Mediating; Meninges; Meningoencephalitis; Methods; Modification; Monitor; Mus; Mycoses; Neurons; Organ Transplantation; Organism; Patients; Peptides; Pharmaceutical Preparations; Pharmacodynamics; Radio; Recombinant Fusion Proteins; Respiratory System; Streptavidin; Symptoms; System; TFRC gene; Testing; Time; Tissues; Toxic effect; Transferrin; Transplant Recipients; Treatment Efficacy; Variant; Veterans; Work; Yeasts; analog; biotin-binding protein; brain parenchyma; brain size; brain tissue; chemotherapy; chronic infection; effective therapy; fluorescence imaging; immunogenicity; immunosuppressed; improved; improved outcome; in vitro Model; in vivo; innovation; monolayer; mortality; mutant; novel; peptidomimetics; protein complex; receptor; systemic toxicity; targeted delivery; transcytosis

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.

背景和意义。新生隐球菌和C. gattii是一种胶囊酵母， 免疫活性和免疫抑制个体的慢性感染， 每年有62.5万人死亡。两性霉素B（AmB）一直是治疗隐球菌性脑膜炎的主要药物， 脑炎（CME）。然而，在这些患者中，使用可用的抗肿瘤药物治疗的显著死亡率（~20%） 抗真菌剂表明需要更好的治疗方法。AmB是一种相对较大的分子，不影响 有效地穿过血脑屏障（BBB）。我们的目标是开发AmB的制剂，其将通过血脑屏障， 在一些实施方案中，本发明提供了能够有效地从脑组织中消除真菌生物体以治疗CME的浓度。 假设。血脑屏障内皮细胞高水平表达转铁蛋白受体（TfR）， 血液进入大脑。已经鉴定了一种肽（CRTIGPSVC），其通过结合转铁蛋白来模拟铁， 可以用来将药物靶向大脑。我们已经产生了一种链霉亲和素（SAm 7）的突变体， 脑靶向肽（BTP）CRTIGPSVC。该融合蛋白（BTP-SAm 7）与TfR结合。此外，BTP- SAm 7与生物素化的AmB* 复合物（BTP-SAm 7-[AmB*]）对C.体外的新生儿， 在体外穿过BBB，并减少鼠CME中的脑CFU。我们假设一种融合蛋白， 生物素结合蛋白（例如，链霉亲和素）和BTP将AmB* 转运到大脑中。我们进一步假设- 脑靶向AmB大小将对CME具有更大的功效。最后，我们假设靶向AmB 将允许使用较低的总剂量，从而减少对AmB全身毒性的担忧。 具体目标。1)为了优化AmB穿过BBB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在鼠CME中的功效， （4）检测脑靶向AmB的毒性。 工作建议。对于目标1，将平行优化转运蛋白BTP-SAm的单个组分。 我们将研究其他肽BTP和其他单体变体的SA，具有不同的生物素的亲和力。我们 将在hCMEC/D3细胞单层上系统地测试这些第二代分子， 与CRTIGPSVC-SAm 7和不相关肽相比， 对照我们将确定BTP-SAm 7是否可以有效地将AmB* 递送到小鼠的脑实质中。 在目标2中，我们将确定AmB的类似物和AmB与抗真菌肽连接是否具有协同作用， AmB对C.新生儿和/或毒性较低。对于目标3，我们将确定疗效 BTP-SAm-[AmB*]在CME小鼠中的表达。对于目标4，我们将评价BTP-SAm-[AmB*]与 原代神经元细胞培养物和未感染小鼠中的AmB。