Substance Use Disorders with drugs such as methamphetamine (METH) and 3,4-methylenediioxymethamphetamine (MDMA) are one of the top public health concerns in the United States. These psychostimulants have many targets in the Central Nervous System (CNS) as well as the periphery. The psychostimulatory effects of these drugs have a wide range of effects on the user, from producing reward to producing toxic neurologic complications such as leakage of the Blood Brain Barrier (BBB) and neuroinflammation. Despite this fact, there are no FDA-approved pharmacological treatment options for METH abuse and dependence other than cognitive behavioral therapies. In an attempt to address this problem, our laboratory developed an anti-METH single chain antibody fragment (scFv7F9) as a potential treatment for METH abuse. However, based on its small size, the half-life would be even shorter than the full monoclonal antibody’s half-life of three weeks. With this potential limitation, the use of this treatment option for a relapse prevention program would be limited due to the tendency of patient noncompliance in drug users. As an attempt to alleviate this concern, we extended the half-life by packaging our construct into the adeno-associated virus vector (AAV8) to produce long-term protection of those toxic effects associated with METH and MDMA abuse. Thus, the three Aims of this dissertation were focused on testing if our longer lasting form of our treatment option (AAV-7F9-Fc) could provide neuroprotection from METH induced effects.
The goal of the Aim 1 was to test whether AAV-7F9-Fc has the ability to prevent METH-induced BBB dysfunction and hyperthermia in a rodent model. Critical tight-junction proteins claudin-5, occludin, and zo-1 were measured 6 hrs. after an acute neurotoxic dose of 10 mg/kg ip. METH can induce hyperthermia in a dose-dependent manner. The implications of hyperthermia and neurotoxicity suggest that the greater extent of hyperthermia, the more likely that the neurotoxic effects will be exhibited. Evans Blue was also used to measure permeability at the 6 hr timepoint to confirm neuroprotection. In our METH treated group, we saw a (< 4°) increase in core body temperature after administration. Our results indicate that AAV-7F9-Fc could effectively shield against the hyperthermic effects of METH.
Aim 2 was to determine if our antibody gene therapy could prevent immune system dysregulation in a low dose, chronic model of METH use. In the CNS, METH can initiate a neuroinflammatory cascade and in the periphery, an immunosuppressive phenotype of cytokines is usually produced. With dissected brain tissue, the expression of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), chemokine monocyte chemoattractant protein-1 (CCL2) and glial cell activation markers glial fibrillary acidic protein (GFAP) and allograft inhibitory factor (Iba1) were measured by qRT-PCR. In the serum, a multiplex analysis of proinflammatory and antiinflammatory cytokines was performed to assess immune dysregulation. qRTPCR results showed a significant reduction in IL-1β, IL-6, TNF, and Aif1 mRNA expression in AAV-7F9-Fc + METH treated groups in the striatum, hippocampus, and frontal cortex. Multiplex results reveal that AAV-7F9-Fc reduced IL-6, IL-10, IL-1β, and KC GRO in serum. However, IL-6 levels were significantly higher than those of the saline treatment group.
The goal of Aim 3 was to determine whether our pharmacokinetic antagonists could mitigate the toxic effects of MDMA. The effects of our antibody gene therapy have not previously been tested on MDMA. Brain tissue was collected 72 hrs after the last injection of METH to mimic early abstinence in a chronic use model. Serum was collected from trunk blood and stored at −80 °C until analysis. In dissected brain tissue, the expression of proinflammatory cytokines IL-1β, IL-6, TNF-α, and glial cell activation markers GFAP and Iba1 were measured by qRT-PCR as they were in the METH studies. Using serumsamples, a multiplex analysis of proinflammatory and anti-inflammatory cytokines was performed to assess immune dysregulation. Serum was also used for measurements of alanine transaminase (ALT), bilirubin, and cortisol for biochemical characterization of our therapy. qRT-PCR results demonstrated a significant reduction in IL-1β, IL-6, TNF-α, and Iba1 in the striatum and hippocampus. Multiplex results reveal that AAV-7F9-Fc resulted in reduced IL-6 and IL-10, and increase in IL-2. These results showed that AAV-7F9-Fc was effective in mitigating the adverse effects of METH and MDMA in a rodent model.
|Advisor:||Peterson, Eric C|
|Commitee:||Owens, S. Michael, Gottschall, Paul E. , Stumhofer, Jason, McGehee, Robert, Jr., Bell, Anthony|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 81/5(E), Dissertation Abstracts International|
|Keywords:||Antibody, Blood brain barrier, MDMA, Methamphetamine, Neuroinflammation|
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