In Vivo Testing of Microtubule-Stabilizing Drugs in Triple Transgenic Mice

描述(申请人提供)：我们的长期目标是开发新的治疗药物，旨在通过保护细胞骨架的完整性来减缓阿尔茨海默病(AD)的神经原纤维病变的发展。基于在暴露于单抗纤维的AD脑和神经元中观察到明显的神经元营养不良和微管(MT)完整性破坏，我们测试了MT稳定药物如紫杉醇对单抗毒性的保护作用。紫杉醇和其他几种MT稳定剂显著提高了单抗处理的神经元在培养中的存活率。然而，这些药物不会进入大脑，因此不能在AD的活体小鼠模型中进行测试。我们已经合成了两种新的化合物，它们在培养中保护神经元，并实际穿过血脑屏障(BBB)。关于这些药物的半衰期和脑分布的药代动力学研究已经完成，有限的毒性实验也已经完成。本项目的目标是使用最近发展起来的同时具有抗体和t病理的三重转基因小鼠模型(3xTg-AD)来进行体内概念验证研究。具体目的是：(1)确定两种药物在慢性给药几周的对照小鼠中的最大耐受量；(2)通过评估3xTg-AD小鼠在整个慢性给药期间的行为表现，测试脑内紫杉烷TX-67及非紫杉烷MT相互作用药物GS-164在3xTg-AD小鼠模型中的体内作用；(3)评价TX-67或GS-164慢性治疗对3xTg-AD小鼠形成的AD样神经病理损害的进展的影响。接受药物治疗的小鼠将与只接受生理盐水注射的对照动物进行比较。在整个治疗期间，行为测试将每两周进行一次。将使用标准的神经病理学、免疫组织化学和生化方法分析大脑中淀粉样蛋白和t损伤的指标。基于我们对原代培养神经元的体外研究，以及最近关于紫杉醇逆转共济失调小鼠脊髓神经元外周轴突运输缺陷的报道，我们预计该药物将缓解3xTg-AD小鼠的脑病理。我们已经确定的药物使我们有可能提供第一次对大脑的体内评估，即稳定MT的药物，因为它们可能减缓大脑中AD样神经病理的进展。如果这些药物确实改变了任何认知变化的进展和/或脑损伤的积累，这一发现将支持对这一非常新颖的治疗策略的进一步研究，以减缓AD中发生的年龄依赖性神经退化。

DESCRIPTION (provided by applicant): Our long-term goal is to develop novel therapeutic agents targeted to slowing the development of t neurofibrillary pathology in Alzheimer's disease (AD) by preserving the integrity of the cytoskeleton. Based on the pronounced neuronal dystrophy and disruption of microtubule (MT) integrity observed in AD brain and neurons exposed to Ab fibrils, we have tested the effects of MT-stabilizing drugs such as Taxol for protection against Ab toxicity. Taxol and several other MT-stabilizing drugs markedly enhanced survival of Ab-treated neurons in culture. However, these drugs do not enter the brain and thus cannot be tested in an in vivo mouse model for AD. We have synthesized two novel compounds that protect neurons in culture and actually cross the blood brain barrier (BBB). Pharmacokinetic studies on the half-lives and the brain distribution of the drugs have already been completed, as have limited toxicity experiments. The goal of this project is to use a recently developed triple transgenic mouse model (3xTg-AD) with both Ab and t pathology to conduct an in vivo proof-of concept study. The Specific Aims are: (1) to determine the maximal tolerated doses of the two drugs in control mice receiving chronic administration for several weeks; (2) to test the in vivo effects of the brain permeant taxane Tx-67 and the non-taxane MT-interacting drug GS-164 in the 3xTg-AD mouse model by assessing the behavioral performance of the mice throughout the period of chronic drug administration; and (3) to evaluate the in vivo effects of chronic treatment with Tx-67 or GS-164 on the progression of the AD-like neuropathological lesions that develop in the 3xTg-AD mic. Drug-treated mice will be compared with control animals receiving only saline injections. Behavioral testing will be done bi-weekly throughout the treatment period. Brains will be analyzed for indicators of both amyloid and t lesions using standard neuropathological, immuno-histochemical, and biochemical strategies. Based on our in vitro studies with primary neurons in culture and a recent report that Taxol reversed peripheral axonal transport deficits in spinal cord neurons of a tauopathy mouse, we anticipate the drugs will moderate the brain pathology in the 3xTg-AD mice. The drugs we have identified make it possible for us to provide the very first in vivo evaluation of brain permeant MT-stabilizing drugs for their potential to slow the progression of AD-like neuropathology in the brain. If the drugs do indeed alter the progression of any cognitive changes and/or the accumulation of the brain lesions, the findings would support further investigation into this very novel therapeutic strategy for slowing age-dependent neurodegeneration such as occurs in AD.