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.

Keywords: Dopamine; Hyperthermia; Methamphetamine; Neurotoxicity; Serotonin; Sigma receptors.

Figure 1

Effects of DTG on lethality and hyperthermia in the absence and presence of methamphetamine (METH). A, male, Swiss Webster mice (N = 10/group) were pretreated i.p. with saline (Sal) or DTG (10 mg/kg) 15 min prior to receiving saline (Sal) or METH (5 mg/kg, i.p.). Dosing was repeated at two hour intervals up to a total of four times. Survival over 24 h was recorded. B, dose response of METH on core body temperature. Male, Swiss Webster mice (N = 5–10/group) were injected with saline or METH (1.25–10 mg/kg, i.p.) at two hour intervals for a total of four times. Core body temperature (BT) was measured via a rectal thermometer 1 h after each injection and data was reported as mean ± SEM. C, effect of DTG pretreatment on basal body temperature in the absence and presence of METH. Male, Swiss Webster mice (N = 5–16/group) were injected with saline or DTG (10 mg/kg, i.p.) 15 min prior to receiving saline or METH (5 mg/kg, i.p.) at two hour intervals for a total of four times. Core BT was measured via a rectal thermometer 1 h after each injection and data was reported as mean ± SEM.*P<0.05, **P<0.01, ***P<0.001 METH vs. saline; $$P<0.01 DTG vs. saline.

Figure 2

Effects of SN79 on body temperature in the absence and presence of METH. Male, Swiss Webster mice (N = 5–10/group) were pretreated with saline (Sal) or SN79 (1, 3, 10 mg/kg, i.p.), and after 15 min, the mice were treated with Sal or METH (5, 10 mg/kg, i.p.). Core body temperature was measured 1 h after each injection combination. This regimen was repeated four times at 2 h intervals. A, effects of SN79 on basal body temperature. B, effects of pretreatment with SN79 on METH (5 mg/kg, i.p.)-induced hyperthermia. C, effects of pretreatment with SN79 on METH (10 mg/kg, i.p.)-induced hyperthermia. Data was reported as mean ± SEM. **P<0.01, ***P<0.001 vs. saline; #P<0.05, ##P<0.01, ###P<0.001 vs. METH.

Figure 3

Effects of methamphetamine (METH) and SN79 on striatal dopamine levels. A, dose response of METH on dopamine (DA) levels in the striatum. Male, Swiss Webster mice (N = 5–9/group) were injected (i.p.) with METH (1.25- 5.0 mg/kg, i.p.) or saline (0 mg/kg, i.p.) at 2 h intervals for a total of four times. DA levels in the striatum were measured one week later. B, effects of SN79 on METH-induced depletion of DA levels in striatum of mouse brain tissue. Male, Swiss Webster mice (N = 5/group) were pretreated with saline or SN79 (1, 3, 10 mg/kg, i.p.). Mice were then treated with saline (-METH 0 mg/kg, i.p.) or METH (+METH 5 mg/kg, i.p.) after 15 min. This treatment regimen was repeated at 2 h intervals for a total of four times. Tissue samples from mouse striatum were collected and DA concentration was measured one week later. Data was reported as mean ± SEM. *P<0.05, ***P <0.001 vs. saline; ###P<0.001 vs. METH.

Figure 4

Effects of methamphetamine (METH) and SN79 on striatal 5-HT levels. A, dose response effects of METH on 5-HT levels in the striatum. Male, Swiss Webster mice (N = 5–10/group) were injected with METH (1.25–10.0 mg/kg, i.p.) or saline (0 mg/kg, i.p.) at 2 h intervals for a total of four times. Striatal tissue samples were collected one week later and measured for 5-HT concentration. B, effect of SN79 pretreatment on METH-induced alteration of 5-HT levels in the striatum of mouse brain. Male, Swiss Webster mice (N = 5–10/group) were pretreated with saline or SN79 (1, 3, 10 mg/kg, i.p.), and 15 min later, the mice were treated with saline (-METH 0 mg/kg, i.p.) or METH (+METH 10 mg/kg, i.p.). These treatment combinations were repeated at 2 h intervals a total of four times. One week later, mouse striatum were collected and 5-HT concentration was measured. Data was reported as mean ± SEM. **P<0.01 vs. saline, #P<0.05, ##P<0.01, ###P<0.001 vs. METH.

Figure 5

Dopamine transporter (DAT) Immunohistochemistry: Effect of SN79 pretreatment on methamphetamine (METH)-induced decrease in striatal DAT levels. Male, Swiss Webster mice (N = 4/group) were pretreated with saline (Sal) or SN79 (SN, 3 mg/kg, i.p.). After 15 min, the mice were then treated with Sal or METH (5 mg/kg, i.p.). This treatment schedule was repeated four times at 2 h intervals. One week later, the brains were removed and stained for DAT immunoreactivity. Average optical density readings (mean ± SEM) are shown. ***P<0.001 vs. saline, ###P<0001 vs. METH.

Figure 6

5-HT transporter (SERT) Immunohistochemistry: Effect of SN79 pretreatment on methamphetamine (METH)-induced decrease in striatal SERT levels. Male, Swiss Webster mice (N = 4/group) were pretreated with saline (Sal) or SN79 (SN, 3 mg/kg, i.p.). After 15 min, the mice were then treated with Sal or METH (5 mg/kg, i.p.). This treatment schedule was repeated four times at 2 h intervals. One week later, the brains were removed and stained for SERT immunoreactivity. Average optical density readings (mean ± SEM) are shown. ***P<0.001 vs. saline, ###P<0001 vs. METH.

Figure 7

Relationship between core body temperature taken after each of the four drug administrations (BT1-4) and A, striatal dopamine (DA) levels or B, striatal 5-HT levels measured one week later. The graphs depict the relationship for individual animals corresponding to the data summarized in Table 1.

Figure 8

Effects of post-treatment with SN79 on methamphetamine (METH)-induced striatal dopamine (DA) depletion. Male, Swiss Webster mice (N = 10/group) were treated (i.p.) with saline (Sal) or METH (5 mg/kg) at 2 h intervals a total of four times. Beginning 3 h after the last injection, mice were orally administered distilled water (H₂O) or SN79 (SN, 10 mg/kg) every 8 h for one week. The striata were dissected from each mouse and DA levels measured. Data was reported as mean ± SEM. ***P<0.005 vs. Sal/H₂O.