Similar 5F-APINACA Metabolism between CD-1 Mouse and Human Liver Microsomes Involves Different P450 Cytochromes

Abstract

In 2019, synthetic cannabinoids accounted for more than one-third of new drugs of abuse worldwide; however, assessment of associated health risks is not ethical for controlled and often illegal substances, making CD-1 mouse exposure studies the gold standard. Interpretation of those findings then depends on the similarity of mouse and human metabolic pathways. Herein, we report the first comparative analysis of steady-state metabolism of N-(1-adamantyl)-1-(5-pentyl)-1H-indazole-3-carboxamide (5F-APINACA/5F-AKB48) in CD-1 mice and humans using hepatic microsomes. Regardless of species, 5F-APINACA metabolism involved highly efficient sequential adamantyl hydroxylation and oxidative defluorination pathways that competed equally. Secondary adamantyl hydroxylation was less efficient for mice. At low 5F-APINACA concentrations, initial rates were comparable between pathways, but at higher concentrations, adamantyl hydroxylations became less significant due to substrate inhibition likely involving an effector site. For humans, CYP3A4 dominated both metabolic pathways with minor contributions from CYP2C8, 2C19, and 2D6. For CD-1 mice, Cyp3a11 and Cyp2c37, Cyp2c50, and Cyp2c54 contributed equally to adamantyl hydroxylation, but Cyp3a11 was more efficient at oxidative defluorination than Cyp2c members. Taken together, the results of our in vitro steady-state study indicate a high conservation of 5F-APINACA metabolism between CD-1 mice and humans, but deviations can occur due to differences in P450s responsible for the associated reactions.

Keywords: 5F-AKB48; 5F-APINACA; CB1 receptor; P450; drug abuse; enzyme kinetics; human; metabolism; mouse; synthetic cannabinoid.

Figure 1

Reported competing and intersecting metabolic pathways of 5F-APINACA. Current experimental design limited metabolism to sequential oxidation of the adamantyl group (blue arrows) and the initial oxidative defluorination (red arrows), avoiding previously reported steps and higher order metabolites, shown in gray [27,28,29,30].

Figure 2

Steady-state metabolism 5F-APINACA by human liver microsomes fit to traditional kinetic models. (A) Kinetic plot for primary metabolites, i.e., 5OH-APINACA from oxidative defluorination of the pentyl group (red), and 5F-APINACA-OH from monohydroxylation of the adamantyl group (light blue), as well as the secondary metabolite, 5F-APINACA-(OH)₂ from another hydroxylation of the adamantyl group (blue). (B) Same data and model fits from (A) highlighting kinetics at lower 5F-APINACA concentrations. (C) Kinetic data from common adamantyl hydroxylation pathway were combined and replotted (dark blue) for comparison to 5OH-APINACA kinetics. (D) Same data and model fits from (C) highlighting kinetics at lower 5F-APINACA concentrations. Steady-state reaction conditions and data analyses were carried out as described in the Materials and Methods. Each data point is an average of 12 replicates, and the displayed curve reflects the best-fit model for the data. The corresponding mechanisms and constants are reported in Table 1.

Figure 3

Steady-state metabolism 5F-APINACA by human liver microsomes fit to differential rate equations. Kinetic plots for 5F-APINACA-OH (A), 5F-APINACA-(OH)₂ (B), and 5OH-APINACA (C) from Figure 2 were refit globally to differential equations for individual rate constants for the mechanism shown in (D) and described in the Materials and Methods. In the kinetic scheme, P1 denotes 5OH-APINACA, P2 denotes 5F-APINACA-OH, and P3 denotes 5F-APINACA-(OH)₂. Each data point is an average of 12 replicates, and corresponding constants are reported in Table 2.

Figure 4

Steady-state metabolism 5F-APINACA by CD-1 mouse liver microsomes fit to traditional kinetic models. (A) Kinetic plot for primary metabolites, i.e., 5OH-APINACA from oxidative defluorination of the pentyl group (red), and 5F-APINACA-OH from monohydroxylation of the adamantyl group (light blue), as well as the secondary metabolite, 5F-APINACA-(OH)₂ from another hydroxylation of the adamantyl group (blue). (B) Same data and model fits from (A) highlighting kinetics at lower 5F-APINACA concentrations. (C) Kinetic data from common adamantyl hydroxylation pathway were combined and replotted (dark blue) for comparison with 5OH-APINACA kinetics. (D) Same data and model fits from (C) highlighting kinetics at lower 5F-APINACA concentrations. Steady-state reaction conditions and data analyses were carried out as described in the Materials and Methods. Each data point is an average of 12 replicates, and the displayed curve reflects the best-fit model for the data. The corresponding mechanism and constants are reported in Table 2.

Figure 5

Steady-state metabolism of 5F-APINACA by CD-1 mouse liver microsomes fit to differential rate equations. Kinetic plots for 5F-APINACA-OH (A), and 5F-APINACA-(OH)₂ (B), and 5OH-APINACA (C) from Figure 4 were refit globally to differential equations for individual rate constants for the mechanism shown in (D) and described in Materials and Methods. Each data point is an average of 12 replicates, and corresponding constants reported in Table 4.

Figure 6

Phenotypic chemical inhibitors used to implicate individual CYPs carrying out observed 5F-APINACA reactions. Human liver microsomal reactions at 25 µM 5F-APINACA were blocked from generating 5F-APINACA-OH (A), 5F-APINACA-(OH)₂ (B), and 5OH-APINACA (C) using inhibitors specific for human CYPs, indicated in parentheses. CD-1 mouse liver microsomal reactions at 25 µM 5F-APINACA were blocked from generating 5F-APINACA-OH (D), 5F-APINACA-(OH)₂ (E), and 5OH-APINACA (F) using inhibitors specific for human CYPs, indicated in parentheses. Rates were normalized to those of the inhibited controls and displayed as an average of 6 replicates. Significance indicated by *, for which p < 0.05, as described in the Materials and Methods.