In vitro and In vivo Effects of Phase 1 Hydroxylated Metabolites of the Synthetic Cannabinoid AB‐PINACA [(S)‐N‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐pentyl‐1H‐indazole‐3‐carboxamide

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Synthetic cannabinoids (SCBs) in abused K2/Spice products exhibit greater toxicity than marijuana. Many SCBs, but not the major psychoactive constituent in marijuana Δ9‐tetrahydrocannabinol (THC), undergo biotransformation to several active metabolites that may contribute to enhanced adverse effects. AB‐PINACA [(S)‐N‐(1‐amino‐3‐methyl‐1‐oxobutan‐2‐yl)‐1‐pentyl‐1H‐indazole‐3‐carboxamide] is a novel illicit SCB for which no information concerning activity of known metabolites has been reported. Therefore, the purpose of this study was to determine if the reported major metabolites of AB‐PINICA (4OH‐ and 5OH‐AB‐PINACA) retain cannabinoid type 1 (CB1) receptor affinity and/or activity in both in vitro and in vivo assays.CHO cells stably expressing human CB1 receptors (CHO‐hCB1) were used for in vitro studies. Receptor affinity was determined by competition receptor binding, and functional activity was assessed by measuring G‐protein activation and inhibition of adenylyl cyclase activity. For in vivo studies, mice were injected (i.p.) with 4OH‐AB‐PINACA at either 30 or 100 mg/kg and examined for effects in the cannabinoid tetrad at 10‐, 30‐, 60‐, 120‐, 180‐, 240‐, and 360‐minutes post injection. Hypothermic effects were assessed by digital rectal thermometer, antinociception via tail immersion in a 50°C water bath, and cataleptic effects via time spent with forepaws on the catalepsy bar. Locomotor activity was also evaluated at either the 30‐ or 60‐minute time points (100 mg/kg and 30 mg/kg doses, respectively) in the open field test using quadrant crossings as a measure of total activity.AB‐PINACA (Ki=4.0 nM) and both major phase I metabolites, 4OH‐AB‐PINACA (Ki=159 nM) and 5OH‐AB‐PINACA (Ki=452 nM), bind to hCB1 receptors with nanomolar affinity. In G‐protein activation assays, AB‐PINACA (ED50=9.1 nM) and metabolites 4OH‐AB‐PINACA (ED50=642 nM) and 5OH‐AB‐PINACA (ED50=2742 nM) all act as agonists, producing concentration‐dependent activation of G‐proteins. Consistent with agonist activity observed in G‐protein assays, in a second functional assay, AB‐PINACA and metabolite 4OH‐AB‐PINACA also inhibit adenylyl cyclase activity with potencies of 3.9 nM and 141 nM, and efficacies of 72% and 87%, respectively. As predicted from in vitro data, 4OH‐AB‐PINACA elicited in vivo effects consistent with that of cannabinoid agonists, producing hypothermia, analgesia and suppression of locomotor activity. Interestingly, with the exception of one animal (30 mg/kg dose at 10‐min), catalepsy was not observed.In conclusion, these data indicate that AB‐PINACA, similar to several previously reported SCBs, is biotransformed to active phase I metabolites that may contribute to toxicity by enhancing and/or prolonging parent drug effects.Support or Funding InformationThis research was supported in part by by NIDA grant: DA039143 and by the UAMS Center for Translational Neuroscience (GM110702). CDW is supported by 3R01DA039143‐02S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.