In 2019 synthetic cannabinoids accounted for more than one-third of new drugs of abuse worldwide. As controlled substances, it is not ethically feasible to assess health risks posed by synthetic cannabinoids, so that exposure studies with CD-1 mice have become the gold standard. The interpretation of those findings will depend on the extent to which the mice exhibit the same metabolic pathways in humans that alter specificity and activity of the drugs. Herein, we report the first analysis of synthetic cannabinoid metabolic pathways in CD-1 mouse. Specifically, we determined and compared steady-state metabolism of N-(1-adamantyl)-1-(5-pentyl)-1H-indazole-3-carboxamide (5F-APINACA/5F-AKB48) by CD-1 mice and humans using hepatic microsomes. Regardless of species, 5F-APINACA metabolism involved two major, highly efficient pathways: (1) sequential adamantyl hydroxylation and (2) oxidative defluorination. Analysis of kinetic profiles indicated equal competition between pathways although the secondary adamantyl hydroxylation was less efficient for mice. At low 5F-APINACA levels, initial pathway rates were comparable for human and mouse microsomal reactions. Nevertheless, at higher 5F-APINACA levels, oxidative defluorination dominated due to substrate inhibition of adamantyl dihydroxylations that may involve occupation of a substrate effector site. For human liver microsomes, CYP3A4 dominated metabolism for both pathways with minor contributions from CYP2C8, 2C19 and 2D6. For CD-1 mouse, Cyp3a11 and a Cyp2c member contributed equally to adamantyl hydroxylation, but Cyp3a11 was more efficient at oxidative defluorination than the Cyp2c member. In following, factors impacting P450 metabolism are likely held in common between species with possible confounding effects from Cyp2c contributions in mouse. Taken together, in vitro steady-state studies indicated high conservation of 5F-APINACA metabolism between CD-1 mice and humans but possible deviations in variability within a population as general support on the translatability of findings from mice studies to humans.

Similar in vitro metabolic kinetics for 5F-APINACA between CD-1 mouse and human but involve different cytochromes P450

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