Pharmacology and anti-addiction effects of the novel κ opioid receptor agonist Mesyl Sal B, a potent and long-acting analogue of salvinorin A

Abstract

Background and purpose: Acute activation of κ opioid (KOP) receptors results in anticocaine-like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi-synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile.

Experimental approach: We evaluated the duration of effects of Mesyl Sal B in vivo utilizing antinociception assays and screened for cocaine-prime induced cocaine-seeking behaviour in self-administering rats to predict anti-addiction effects. Cellular transporter uptake assays and in vitro voltammetry were used to assess modulation of dopamine transporter (DAT) function and to investigate transporter trafficking and kinase signalling pathways modulated by KOP receptor agonists.

Key results: Mesyl Sal B had a longer duration of action than SalA, had anti-addiction properties and increased DAT function in vitro in a KOP receptor-dependent and Pertussis toxin-sensitive manner. These effects on DAT function required ERK1/2 activation. We identified differences between Mesyl Sal B and SalA, with Mesyl Sal B increasing the Vmax of dopamine uptake without altering cell-surface expression of DAT.

Conclusions and implications: SalA analogues, such as Mesyl Sal B, have potential for development as anticocaine agents. Further tests are warranted to elucidate the mechanisms by which the novel salvinorin-based neoclerodane diterpene KOP receptor ligands produce both anti-addiction and adverse side effects.

Linked articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Keywords: Salvia divinorum; addiction; cocaine self-administration; dopamine transporter; drug seeking; nociception; salvinorin A; κ opioid receptor.

Figure 1

Chemical structures and in vitro functional potencies (EC₅₀) for SalA (A) and Mesyl Sal B (B). Reported previously in Harding et al. (2005). [³⁵S]GTPγS assays in CHO cells stably expressing human KOP receptors. EC₅₀ SalA 40 ± 10 nM (EC₅₀ ± SD), Emax 120 ± 2% compared with U50, 488H [% stimulation of binding compared with U50, 488H (500 nM)]. Mesyl Sal B: EC₅₀ 30 ± 5; Emax 112%.

Figure 2

Duration of action of Mesyl Sal B in vivo in the hot water tail immersion assay in mice. Tail-withdrawal latency is expressed as percent maximal possible effect (% MPE ± SEM) versus time. SalA (1 mg·kg^–1) has rapid analgesic effects compared with vehicle at 5, 10 and 15 min. Mesyl Sal B (1 mg·kg⁻¹) has a slower onset and longer duration of action with increased tail-withdrawal latencies at 30, 45 and 60 min. SalA and Mesyl Sal B showed significant differences at 10 min (SalA compared to veh *P < 0.05, ***P < 0.001 ****P < 0.0001, Mesyl Sal B compared to veh #P < 0.05, SalA compared with Mesyl Sal B ^∧∧∧∧P < 0.0001). Repeated measures anova revealed a significant effect of time [F(3603) = 4.952; P < 0.0001], drug [F(6625) = 19.88; P < 0.0001] and a drug and time interaction [F(3580) = 3.69; P < 0.001]. Two-way anova followed by Bonferroni post test (n = 9).

Figure 3

Mesyl Sal B attenuates cocaine-prime induced cocaine-seeking behaviour in rats. (A) Active lever press responses following Mesyl Sal B 45 min prior to a priming injection of cocaine (20 mg·kg⁻¹) ***P < 0.001, data compared with vehicle treated controls (0 mg·kg⁻¹): one-way anova followed by Tukey's post hoc test: n = 5–6 for each group. (B) norBNI (30 min pretreatment, 2 mg·kg⁻¹, s.c.) followed by Mesyl Sal B (0.3 mg·kg⁻¹, ip) during phase 3 of reinstatement tests (mean ± SEM). (C) Spontaneous locomotor activity in vehicle and Mesyl Sal B treated rats over 60 min (mean ± SEM) (Student's t-test) (n = 7 for each group).

Figure 4

Mesyl Sal B modulates DAT function in vitro via a KOP receptor-dependent and PTX-sensitive mechanism in cells transiently expressing YFP-hDAT and Myc-rKOP receptor (A) Mesyl Sal B, (B) U50,488H and (C) SalA cause concentration-dependent increases in ASP+ uptake. These effects (at 10 μM Mesyl Sal B) were norBNI reversible (30 min pretreatment, 1 μM compared to 10 μM drug, #P < 0.05, ##P < 0.01, ###P < 0.001). Mesyl Sal B (10 μM) also caused an increase in ASP+ uptake in (D) N2A and (E) COS7 cells. (F) ASP+ binding following Mesyl Sal B (10 μM). The addition of DMSO (at time 0) shows that the vehicle control did not lead to a significant decrease in uptake from vehicle for any of the agonists (one sample Student's t-test). Experiments were carried out over three transfections with at least two dishes used per transfection (n = 46–64 cells). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the vehicle control without KOP receptor agonist (mean ± SEM).

Figure 5

Mesyl Sal B modulates DAT function through an ERK1/2-dependent pathway. (A) Fluorescent scan of a representative gel showing YFP-hDAT expression (top), P-ERK1/2 expression (middle) and total ERK1/2 expression (bottom) after Mesyl Sal B treatment (10 μM). (B) P-ERK1/2 expression was normalized to total ERK1/2 and expressed graphically as a fold change compared with time 0 (one-way anova followed by Bonferroni post test, *P < 0.01, n = 4). (C & E) Inhibition of ERK1/2 activation by U0126 pretreatment (20 μM, 30 min) one-way anova followed by Bonferroni post test, *P < 0.05, **P < 0.01 compared with appropriate control, ###P < 0.001 compared with appropriate Mesyl Sal B treated control, n = 6. (D) A representative Western blot of the effect of U0126 on the Mesyl Sal B-induced phosphorylation of ERK1/2. (E) The increase in uptake caused by Mesyl Sal B was inhibited by U0126; one-way anova followed by Bonferroni post test, ***P < 0.001 compared with DMSO control, ###P < 0.001 compared with Mesyl Sal B treated control respectively (n = 32–50). The addition of DMSO as a vehicle control did not lead to a significant decrease in uptake from 0 (one sample Student's t-test).

Figure 6

Mesyl Sal B modulates ERK phosphorylation in vivo. (A) P-ERK1/2 expression in the dStr, (C) NAcb and (E) prefrontal cortex was normalized to total ERK1/2 and expressed as fold change (mean ± SEM) compared with time 0. ERK1/2 phosphorylation following Mesyl Sal B administration (Student's t-test, *P < 0.05, n = 6–17). Representative Western blot showing P-ERK1/2 expression (top) and ERK1/2 expression (bottom) after Mesyl Sal B administration in (B) dStr, (D) NAcb and (F) prefrontal cortex. MM = molecular weight marker.

Figure 7

Mesyl Sal B modulates DAT function ex vivo via a KOP receptor dependent mechanism. (A) NAcb tissue was pre-incubated with vehicle (DMSO matched), or KOP receptor agonist U50,488H or Mesyl Sal B before sequential addition of dopamine. Data points shown are the mean ± SEM (n = 6–8 tissue homogenates). Mesyl Sal B significantly increased V_max (1567 ± 228, pmol·s^–1·g^–1) (**P < 0.01) with no change in K_m (1.9 ± 0.6 μM) compared with vehicle control. U50,488H also showed an increase in V_max [1837 ± 406 pmol·s⁻¹·g⁻¹ (*P < 0.05)] with no change in K_m (1.9 ± 0.9 μM). (B) Mesyl Sal B (500 nM) and U50,488H showed a significant increase in dopamine clearance in the NAcb (P < 0.05 and P < 0.01, respectively), dorsal striatum (P < 0.01 and P < 0.01, respectively) and prefrontal cortex (P < 0.01 and P < 0.01, respectively) following a single addition of dopamine (2 μM). (C) This increase in dopamine clearance was inhibited by prior incubation of dStr tissue with norBNI (1 μM, 30 min) (#P < 0.05). Incubation with norBNI alone had no effect on dopamine uptake. Pre-incubation of dStr tissue with U0126 (10 μM) prevented Mesyl Sal B from increasing DAT function (Mesyl Sal B + U0126 compared with DMSO + U0126 (P = 0.9368); Mesyl Sal B/vehicle compared with Mesyl Sal B + U0126 (***P < 0.001); one-way anova followed by Bonferroni post test, (n = 6–7). DA = dopamine.

Figure 8

The increase in DAT function following Mesyl Sal B is not via an increase in DAT cell-surface expression in HEK-293 cells. (A–C) HEK-293 cells were incubated with KOP receptor agonists (10 μM, 30 min) before biotinylation studies. (D–F) TIRFM studies were carried out on HEK-293 cells expressing YFP-hDAT and myc-rKOP receptor with drug addiction occurring at 10 min. (A) Mesyl Sal B biotinylation (one-way anova followed by Bonferroni post test; n = 12–13), (D) Mesyl Sal B TIRFM experiments (two-way anova followed by Bonferroni post test; n = 9–10). (B) U69,593 biotinylation (one-way anova followed by Bonferroni post test, *P < 0.05; n = 6–8), (E) TIRFM studies with U69,593 (two-way anova followed by Bonferroni post test, *P < 0.05, **P < 0.01; n = 12). (C) SalA biotinylation experiments (one-way anova followed by Bonferroni post test, **P < 0.01; n = 12–16) and (F) TIRFM studies with Sal A (two-way anova followed by Bonferroni post test, *P < 0.05; n = 7–9). T = total, I = internal, CS = cell surface.