A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

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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.