Evaluation of Biased and Balanced Salvinorin A Analogs in Preclinical Models of Pain

Abstract

In the search for safer, non-addictive analgesics, kappa opioid receptor (KOPr) agonists are a potential target, as unlike mu-opioid analgesics, they do not have abuse potential. Salvinorin A (SalA) is a potent and selective KOPr agonist, however, clinical utility is limited by the short duration of action and aversive side effects. Biasing KOPr signaling toward G-protein activation has been highlighted as a key cellular mechanism to reduce the side effects of KOPr agonists. The present study investigated KOPr signaling bias and the acute antinociceptive effects and side effects of two novel analogs of SalA, 16-Bromo SalA and 16-Ethynyl SalA. 16-Bromo SalA showed G-protein signaling bias, whereas 16-Ethynyl SalA displayed balanced signaling properties. In the dose-response tail-withdrawal assay, SalA, 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail-withdrawal assay, and 16-Ethynyl had greater antinociceptive effect in the hot-plate assay, compared to SalA. In the intraplantar 2% formaldehyde test, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced both nociceptive and inflammatory pain-related behaviors. Moreover, 16-Ethynyl SalA and 16-Bromo SalA had no anxiogenic effects in the marble burying task, and 16-Bromo SalA did not alter behavior in the elevated zero maze. Overall, 16-Ethynyl SalA significantly attenuated acute pain-related behaviors in multiple preclinical models, while the biased KOPr agonist, 16-Bromo SalA, displayed modest antinociceptive effects, and lacked anxiogenic effects.

Keywords: Salvinorin A; antinociception; anxiety; biased agonism; kappa opioid receptor.

FIGURE 1

Chemical structures of Salvinorin A, 16-Ethynyl Salvinorin A, and 16-Bromo Salvinorin A.

FIGURE 2

The activity of U50,488, 16-Ethynyl SalA, and 16-Bromo SalA in the cyclic adenosine monophosphate (cAMP) and β-arrestin recruitment assays. (A) The KOPr agonists were measured for inhibition of forskolin-induced cAMP (HitHunter^TM): U50,488 n = 29 across 11 experiments, 16-Ethynyl SalA n = 36 across 16 experiments, 16-Bromo SalA n = 18 across six experiments and (B) β-arrestin recruitment (PathHunter^TM): U50,488 n = 26 across nine experiments, 16-Ethynyl SalA n = 12 across three experiments, 16-Bromo SalA n = 15 across five experiments. Values presented as mean ± SEM.

FIGURE 3

Antinociceptive effects of KOPr agonists in acute thermal pain-related behavioral models. (A) Cumulative dose-response effects of 16-Ethynyl SalA and 16-Bromo SalA in the warm water (50°C) tail-withdrawal assay. The maximal possible effect (%MPE) at each dose was calculated as a percentage based on the pre-treatment baseline latencies. Non-linear regression analysis showed 16-Ethynyl SalA and 16-Bromo SalA both exerted antinociceptive effects (n = 6). (B,C) The warm water (50°C) tail-withdrawal latencies were measured over a time course, up to 120 min (n = 7). (B) 16-Ethynyl SalA showed a significant effect up to 15 min for the 1 mg/kg dose and 60 min for the 2 mg/kg dose. (C) 16-Bromo SalA showed a significant duration of action for up to 60 min. (D) The %MPE of the paw withdrawal time on the hot-plate (50°C) was calculated based on pre-treatment baseline latencies (n = 6). Mice were treated with either vehicle, SalA (2 mg/kg), 16-Ethynyl SalA (2 mg/kg) or 16-Bromo SalA (2 mg/kg) and the withdrawal latencies measured up to 60 min. All compounds had a duration of action of 30 min, and 16-Ethynl SalA was more potent than SalA and 16-Bromo SalA at the 30 min time point. Two-way repeated-measures ANOVA followed by Bonferroni post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for 2 mg/kg doses vs. vehicle control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 for 1 mg/kg doses vs. vehicle control. ^∧∧∧p < 0.001 for 16-Ethynyl SalA vs. both SalA and 16-Bromo SalA. Values presented as mean ± SEM.

FIGURE 4

Antinociceptive effect of 16-Ethynyl SalA and 16-Bromo SalA in the intraplantar 2% formaldehyde test. (A) Time course of pain-related behavior following intraplantar 2% formaldehyde injection into the right hind paw. 16-Ethynyl SalA (1–2 mg/kg i.p.) treatment showed a significant reduction in pain-related behavior compared to the vehicle/formaldehyde-treated control group. (B,C) The area under the curve (AUC) was calculated for phase I nociceptive pain-related behavior (0–15 min, B) and phase II inflammatory pain-related behavior (20–60 min, C). 16-Ethynyl SalA showed a significant reduction in both phases compared to the vehicle/formaldehyde treatment, which was reversed using the KOPr antagonist nor-binaltorphimine (nor-BNI). (D) Time course of pain-related behavior for 16-Bromo SalA (1–2 mg/kg i.p.) treatment showed a significant reduction in pain-related behavior compared to the vehicle/formaldehyde-treated positive control group. (E,F) AUC for phase I nociceptive pain-related behavior (0–15 min, E) and phase II inflammatory pain-related behavior (20–60 min, F). 16-Bromo SalA did not show a significant reduction in both phases of pain-related behavior compared to the vehicle/formaldehyde control group. (A,D) Two-way repeated-measures ANOVA with Bonferroni post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for 2 mg/kg dose vs. vehicle/formaldehyde control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for 1 mg/kg dose vs. vehicle/formaldehyde control. ^∧p < 0.05, ^∧∧p < 0.01, ^∧∧∧p < 0.001, ^∧∧∧∧p < 0.0001 for comparison between the 2 mg/kg with and without nor-BNI pre-treatment. (B,C,E,F) Kruskal–Wallis test with Dunn’s post-tests. Values presented as mean ± SEM, n = 6–7 and n = 5 for nor-BNI antagonist group. Number in brackets indicates dose in mg/kg and F = formaldehyde i.pl. administration.

FIGURE 5

Local adminstration of 16-Ethynyl SalA produced antinocieptive effects in the intraplantar 2% formaldehyde assay. (A) Time course of pain-related behavior following intraplantar 2% formaldehyde injection into the right hind paw. 16-Ethynyl SalA (2 mg/kg i.pl.) treatment showed a significant reduction in pain-related behavior compared to the vehicle/formaldehyde-treated control. Two-way repeated-measures ANOVA with Bonferroni post-tests. (B,C) Area under the curve (AUC) analysis of phase I (B) and II (C) pain-related behaviors. 16-Ethynyl SalA reduced both phases of pain-related behavior. Kruskal–Wallis test with Dunn’s post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for 2 mg/kg doses vs. vehicle/formaldehyde control. Values presented as mean ± SEM, n = 6. Number in brackets indicates dose in mg/kg and F = formaldehyde i.pl. administration.

FIGURE 6

KOPr agonists cause motor incoordination in the rotarod performance assay. Using a rotarod apparatus set to accelerate from 4 to 40 rpm over 300 s, SalA showed a significant effect at 15–30 min. (A) 16-Ethynyl showed a significant effect at 15–30 min. (B) 16-Bromo SalA had a significant impairment at 15–30 min at 1 mg/kg. Two-way repeated-measures ANOVA, **p < 0.01, ****p < 0.0001 for 2 mg/kg dose (above) or SalA (below) vs. vehicle. ^#p < 0.05, ^###p < 0.001 for 1 mg/kg dose vs. vehicle. ^∧indicates time point where SalA is more sedative than both doses of the novel analog. Values presented as mean ± SEM, n = 6.

FIGURE 7

Biased agonist 16-Bromo SalA does not have anxiogenic side effects compared to balanced agonist 16-Ethynyl SalA. Mice were tested on the elevated zero maze for 5 min with recordings made for the (A) time in the open area, (B) the number of open entries, and (C) number of head dips. SalA (2 mg/kg i.p.) and 16-Ethynyl SalA (1–2 mg/kg i.p.) reduced the all the behaviors, whereas, 16-Bromo SalA (1–2 mg/kg i.p.) had no effect. (D) The marble-burying experiment showed SalA administration lead to a significant increase in the percentage of marbles buried, whereas, the novel analogs did not alter the marble count. One-way ANOVA with Bonferroni post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to vehicle control. Values presented as mean ± SEM, n = 8.