An in vitro Model of Antiretroviral Toxic Neuropathy

描述（由申请人提供）：核苷类似物逆转录酶抑制剂（NRTIs）是HAART（高活性抗逆转录病毒治疗）的重要组成部分，可显著降低HIV-1感染的发病率和死亡率。然而，许多这些药物的使用，包括ddC、ddl和d4T，与疼痛的感觉多神经病变（抗逆转录病毒毒性神经病，ATN）有关，这通常需要患者停药。目前还没有可靠的NRTI神经毒性体外或体内动物模型。我们建议建立一个强大可靠的NRTI神经毒性体外模型，以研究毒性的细胞机制，并开始筛选预防或治疗药物。背根神经节感觉神经元是NRTI毒性作用的主要靶点。因此，我们将利用大鼠的原代DRG感觉神经元来建立该模型。我们将研究各种已知会导致人类ATN的nrti的影响。已建立的形态学标准将作为神经毒性的主要预后指标。此外，我们将利用轴突生长相关基因的表达开发神经毒性的生化和分子分析。这些将根据主要形态学标准进行验证，并用于开发高通量分析。我们的初步数据表明nrti的毒性可能是通过线粒体介导的。我们观察到与坏死细胞死亡相关的线粒体能量衰竭。我们将通过检查ATP生成、活性氧和线粒体特异性热休克蛋白的作用来研究这种神经毒性的机制。这些研究的最终目标将是找到可能改善nrti神经毒性的化合物。非免疫抑制性神经亲免疫蛋白配体将用于本次筛选。我们的筛选计划可能会产生临床上有用的神经保护化合物，这些化合物可以与nrti联合使用，作为HAART治疗方案的一部分。这是神经保护范例的理想情况，因为神经保护药物可以提前给予或与神经毒性药物一起给予。此外，该检测系统可用于开发无神经毒性副作用的nrti。

DESCRIPTION (provided by applicant): Nucleoside analogue Reverse Transcriptase Inhibitors (NRTIs) are an essential component of HAART (Highly Active AntiRetroviral Therapy), substantially reducing the morbidity and mortality of HIV-1 infection. However, the use of many of these drugs, including ddC, ddl and d4T has been associated with a painful sensory polyneuropathy (Antiretroviral Toxic Neuropathy, ATN) that often necessitates drug discontinuation by the patient. There has been no reliable in vitro or in vivo animal models of NRTI neurotoxicity. We propose to develop a robust and reliable in vitro model of NRTI neurotoxicity to study the cellular mechanism of toxicity and initiate screening of preventative or therapeutic drugs. Dorsal root ganglion (DRG) sensory neurons are the main targets for NRTI toxicity in humans. Therefore, primary DRG sensory neurons from rats will be used to develop this model. We will examine the effects of various NRTIs that are known to cause ATN in humans. Established morphological criteria will be used as primary outcome measures of neurotoxicity. Additionally we will develop biochemical and molecular assays of neurotoxicity using the expression of axonal outgrowth associated genes. These will be validated against the primary morphological criteria and used in development of high-throughput assays. Our preliminary data suggests that the toxicity from NRTIs may be mediated through mitochondria. We observed mitochondrial energy failure associated with necrotic cell death. We will study the mechanism of this neurotoxicity by examining the roles of ATP generation, reactive oxygen species and mitochondria-specific heat-shock proteins. The ultimate goal of these studies will be to find compounds that may ameliorate the neurotoxicity of NRTIs. Nonimmunosuppressive neuroimmunophilin ligands will be used in this screening. Our screening program may yield clinically useful neuroprotective compounds that can be given in conjunction with the NRTIs as part of their HAART regimen. This is an ideal situation for a neuroprotective paradigm, because the neuroprotective drugs can be given in advance or in conjunction with the neurotoxic drugs. Furthermore, this assay system may be useful in development of NRTIs without the neurotoxic side effects.