Xylazine is an agonist at kappa opioid receptors and exhibits sex-specific responses to opioid antagonism

Abstract

Xylazine is in the unregulated drug supply at increasing rates, usually combined with fentanyl, necessitating understanding of its pharmacology. Despite commentary from politicians, and public health officials, it is unknown how xylazine impacts naloxone efficacy, and. few studies have examined it alone. Here, we examine the impact of xylazine alone and in combination with fentanyl on several behaviors in mice. Surprisingly, naloxone precipitates withdrawal from xylazine and fentanyl/xylazine coadministration, with enhanced sensitivity in females. Further, xylazine is a full agonist at kappa opioid receptors, a potential mechanism for its naloxone sensitivity. Finally, we demonstrate surprising effects of xylazine to kappa opioid antagonism, which are relevant for public health considerations. These data address an ongoing health crisis and will help inform critical policy and healthcare decisions.

Keywords: Fentanyl; Mouse; Opioid; Pharmacology; Xylazine.

Fig. 1.

Effect of acute IP xylazine administration on locomotor activity. (A) Cumulative distance traveled, (B) % ambulatory time, and (C) average velocity of male and female mice administered saline or xylazine (0.5, 1.0, or 3.0 mg/kg). (D) Distance traveled, (E) % ambulatory time, and (F) velocity split into 30 min bins. (D-F) 3-way ANOVAs (Time x Sex x Dose).

Fig. 2.

Naloxone- and atipamezole-precipitated withdrawal. (A) 3-day precipitated withdrawal paradigm. Global scores are shown as average z-score ± SEM. (B) Average z-scores of female and male mice over three days of naloxone-precipitated withdrawal. (C) Heatmap of average z-scores on day three of withdrawal for individual behaviors. (D) Average z-scores of female and male mice over three days of atipamezole-precipitated withdrawal. (E) Heatmap of average z-scores on day three of atipamezole-precipitated withdrawal for individual behaviors. (B&D) 3-way ANOVAs (Day X Addition of Fentanyl X Addition of Xylazine) P = 0.05*, 0.01**, 0.001***, 0.0001****. Main effects p-values and Tukey’s post-hoc shown in Fig. S2. (C&E) 2-way ANOVAs (Tx Group X Behavior) with Tukey’s post-hoc, P = 0.05 where * (vs. saline), # (vs. fentanyl), @ (vs. fentanyl/xylazine), and $ (vs. xylazine). SN=saline-naloxone; FN=fentanyl-naloxone; FXN=fentanyl/xylazine-naloxone; XN=xylazine-naloxone; SA=saline-atipamezole; FA=fentanyl-atipamezole; FXA=fentanyl/xylazine-atipamezole; XA=xylazine-atipamezole.

Fig. 3.

Quantification of c-Fos expression following naloxone-precipitated withdrawal. (A) Female and male c-Fos expression displayed as number of positive cells per mm2 in various regions of interest. (B) Representative images of female regions of interest. (C) Representative images of male regions of interests. (A) 2-way ANOVAs (Tx group X Sex) with Tukey’s post-hoc. P = 0.05*, 0.01**, 0.001***, 0.0001****.

Fig. 4.

Xylazine acts as a G-protein biased agonist at κOR and α_2A-AR. (A-B) Radioligand competitive binding assay confirms xylazine activity at κOR (A) and α₂-AR (B), shown with known reference agonists. (C-D) Gi-GloSensor cAMP assays at κOR (C) and α₂-AR (D). (E-L) TRUPATH BRET2 assays for Gi1 (κOR (E) and α₂-AR (F)), GoA (κOR (G) and α₂-AR (H)), Gz (κOR (I) and α₂-AR (J)), and Barr2 (κOR (K) and α₂-AR (L)).

Fig. 5.

Female mice exhibit increased responses to kOR antagonism. (A) nor-BNI-precipitated withdrawal paradigm. Mice received agonist injections for 3 days and nor-BNI 2 h later only on the 3rd day. (B) nor-BNI pretreated withdrawal paradigm. Mice received nor-BNI or saline 7 days prior to xylazine and naloxone-precipitated withdrawal. (C) Average z-score of nor-BNI precipitated withdrawal for female and male mice. 2-way ANOVA (Treatment Group X Sex) (D) Average z-score of nor-BNI pretreated naloxone withdrawal for individual behaviors. (E) Average z-score of nor-BNI pretreated naloxone withdrawal for female and male mice across 3 days. 2-way ANOVAs (Day X Pretreatment Condition). Tukey’s post-hoc Day 1 vs Day 3: P = P = 0.05⁺, 0.01⁺⁺, 0.001⁺⁺⁺, 0.0001⁺⁺⁺⁺. (F) Average z-score of nor-BNI pretreated naloxone withdrawal for individual behaviors on day 3. P = 0.05*, 0.01**, 0.001***, 0.0001****. Main effects p-values and Tukey’s post-hoc shown in Fig. S6. (D & F) 2-way ANOVAs (Tx Group X Behavior) with Tukey’s post-hoc, P = 0.05 where * (vs. saline), # (vs. fentanyl), @ (vs. fentanyl/xylazine), and $ (vs. xylazine). XN=xylazine-naloxone; SB=saline-nor-BNI; FB=fentanyl-nor-BNI; FXB=fentanyl/xylazine-nor-BNI; XB=xylazine-nor-BNI.