Pharmacological evaluation of SN79, a sigma (σ) receptor ligand, against methamphetamine-induced neurotoxicity in vivo

Abstract

Methamphetamine 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 methamphetamine, necessitating identification of potential novel therapeutic targets. Earlier studies showed that methamphetamine binds to sigma (σ) receptors in the brain at physiologically relevant concentrations, where it “acts in part as an agonist.” SN79 (6-acetyl-3-(4-(4-(4-florophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one) was synthesized as a putative σ receptor antagonist with nanomolar affinity and selectivity for σ receptors over 57 other binding sites. SN79 pretreatment afforded protection against methamphetamine-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). In contrast, di-o-tolylguanidine (DTG), a well established σ receptor agonist, increased the lethal effects of methamphetamine, although it did not further exacerbate methamphetamine-induced hyperthermia. Together, the data implicate σ receptors in the direct modulation of some effects of methamphetamine such as lethality, while having a modulatory role which can mitigate other methamphetamine-induced effects such as hyperthermia and neurotoxicity.

Introduction

High or repeated methamphetamine administration results in hyperthermia, neurotoxicity, and even mortality (Cruickshank and Dyer, 2009). In response to high doses of methamphetamine, cellular neurotoxic cascades are activated due to excessive dopamine and 5-HT release into the cytoplasm and synapse (Baldwin et al., 1993, Kuczenski et al., 1995, Gough et al., 2002). Damage to dopaminergic and serotonergic neurons, which can be measured as reductions in dopamine transporters (DAT), serotonin transporters (SERT), dopamine and 5-HT levels, is observed in several brain regions in human methamphetamine users, as well as in laboratory animals (Krasnova and Cadet, 2009).

Numerous clinical and imaging studies have indicated an association between neurotoxicity and several neuropsychiatric disorders, including psychosis (Scott et al., 2007). Motor and cognitive deficits have also been reported (Hart et al., 2012, Scott et al., 2007), including a recent study showing that long term methamphetamine abuse can increase the risk of Parkinson's disease (Callaghan et al., 2012). Clinical cases of methamphetamine-related fatalities are also rising (Krasnova and Cadet, 2009, Hart et al., 2012), and these lethal effects have been linked to methamphetamine-induced hyperthermia (Bowyer et al., 1994).

Recent evidence has shown that methamphetamine binds to and produces some of its behavioral effects through sigma (σ) receptors, and these proteins can be targeted to mitigate the effects of methamphetamine (Kaushal and Matsumoto, 2011, Nguyen et al., 2005, Robson et al., 2012). In addition to methamphetamine, the psychostimulant cocaine also binds to σ receptors at physiologically relevant concentrations (Robson et al., 2012). With the aim of developing a medication to counteract the harmful effects of psychostimulant abuse, SN79 (6-acetyl-3-(4-(4-(4-florophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one), a putative σ receptor antagonist with druggable properties, including a long half life and good oral bioavailability, was developed (Kaushal et al., 2011a). SN79 interacts with both σ₁ and σ₂ receptors (K_i 27 and 7 nM, respectively; Kaushal et al., 2011a). It was also shown to attenuate the acute and subchronic effects of cocaine in mice upon intraperitoneal as well as oral administration, making it a viable preclinical drug candidate (Kaushal et al., 2011a).

In the present study, the involvement of σ receptors in methamphetamine-induced toxicity was further evaluated, with an emphasis on hyperthermia, neurotoxicity, and lethality. Modulation of methamphetamine-induced effects was assessed using the well established σ receptor agonist, di-o-tolylguanidine (DTG), and the novel σ receptor putative antagonist SN79. In the first part of the study, it was determined if DTG worsens the effects of methamphetamine. In the second part of the study, it was determined if SN79 attenuates the hyperthermia, neurotoxicity, and lethality caused by methamphetamine. Four well known markers of methamphetamine-induced neurotoxicity were measured: reductions in striatal dopamine and 5-HT levels, as well as decreases in striatal DAT and SERT expression (Krasnova and Cadet, 2009). Striatal tissue was evaluated in the present investigation because it contains the terminals of monoaminergic neurons that are primarily affected by methamphetamine-induced neurotoxicity (Brunswick et al., 1992, Kovachich et al., 1989, Ricaurte et al., 1980, Seiden et al., 1988).

Methamphetamine also causes an elevation in body temperature (Fukumura et al., 1998, Numachi et al., 2007). Earlier studies have shown that hyperthermia potentiates methamphetamine-induced dopamine and 5-HT depletions and exacerbates oxidative stress in the brain (Bowyer et al., 1994, Fukumura et al., 1998, Hirata et al., 1995), whereas hypothermia protects against these effects (Bowyer et al., 1994). Therefore, in the third part of the study, to determine if the neuroprotective effects of SN79 are associated with its ability to decrease the hyperthermic effects of methamphetamine, correlation analysis compared the body temperatures of mice in the various treatment groups with their corresponding striatal dopamine and 5-HT levels.

In addition to establishing an important involvement of σ receptors in the effects of methamphetamine, the ability of SN79 to be clinically developed as a drug candidate against methamphetamine-induced neurotoxicity was also evaluated as a post-treatment following methamphetamine exposure. Previous post-treatment studies have shown σ receptor antagonists to be effective against cocaine-induced lethality (Matsumoto et al., 2001). Therefore, in the fourth part of this study, post-treatment experiments were conducted to evaluate the effectiveness of orally administered SN79 in attenuating methamphetamine-induced striatal dopaminergic neurotoxicity.