A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

一种新的抗氧化剂可防止 HIV 蛋白与甲基苯丙胺的毒性

• 批准号: 7418174
• 负责人: NURAN ERCAL
• 金额: $ 22.58万
• 依托单位: MISSOURI UNIVERSITY OF SCIENCE & TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2009-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418174
• 关键词: AIDS Dementia Complex; Acetylcysteine; Acquired Immunodeficiency Syndrome; Address; Affect; Albumins; Alcohols; Amides; Animals; Antioxidants; Apoptosis; Astrocytes; Behavioral; Biological Assay; Biological Availability; Blood - brain barrier anatomy; Brain; Cell membrane; Cells; Characteristics; Charge; Cognitive deficits; Complication; Cysteine; Deacetylation; Dementia; Development; Disease; Disruption; Dose; Drug or chemical Tissue Distribution; End Point; Endocytosis; Endothelial Cells; Enzymes; Equilibrium; Event; Exposure to; Extravasation; Failure; Free Radical Scavenging; Free Radicals; Functional disorder; Glutathione; Glutathione Disulfide; Glutathione Reductase; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Seropositivity; HIV-1; High Pressure Liquid Chromatography; Human; In Vitro; Individual; Infection; Laboratories; Lipid Peroxidation; Measures; Methamphetamine; Microglia; Modeling; Modification; Morphine; Motor; N-Acetylcysteinamide; Neurologic; Neurons; Neuropathogenesis; Nitrogen; Numbers; Oral; Oxidative Stress; Oxygen; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Play; Production; Protein Overexpression; Proteins; Rate; Rattus; Reactive Oxygen Species; Recovery; Risk; Role; Solubility; Sulfhydryl Compounds; Syndrome; Techniques; Testing; Therapeutic Agents; Time; Tissues; Toxic effect; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Wild Type Mouse; Work; antiretroviral therapy; carboxyl group; caspase-3; catalase; cytokine; design; ecstasy; glutathione peroxidase; gp-120 Antigen; human tissue; in vitro Model; in vivo; in vivo Model; neurotoxicity; prevent; research study; response; stress related disorder; tat Protein; transcytosis; AIDS Dementia Complex; Acetylcysteine; Acquired Immunodeficiency Syndrome; Address; Affect; Albumins; Alcohols; Amides; Animals; Antioxidants; Apoptosis; Astrocytes; Behavioral; Biological Assay; Biological Availability; Blood - brain barrier anatomy; Brain; Cell membrane; Cells; Characteristics; Charge; Cognitive deficits; Complication; Cysteine; Deacetylation; Dementia; Development; Disease; Disruption; Dose; Drug or chemical Tissue Distribution; End Point; Endocytosis; Endothelial Cells; Enzymes; Equilibrium; Event; Exposure to; Extravasation; Failure; Free Radical Scavenging; Free Radicals; Functional disorder; Glutathione; Glutathione Disulfide; Glutathione Reductase; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Seropositivity; HIV-1; High Pressure Liquid Chromatography; Human; In Vitro; Individual; Infection; Laboratories; Lipid Peroxidation; Measures; Methamphetamine; Microglia; Modeling; Modification; Morphine; Motor; N-Acetylcysteinamide; Neurologic; Neurons; Neuropathogenesis; Nitrogen; Numbers; Oral; Oxidative Stress; Oxygen; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Play; Production; Protein Overexpression; Proteins; Rate; Rattus; Reactive Oxygen Species; Recovery; Risk; Role; Solubility; Sulfhydryl Compounds; Syndrome; Techniques; Testing; Therapeutic Agents; Time; Tissues; Toxic effect; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Wild Type Mouse; Work; antiretroviral therapy; carboxyl group; caspase-3; catalase; cytokine; design; ecstasy; glutathione peroxidase; gp-120 Antigen; human tissue; in vitro Model; in vivo; in vivo Model; neurotoxicity; prevent; research study; response; stress related disorder; tat Protein; transcytosis

DESCRIPTION (provided by applicant): A major complication of HIV-1 infection is the development of HIV-1-associated dementia (HAD). The mechanisms behind HAD are not yet known. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. In particular, these proteins affect the integrity of the blood- brain barrier (BBB) by crossing the BBB, altering BBB transporters, and disrupting the BBB. Our studies show that these proteins also induce oxidative stress in RBE4 cells, an in vitro BBB model. We determined that gp120 and Tat induced oxidative stress in RBE4 cells by measuring selected oxidative stress parameters. Subsequently, the levels of glutathione (GSH), the principal intracellular thiol responsible for maintaining the oxidative balance in cells, significantly decreased, indicating that the cells were undergoing oxidative stress. Antioxidants are becoming increasingly popular in oxidative stress-related disorders and hold promise as therapeutic agents. We have also found that N-acetylcysteine amide (NACA), a new thiol antioxidant, significantly increased the levels of GSH in gp120 and Tat-exposed RBE4 cells. Furthermore, many AIDS/HIV-positive patients use addictive drugs, including methamphetamines (METH). Since METH induces oxidative stress as well, drug abusing patients might be at risk of a synergistic effect and increased damage. Our preliminary results showed that METH does indeed work synergistically with gp120 and Tat to induce oxidative stress in RBE4 cells. Since work with RBE4 cells is open to the criticism, discussed under the "Specific Aims" section, we plan to use a more appropriate in vitro BBB model. Human brain microvascular endothelial cells (HBMVECs) will be used to evaluate the synergistic effect of METH and the two HIV proteins in inducing oxidative stress. Transgenic mice overexpressing gp120 or Tat will serve as our in vivo model. Permeability experiments in BBB models (both in HBMVECs and isolated brain microvascular endothelial cells from the transgenic animals) will also be performed to serve as functional endpoints. Therefore, we propose to determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination).Despite the development and use of effective antiretroviral therapy, HIV-1 associated dementia (HAD) still persists. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. One proposed mechanism is that HIV-1 induced neurotoxicity may be due to an increased production of reactive oxygen species (ROS) by HIV-1 proteins (gp120 and Tat). Furthermore, many AIDs/HIV-positive patients use addictive drugs such as methamphetamine (METH). Among these addictive drugs, METH, in particular, induces free radicals in various cells. If neuropathogenesis of HIV infection is found to be caused by a significant increase in oxidative damage by METH and gp120 and/or Tat, antioxidants should be included in the treatment to prevent HIV-induced dementia. Glutathione (GSH) is the principal intracellular thiol responsible for scavenging reactive oxygen species (ROS) and maintaining the oxidative balance in tissues. Cysteine and glutathione delivery compounds protect cells from free radical damage. For example, N-acetylcysteine (NAC) indirectly replenishes GSH through deacetylation to cysteine and prevents oxidative stress. However, NAC has a very low oral bioavailability (30%) and its efficacy is only significant when given intravenously. NAC's failure to provide significant antioxidant effects in vivo might originate from its low solubility and tissue distribution. This inability to provide antioxidant defense may result from NAC's negatively charged carboxyl group. Recently, an amide form of NAC has been synthesized and our laboratory has tested this new compound in various oxidative stress models. Because the carboxylic group in NACA is neutralized, NACA is more lipophilic than NAC and can easily cross the cell membranes. Therefore, we hypothesize that addictive drugs, as exemplified by METH, potentiate the oxidative stress induced by gp120 and Tat at the blood brain barrier (BBB) and that NACA blocks oxidative stress and protects the BBB.

描述（由申请人提供）：HIV-1感染的主要并发症是HIV-1相关痴呆症（HAD）的发展。背后的机制尚不清楚。某些研究表明，HIV-1包膜糖蛋白（GP120）和转质蛋白（TAT）在HAD的发育中起作用。特别是，这些蛋白质通过越过BBB，改变BBB转运蛋白并破坏BBB来影响血脑屏障（BBB）的完整性。我们的研究表明，这些蛋白还会诱导体外BBB模型RBE4细胞中的氧化应激。我们确定GP120和TAT通过测量选定的氧化应激参数来诱导RBE4细胞中的氧化应激。随后，谷胱甘肽（GSH）的水平，主要的细胞内硫醇负责维持细胞的氧化平衡，显着降低，表明细胞正在经历氧化应激。抗氧化剂在与氧化应激有关的疾病中越来越流行，并成为治疗剂的希望。我们还发现，一种新型硫醇抗氧化剂N-乙酰半胱氨酸酰胺（NACA）显着提高了GP120和TAT暴露的RBE4细胞中GSH的水平。此外，许多艾滋病/艾滋病毒阳性患者使用上瘾的药物，包括甲基苯丙胺（METH）。由于MET也诱导氧化应激，因此滥用药物的患者可能有协同作用和增加损害的风险。我们的初步结果表明，METH确实确实与GP120和TAT协同起作用，以诱导RBE4细胞中的氧化应激。由于在“特定目的”部分中讨论的RBE4细胞的工作对批评开放，因此我们计划使用更合适的体外BBB模型。人脑微血管内皮细胞（HBMVECs）将用于评估甲基甲基和两种HIV蛋白在诱导氧化应激中的协同作用。过表达GP120或TAT的转基因小鼠将用作我们的体内模型。在BBB模型中（在HBMVEC和分离的脑微血管内皮细胞中，来自转基因动物）的渗透性实验也将作为功能性终点。因此，我们建议确定有效的抗氧化剂NACA是否可以保护BBB免受GP120，TAT或METH的影响（单独和/或组合）。尽管开发和使用有效的抗逆转录病毒疗法，但HIV-1相关的痴呆症（HIV-1相关痴呆症）仍然存在。某些研究表明，HIV-1包膜糖蛋白（GP120）和转质蛋白（TAT）在HAD的发育中起作用。提出的一种机制是，HIV-1诱导的神经毒性可能是由于HIV-1蛋白（GP120和TAT）增加了活性氧（ROS）的产生。此外，许多艾滋病/艾滋病毒阳性患者使用上瘾的药物，例如甲基苯丙胺（METH）。在这些成瘾性药物中，尤其是甲基苯丙胺可诱导各种细胞的自由基。如果发现HIV感染的神经病发生是由METH和GP120和/或TAT氧化损伤显着增加引起的，则应将抗氧化剂包括在治疗中，以防止HIV诱导的痴呆症。谷胱甘肽（GSH）是负责清除活性氧（ROS）并维持组织中氧化平衡的主要细胞内硫醇。半胱氨酸和谷胱甘肽递送化合物可保护细胞免受自由基损伤。例如，N-乙酰半胱氨酸（NAC）间接地通过脱乙酰化为半胱氨酸补充GSH，并防止氧化应激。但是，NAC具有非常低的口服生物利用度（30％），并且其功效仅在静脉内给予时才显着。 NAC无法在体内提供明显的抗氧化作用，可能源于其低溶解度和组织分布。这种无法提供抗氧化防御的可能是由NAC带负电荷的羧基造成的。最近，已合成了一种NAC的酰胺形式，我们的实验室在各种氧化应激模型中测试了这种新化合物。由于NACA中的羧基被中和，因此NACA比NAC更具亲脂性，并且可以轻松地越过细胞膜。因此，我们假设通过甲基甲基苯丙胺的例证，上瘾的药物增强了由GP120和TAT在血脑屏障（BBB）处诱导的氧化应激，NACA会阻止氧化应激并保护BBB。