Xylazine is a Kappa-Opioid Agonist with Gender-Specific Responses to Opioid Antagonists (e.g. Naloxone)

"Here, we report the first xylazine dose-response locomotor study in male and female mice as well as the first assessment of adrenergic- and opioid-receptor antagonist-precipitated withdrawal symptoms following, xylazine, fentanyl, and xylazine/fentanyl administration in mice. These experiments show that male and female mice are differentially sensitive to xylazine. We find female mice are less sensitive to the motor-suppressing effects of xylazine contrary to the recent findings in rats reported by Khatri et al. (2023), potentially due to their use of repeated dosing of xylazine or species differences [27]. Using a modified version of our 3-day precipitated withdrawal model [40,41,46], we show xylazine is indeed responsive to naloxone, contrary to common assumptions made by both health professionals and in the media [7]. Both sexes exhibited some level of somatic withdrawal behaviors to xylazine and naloxone, though females showed sensitized behavioral responding. Indeed, females appear to be as sensitive, if not more sensitive to xylazine withdrawal than fentanyl withdrawal at tested doses, while males remain much more responsive to fentanyl withdrawal conditions. At the doses tested in our study, the effect of naloxone precipitated withdrawal on xylazine/fentanyl combination was synergistic as compared to each drug in isolation. This was especially apparent when examining increased bouts of paw tremors, which may represent a more passive coping behavior that we have previously observed is sexually dimorphic in opioid withdrawal [41]. In contrast, we did not observe similar findings when withdrawal was precipitated by atipamezole, an α2-AR antagonist used anesthesia reversal in veterinary medicine. These intriguing findings led us to consider the possibility of direct xylazine activity on opioid receptors. Previous studies have shown that xylazine is antinociceptive, results in a cross-tolerance to some mechanisms of opioid induced antinociception, and that these effects are naloxone-sensitive, but surprisingly not sensitive to the κOR selective antagonist nor-BNI [57–60]. Congruent with this data, we did not observe significant expression of withdrawal behavior to nor-BNI precipitated withdrawal, and pretreatment with nor-BNI exacerbated naloxone precipitated withdrawal in female mice. Until now, xylazine was thought to exert these effects through promotion of endogenous opioid release and xylazine has not been directly tested as a potential opioid agonist. We are the first to report definitive evidence that xylazine acts as a full agonist at κOR and is biased towards G-protein signaling pathways."