Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats

Abstract

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.

Keywords: RXFP3; alcohol; relaxin-3; self-administration.

Figure 1.

Experimental timeline and subject details for Experiments 1 and 2 (A) and Experiment 3 (B).

Figure 2.

A. RLX-33 reduced alcohol lever responses in male (n = 11) and female (n = 12) Wistar rats. B. All doses of RLX-33 reduced alcohol lever responses at each time point. C. RLX-33 had no effect on locomotor rate during alcohol self-administration period. D. RLX-33 reduced sucrose self-administration though no differences were found in post-hoc analyses (n = 10 males, 8 females). E. RLX-33 at the 20 mg/kg dose reduced sucrose self-administration after minute 15 (p<0.05) through the remainder of the session relative to vehicle in males, with no effects in females. F. RLX-33 had no effect on locomotor rate during sucrose self-administration period. X-axis break signified that the sexes were analyzed separately. Data are shown as mean ± SEM. *p<0.05, **p<0.01 compared with same sex vehicle. ^@5mg/kg, ^#10 mg/kg, ^20 mg/kg: p<0.05 compared with same-sex vehicle. ^&Main effect of RLX-33 dose. X-axis break indicates a main effect of sex and thus sexes were analyzed separately.

Figure 3

A. RLX-33 reduced alcohol self-administration in male (n = 13), but not female (n = 8) P rats B. RLX-33 reduced the cumulative alcohol lever responses at all time points in males but not in females. C. RLX-33 had no effect on locomotor rate during alcohol self-administration. D/E. RLX-33 had no effect on sucrose lever responses during sucrose self-administration. F. RLX-33 had no effect on locomotor rate during sucrose self-administration. Data are shown as mean ± SEM. ****p<0.0001 compared with same sex vehicle. ^@5mg/kg, ^#10 mg/kg, ^20 mg/kg: p<0.001 compared with same-sex vehicle. X-axis break indicates a main effect of sex and thus sexes were analyzed separately.

Figure 4.

A. Timeline of Experiment 3. B. RLX-33 had no effect on total distance traveled in the open field test. C. RLX-33 decreased percentage of time spent in center of open field in males (n = 7–8 per treatment, N = 31), but not in females (n = 8 per treatment, N = 32). D. Time spent in open area in the zero maze was unaffected by RLX-33. E. The 5 mg/kg dose of RLX-33 increased acoustic startle response to the 110 and 120 dB stimulus in males, but not in females. F. RLX-33 (20 mg/kg) increased prepulse inhibition of the acoustic startle response in females, but not in males. G. Rats received RLX-33 (IP) then 1 hour later were given of 1 g/kg alcohol (IG). Alcohol clearance rate were increased following of the highest dose (20 mg/kg) of RLX-33 in male, but not in female rats. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared with same sex vehicle. X-axis break indicates a main effect of sex and thus sexes were analyzed separately.