In vitro approaches for the identification and characterization of urinary metabolite biomarkers of synthetic cannabinoid ADB-BUTINACA

Abstract

To identify the major metabolites and investigate the key disposition kinetics of synthetic cannabinoid ADB-BUTINACA so as to establish the optimal urinary biomarkers for forensic surveillance. Novel synthetic cannabinoids (SCs) continue to evolve and cause widespread intoxications globally. Despite this increasing prevalence of abuse, severe toxicities associated with SC intake coupled with uncertainties in identifying the parent drug in biological samples remain perennial problems undermining forensic management. ADB-BUTINACA is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene with a total of 40 and 107 occurrences in 2020 and 2021, respectively. Due to the unavailability of toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. The metabolites of ADB-BUTINACA were identified in human liver microsomes (HLM) using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. Recombinant enzymes were used to generate a pool of key Phase 1 metabolites in situ and the metabolic stability of these metabolites were determined. By coupling in vitro metabolism and authentic human urine analyses, a panel of urinary metabolite biomarkers for the detection of ADB-BUTINACA abuse was proposed. A total of fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. From the reaction phenotyping studies, it was established that ADB-BUTINACA was rapidly eliminated via CYP3A4-, CYP2C19-, and CYP3A5-mediated metabolism pathways. Three major monohydroxylated metabolites were generated in situ, and they demonstrated greater metabolic stability compared to the parent drug, ADB-BUTINACA. Four urinary biomarker metabolites of ADB-BUTINACA, namely 3 hydroxylated metabolites and 1 oxidative deaminated metabolite, were identified from in vitro metabolite profiling coupled with authentic urinary analyses. The 2-pronged approach of using specific enzyme inhibitors and recombinant enzymes have provided adequate evidence to ascertain the metabolic routes of ADB-BUTINACA. Findings also confirmed that cytochrome P450 (CYP450) is the main class of enzymes contributing to the elimination of ADB-BUTINACA. Through this study, a feasible approach of combining in vitro HLM metabolite profiling with authentic human urine analyses for the comprehensive identification of urinary metabolite biomarkers of ADB-BUTINACA is established. The metabolic profile of ADB-BUTINACA and its key metabolizing enzymes and biotransformation pathways had been characterized in this study. This innovative approach used to identify the biomarkers can be applied to other existing and new prevailing SCs for diagnosing abuse.