In vitro Phase I metabolism of indazole carboxamide synthetic cannabinoid MDMB‐CHMINACA via human liver microsome incubation and high‐resolution mass spectrometry

Abstract

Synthetic cannabinoids have proliferated over the last decade and have become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide class synthetic cannabinoids have been particularly rampant, and exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB1 and CB2). MDMB‐CHMINACA, methyl 2‐[1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate, a compound of this chemical class, has been identified in forensic casework and is structurally related to several other synthetic cannabinoids. This study presents the first extensive report on the Phase I metabolic profile of MDMB‐CHMINACA, a potent synthetic cannabinoid. The in vitro metabolism of MDMB‐CHMINACA was determined via incubation with human liver microsomes and high‐resolution mass spectrometry. The accurate masses of precursor and fragments, mass error (ppm), and chemical formula were obtained for each metabolite. Twenty‐seven metabolites were identified, encompassing twelve metabolite types. The major biotransformations observed were hydroxylation and ester hydrolysis. Hydroxylations were located predominantly on the cyclohexylmethyl (CHM) moiety. Ester hydrolysis was followed by additional biotransformations, including dehydrogenation; mono‐ and dihydroxylation and ketone formation, each with dehydrogenation. Minor metabolites were identified and reported. The authors propose that CHM‐monohydroxylated metabolites specific to MDMB‐CHMINACA are the most suitable candidates for implementation into bioanalytical assays to demonstrate consumption of this synthetic cannabinoid. Due to the structural similarity of MDMB‐CHMINACA and currently trending synthetic cannabinoids whose metabolic profiles have not been reported, the results of this study can be used as a guide to predict their metabolic pathways.

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

“…The methodology described in this paper has been used to determine the metabolic profile of compounds of numerous NPS classes including opioid receptor agonists, cathinone derivatives, phenethylamine derivatives, and synthetic cannabinoids (Krotulski et al, ; Krotulski, Mohr, Papsun, & Logan, , ; Krotulski, Papsun, De Martinis, Mohr, & Logan, c; Presley et al, ; Temporal, Scott, Mohr, & Logan, ). This demonstrates the applicability of the methods to a host of compounds, including non‐drug substances to predict their metabolic fate.…”

“…The in vitro Phase I metabolic profile of PX‐1 was determined via incubation with HLM, using a method similar to one previously described (Presley, Logan, & Jansen‐Varnum, ). PX‐1 stock standard was prepared at a concentration of 1 mg/mL from reference material.…”

“…The in vitro Phase I metabolic profile of PX-1 was determined via incubation with HLM, using a method similar to one previously described (Presley, Logan, & Jansen-Varnum, 2019 Incubations were terminated by adding 500 μL of LC-MS-grade acetonitrile followed by vortex mixing. Samples were then transferred to microcentrifuge tubes and centrifuged at 10,000g for 5 min.…”

Metabolic profiling of synthetic cannabinoid 5F‐ADB and identification of metabolites in authentic human blood samples via human liver microsome incubation and ultra‐high‐performance liquid chromatography/high‐resolution mass spectrometry

“…67 Hydrolyzed metabolic products have been reported in other synthetic cannabinoid metabolism studies involving compounds with ester and amide functional groups. [68][69][70][71][72] Thomsen et al 69 reported that synthetic cannabinoids can be hydrolyzed by carboxylesterase type-1 (CES1) and type-2 (CES2) enzymes.…”

Differential activation of G protein‐mediated signaling by synthetic cannabinoid receptor agonists

“…Although it is speculated that the adverse effects of SCRAs are mediated by CB1, based on the results presented here we wonder how the differential responses of SCRAs are related to the physiological effects resulting from the activation of each intracellular pathway, and if these may be correlated with the in vivo toxicity of SCRAs. The unique toxicological profile of SCRAs may result from a combination of factors; pharmacokinetic differences, activity at both cannabinoid and noncannabinoid targets, pharmacological activity of metabolites and thermolytic degradants . These findings may provide a starting point to help predict the pharmacological characteristics of SCRAs that demonstrate differential activation of Gα i vs Gα s coupling to CB1.…”