Semester

Spring

Date of Graduation

2012

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Rae R Matsumoto

Abstract

Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse, causing hyperthermia and neurotoxicity at high doses. Currently, there is no clinically proven pharmacotherapy to treat these effects of METH, necessitating identification of potential novel therapeutic targets. Earlier studies showed that METH binds to sigma receptors in the brain at physiologically relevant concentrations, where it acts in part as an agonist. AC927 (1-(2-phenethyl)piperidine oxalate), CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d]thiazole-2(3H)-thione) and SN79 (6-acetyl-3-(4-(4-(4-florophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one) were synthesized as putative sigma receptor antagonists with nanomolar affinity and selectivity for sigma receptors over 29-80 other binding sites. AC927, CM156 and SN79 pretreatment afforded protection against METH-induced hyperthermia and striatal dopaminergic and serotonergic neurotoxicity in male Swiss Webster mice (measured as depletions in striatal dopamine and serotonin levels, and reductions in striatal dopamine and serotonin transporter expression levels). To elucidate the mechanism of neuroprotection provided by sigma receptor antagonists, differentiated NG108-15 cells were used as a cell culture model. METH, at physiologically relevant micromolar concentrations, caused apoptosis in NG108-15 cells. At higher concentrations necrotic cell death was observed. At earlier time points, METH caused generation of reactive oxygen/nitrogen species like hydrogen peroxide and nitric oxide and also release of dopamine from the cells. At later time points, caspase-3, -8 and -9 activation was observed indicating activation of both extrinsic and intrinsic caspase-dependent apoptotic pathways. Sigma receptor antagonism attenuated these neurotoxic mediators and cell death. Additionally, DTG, a sigma receptor agonist shifted the dose response curve of METH-induced cell death (especially necrosis) towards left further confirming the involvement of sigma receptors in the neurotoxic effect of METH. Among the two subtypes of sigma receptors, sigma-2 receptors are highly upregulated in tumor cells and are implicated in cell death mechanism. Both CM398 and CM775, sigma-2 receptor antagonists also attenuated METH-induced neurotoxicity, providing further evidence for involvement of this subtype in METH-induced cell death mechanisms. In addition to activation of cell death mediators, METH also causes increase in body temperature. METH treatment in NG108-15 cells maintained at 40 ºC versus 37 ºC caused a significant and synergistic increase in cell death, indicating that increase in temperature exacerbates METH-induced toxicity. SN79 was able to attenuate the enhanced cell death observed in the METH treated cells at 40 ºC. This can have important implications in in vivo systems where sigma receptor antagonism can protect against toxicity induced by METH at elevated temperature. This study provided evidence for the potential application of sigma receptor antagonists as drug development against METH-induced neurotoxicity, in addition to providing a mechanistic basis of neuroprotection.

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