Synthetic cannabinoids (SCs) are novel psychoactive substances (NPS) eliciting cannabimimetic psychoactive effects [1]. SCs were originally produced for biomedical research but now are abused, leading many countries to schedule these drugs as controlled substances [2-5]. Clandestine laboratories continue producing new compounds that mimic effects of scheduled NPS to circumvent legislation. More than 130 SCs were identified to date [6]. Little pharmacological or toxicological data are available when substances first appear. Analytical methods require constant updating, and pharmacodynamic and pharmacokinetic studies must be conducted for the continual emergence of NPS. With its patent filed only in 2009, ADB-FUBINACA, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (Fig. 1) is one of the newest SC [7]. In 2013, the drug was identified for the first time in illegal herbal blends in Japan, in association with two other SCs ADBICA and XLR-11 [8]. The drug was reported for the first time in Europe in the same year in Hungary [6] as Facebook logo shaped tablets, and in biological samples from patients who swallowed or crushed and insufflated the product [9, 10].

There are currently no in vivo human or animal ADB-FUBINACA pharmacodynamic data. In vitro experiments demonstrated potent CB₁ cannabinoid receptor binding (K_i = 0.36 nM, EC₅₀ = 0.98 – 1.2 nM) [7, 11] and CB₂ (EC₅₀ = 3.5 nM) [11]. CB₁ binding affinity that correlates with cannabinoid psychoactive effects [12] is 140 times stronger than for THC, comparable to other indole and indazole SCs with an aminomethylbutanamide or aminodimethylbutanamide structure [11]. Affinity for CB₁ receptors is slightly lower for the desmethyl and indole analogs, AB-FUBINACA (scheduled) [13] and ADB-FUBICA, and higher with the N-pentyl and N-fluoropentyl analogs, ADB-PINACA (scheduled) [13] and 5F-ADB-PINACA [11]. ADB-FUBINACA reportedly produces drowsiness, respiratory deprivation, CO₂ retention, hypotonia, hypothermia, vomiting, sinus tachycardia, mydriasis, extrapyramidal movement disorder, hyperkinesis, acoustic and visual hallucinations, agitation and mixed symptoms [2, 10]. Structural analogs were involved in several intoxication and fatalities [14-19], with the first ADB-FUBINACA death reported in 2016 [20].

ADB-FUBINACA pharmacodynamic and pharmaco-kinetic studies are still lacking. Many SCs are active at low doses and extensively metabolized with little to no parent compound found in urine, making urinary metabolite detection critical for documenting consumption in forensic and clinical cases [21-25]. One ADB-FUBINACA human liver microsome (HLM) in vitro metabolism study identified a single hydroxyalkyl metabolite [26]. Identifying the SC responsible for resultant toxicities also is important for educating the public on the drug’s dangers.

Our aim was to determine ADB-FUBINACA human metabolism to improve analytical identification of this new SC in biological samples. In vitro HLM [27-29] and human hepatocytes incubations [30-33] with high resolution mass spectrometry (HRMS) analysis proved useful for predicting human SC metabolism. Human hepatocytes contain all required hepatic metabolic enzymes and endogenous cofactors and provide the natural orientation of membrane enzymes, better predicting metabolite production in in vivo conditions than enzymes alone or HLM [34].