Brain Drug Delivery Using Parkinson as a Disease Model

描述（由申请人提供）：骨结构头骨的保护，缺乏靶向或保留特异性，以及不可渗透血脑屏障（BBB）的存在使大脑成为药物干预最没有希望的领域。在这项R21应用中，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磁共振成像剂，超超磁性氧化铁纳米颗粒（MION）作为药物载体，利用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，以定位和保留目标MION到脑毛细血管上。TAT是一种从HIV蛋白中提取的有效的细胞转导肽，它也将与葡聚糖包被连接在一起，作为覆盖血脑屏障和细胞膜屏障的装置。在动物研究中已经证明，通过共价连锁，TAT能够将MION转导到包括大脑在内的器官组织中。为了减少正常组织对MION的非特异性摄取，TAT的跨膜活性将通过与肝素的结合而被掩盖。肝素在体内已被证实能完全抑制tat介导的细胞转导。将进行一项药代动力学研究，以确定MION在大脑部位达到最大定位和最小全身分布所需的时间。鱼精蛋白是一种临床肝素解毒剂，将在这个预先确定的时间框架内施用，以将肝素从其与TAT的静电结合中分离出来。一旦肝素抑制解除，TAT将恢复其有效的跨膜活性，使MION穿过血脑屏障进入脑细胞。在大脑内部，药物分子通过可水解键与右旋糖酐涂层连接，从MION中缓慢释放，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的可行性。这主要是因为PD提供了一种敏感和临床相关的动物模型（即6-OHDA大鼠模型），该模型产生与脑多巴胺浓度和活性直接相关的物理（如动力学）和化学（如TH免疫组织化学）反应。因此，这种大脑给药方式的成功或失败可以毫无疑问地从实验结果中得到证实。由于6-OHDA大鼠PD模型可以反向应用于检查过氧化物酶的神经保护作用，过氧化物酶是一种有效的H202清除剂，可以保护神经元免受自由基的攻击，因此也将尝试递送过氧化物酶，看看它是否可以延缓PD的进展。由于预算有限，R21拨款期限短，本申请计划采取霰弹枪方法，通过主要进行体内动物研究来实现该项目的概念验证。然而，如果该方法在传递亲水性多巴胺和大过氧化物酶蛋白（两种不能穿过血脑屏障的药物）方面被证明是可行的，那么基于脑传递神经营养因子以促进神经元存活、刺激轴突生长和改变潜在疾病进程的R01应用将被进一步扩展，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.