Brain Drug Delivery Using Parkinson as a Disease Model

描述（由申请人提供）： 骨结构颅骨的保护，缺乏靶向或保留特异性，以及不可渗透的血脑屏障（BBB）的存在，使大脑成为药物干预的最有希望的领域。在这项R21申请中，我们提出了一种创新的脑药物递送方法，利用可生物降解的临床MR成像剂，超顺磁性氧化铁纳米颗粒（MION），作为药物载体和临床磁场作为克服颅骨屏障的工具，从而实现特定的脑部位靶向。转铁蛋白配体将固定在MION颗粒的葡聚糖涂层上，用于将靶向MION定位和保留在脑毛细血管上。达特是一种来自HIV蛋白的有效细胞转导肽，也将与葡聚糖涂层连接，以作为克服BBB和细胞膜屏障的发明。在动物研究中已经证明，通过共价连接，达特能够将MION结合到包括脑在内的器官组织中。为了减弱正常组织对MION的非特异性摄取，达特的跨膜活性将通过与肝素的结合来掩蔽。已证实肝素在体内可完全抑制TAT介导的细胞转导。将进行药代动力学研究，以确定MION在脑部位达到最大定位但达到最小全身分布所需的时间。鱼精蛋白（一种临床肝素解毒剂）将在该预定时间范围内给药，以使肝素与达特的静电结合解离。一旦解除肝素抑制，达特将恢复其有效的跨膜活性，使MION能够穿过BBB并进入脑细胞。在大脑内部，药物分子将通过可水解的键与葡聚糖涂层连接，并将从MION中缓慢释放，从而在较长时间内维持药物的治疗浓度。将选择帕金森病（PD）作为疾病模型，以评估这种方法将多巴胺输送到大脑中的可行性。这主要是因为PD提供了一种敏感且临床相关的动物模型（即6-OHDA大鼠模型），其产生与脑多巴胺浓度和活性直接相关的物理（例如运动）和化学（例如TH免疫组织化学）反应。因此，从实验结果中可以毫无疑问地证实这种脑药物递送途径的成功或失败。由于6-OHDA大鼠PD模型可以以相反的方式应用以检查过氧化物酶（一种可以保护神经元免受自由基攻击的有效H2 O2清除剂）的神经保护作用，因此还将尝试递送过氧化物酶以观察这是否可以延缓PD进展。由于R21赠款的预算有限和期限较短，该申请计划通过主要进行体内动物研究，采用鸟枪法来实现该项目的概念验证。然而，如果证明该方法在递送亲水性多巴胺和大过氧化物酶蛋白（两种不能穿过BBB的药物）方面是可行的，则将遵循基于神经营养因子的脑递送的大大扩展的R 01应用，以促进神经元存活、刺激轴突生长和改变潜在疾病的病程，以实现最终的PD治疗。

DESCRIPTION (provided by applicant): The protection by the bone-structured skull, the lack of targeting or retention specificity, and the presence of the impermeable blood brain barrier (BBB) render the brain the least promising territory for drug intervention. In this R21 application, we propose an innovative brain drug delivery approach by utilizing the biodegradable clinical MR imaging agent, the superpara-magnetic iron oxide nanoparticles (MION), as the drug carrier and a clinical magnetic field as the tool for overcoming the skull barrier thereby achieving the specific brain-site targeting. Transferrin ligands will be immobilized onto the dextran coating of the MION particles for localization and retention of the targeted MION onto brain capillaries. TAT, a potent cell transduction peptide derived from the HIV protein, will also be linked to the dextran coating to serve as the contrivance for overriding the BBB and cell membrane barriers. It has been demonstrated in animal studies that via covalent linkage, TAT was able to transduce MION into organ tissues including the brain. To attenuate the non-specific uptake of MION by normal tissues, the trans-membrane activity of TAT will be masked via the binding with heparin. It has been confirmed that heparin can completely inhibit TAT-mediated cell transduction in vivo. A pharmacokinetic study will be conducted to determine the time required for MION to reach the maximum localization at the brain site but minimum systemic distribution. Protamine, a clinical heparin antidote, will be administered at this pre-determined time frame to dissociate heparin from its electrostatic binding to TAT. Once relieved from heparin inhibition, TAT will resume its potent trans-membrane activity, enabling MION to cross BBB and enter brain cells. Inside the brain, drug molecules, which will be linked to the dextran coating via hydrolysable bonds, will be slowly released from MION, sustaining a therapeutic concentration of the drug over an extended period of time. Parkinson's disease (PD) will be selected as the disease model to assess the feasibility of this approach in delivering dopamine into the brain. This is primarily because that PD offers a sensitive and clinically relevant animal model (i.e. the 6-OHDA rat model) that produces both physical (e.g. kinesic) and chemical (e.g. TH immunohistochemistry) responses in a direct correlation to the brain dopamine concentration and activity. Therefore, the success or failure of this brain drug delivery approach can be unquestionably confirmed from the experimental results. Since the 6-OHDA rat PD model can be applied in a reverse manner to examine the neuroprotective effects of peroxidase, a potent H202 scavenger that can protect neurons from attack by free radicals, delivery of peroxidase will also be attempted to see if this can retard PD progression. Because of the restricted budget and short duration of the R21 grant, this application plans to take a shotgun approach to achieve the proof-of-concept of this project, by conducting primarily in vivo animal studies. However, if the approach proves feasible in delivering both the hydrophilic dopamine and large peroxidase protein (two drugs that cannot cross BBB), a greatly extended R01 application basing on brain delivery of neurotrophic factors for promoting neuronal survival, stimulating axonal growth, and altering the course of the underlying 9 disease, will be followed to achieve the ultimate PD treatment.