Abstract
Within New Zealand alone, the synthetic cannabinoid AMB-FUBINACA has been associated with at least 64 deaths between May 2017 and February 2019. Little is known about the mechanism of synthetic cannabinoid toxicity and while it is hypothesised to be mediated through cannabinoid receptor type-1 (CB1), it may not be as simple as activation of the receptor. High amounts of other drugs have also been identified in these fatal cases, in particular the “party pill” p-fluorophenylpiperazine (pFPP), which was detected post-mortem in the blood of 43% of those tested; Cannabis and alcohol use were also common. The enzyme responsible for the rapid hepatic conversion of AMB-FUBINACA to the hydrolysed metabolite, AMB-FUBINACA acid, is currently unknown. As AMB-FUBINACA acid is inactive at CB1, inhibition of this metabolic pathway may result in additional CB1 activation by AMB-FUBINACA and other non-specific effects. This study therefore aimed to characterise the metabolism of AMB-FUBINACA in human and rat liver microsomes and investigate if interactions with pFPP, the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) or ethanol were likely to occur in vivo. Concentrations of AMB-FUBINACA and AMB-FUBINACA acid were determined using high-performance liquid chromatography. Initial experiments with rat liver microsomes suggested that AMB-FUBINACA hydrolysis was largely cytochrome P450 (CYP450)-mediated (half-life = 2.26 min). On the other hand, AMB-FUBINACA hydrolysis with human liver microsomes occurred extremely rapidly (half-life = 0.21 min) and was largely non-CYP450-mediated. Additionally, no interactions with pFPP were noted in either rat or human liver microsomes. It was hypothesised that carboxylesterase 1 (CES1) was responsible for the metabolism of AMB-FUBINACA. Metabolism was assessed in vitro with recombinant carboxylesterases. AMB-FUBINACA metabolism by CES1 was confirmed (half-life = 120 s) and was significantly inhibited by the steroidal saponin digitonin (100 μM: 56.3 ± 4.6% maximal inhibition). Previous literature indicates that Δ9-THC and ethanol also inhibit CES1; however, AMB-FUBINACA metabolism with human liver microsomes was not significantly inhibited in the presence of Δ9-THC (10 μM: 10.8 ± 7.7% maximal inhibition) or ethanol (100 mM: 3.1 ± 1.6% maximal inhibition). Further studies should assess AMB-FUBINACA metabolism in the presence of xenobiotics also detected within the blood of these fatal New Zealand AMB-FUBINACA poisonings to establish a link between epidemiological data and in vitro metabolism research. The rapid in vitro metabolism of AMB-FUBINACA suggests minor inhibition of this pathway could prove toxic. Understanding not only the metabolic interactions, but absorption, distribution and excretion interactions may give insight into why some users experience high levels of harm.