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Synthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3- carboxamide (STS-135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS-135. In this study, STS-135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra-performance liquid chromatography. STS-135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS-135's oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a Km value of 11.4 μmol/L. Thus, it was concluded that STS-135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS-135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS-135-induced cardiovascular toxicity. [ABSTRACT FROM AUTHOR]

Recently, there has been a rise in abuse of synthetic cannabinoids (SCBs). The consumption of SCBs results in various effects and can induce toxic reactions, including paranoia, seizures, tachycardia and even death. 1-Naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22) is a third generation SCB whose metabolic pathway has not been fully characterized. In this study, we conducted in vitro pharmacokinetic analysis of FDU-PB-22 metabolism. Metabolic reactions containing FDU-PB-22 and human liver microsomes (HLMs) were independent of NADPH but not UDP-glucuronic acid (UDPGA), suggesting that UDP-glucuronosyltransferases (UGTs) are the primary enzymes involved in this metabolism. It was further determined that the metabolite extensively formed after incubating FDU-PB-22 with UDPGA in HLMs was the glucuronide of FDU-PB-22 3-carboxyindole (FBI-COOH). Various hepatic UGTs showed enzymatic activity for FBI-COOH. A series of UGT inhibitors showed moderate to strong inhibition of FBI-COOH-glucuronidation in HLMs, suggesting that multiple UGT isoforms are involved in FBI-COOH-glucuronidation in the liver. Interestingly, an extra-hepatic isoform, UGT1A10, exhibited the highest activity with a Km value of 38 µM and a Vmax value of 5.90 nmol/min/mg. Collectively, these results suggest that both genetic mutations of and the co-administration of inhibitors for FDU-PB-22-metabolizing UGTs will likely increase the risk of FDU-PB-22-induced toxicity. [ABSTRACT FROM AUTHOR]

In 2020, nearly one-third of new drugs on the global market were synthetic cannabinoids including the drug of abuse N-(1-adamantyl)-1-(5-pentyl)-1H-indazole-3-carboxamide (5F-APINACA, 5F-AKB48). Knowledge of 5F-APINACA metabolism provides a critical mechanistic basis to interpret and predict abuser outcomes. Prior qualitative studies identified which metabolic processes occur but not the order and extent of them and often relied on problematic "semi-quantitative" mass spectroscopic (MS) approaches. We capitalized on 5F-APINACA absorbance for quantitation while leveraging MS to characterize metabolite structures for measuring 5F-APINACA steady-state kinetics. We demonstrated the reliability of absorbance and not MS for inferring metabolite levels. Human liver microsomal reactions yielded eight metabolites by MS but only five by absorbance. Subsequent kinetic studies on primary and secondary metabolites revealed highly efficient mono- and dihydroxylation of the adamantyl group and much less efficient oxidative defluorination at the N-pentyl terminus. Based on regiospecificity and kinetics, we constructed pathways for competing and intersecting steps in 5F-APINACA metabolism. Overall efficiency for adamantyl oxidation was 17-fold higher than that for oxidative defluorination, showing significant bias in metabolic flux and subsequent metabolite profile compositions. Lastly, our analytical approach provides a powerful new strategy to more accurately assess metabolic kinetics for other understudied synthetic cannabinoids possessing the indazole chromophore. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoids (SCBs), synonymous with ‘K2’, ‘Spice’ or ‘synthetic marijuana’, are psychoactive drugs of abuse that frequently result in clinical effects and toxicity more severe than those classically associated with Δ 9 -tetrahydrocannabinol such as extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. JWH-018 is one of the earliest compounds identified in various SCB products, and our laboratory previously demonstrated that JWH-018 undergoes extensive metabolism by cytochromes P450 (P450), binds to, and activates cannabinoid receptors (CBRs). The major enzyme involved in the metabolism of JWH-018 is CYP2C9, a highly polymorphic enzyme found largely in the intestines and liver, with *1 being designated as the wild type, and *2 and *3 as the two most common variants. Three different major products have been identified in human urine and plasma: JWH-018 (ω)-OH, JWH-018 (ω-1)-OH(R), and JWH-018 (ω-1)-OH(S). The (ω-1)-OH metabolite of JWH-018 is a chiral molecule, and is thus designated as either (ω-1)-OH(R) or (ω-1)-OH(S). Here, in vitro enzyme kinetic assays performed with human recombinant CYP2C9 variants (*1, *2, and *3) revealed that oxidative metabolism by CYP2C9*3 resulted in significantly less formation of (ω)-OH and (ω-1)-OH metabolites. Surprisingly, CYP2C9*2 was roughly 3.6-fold more efficient as the CYP2C9*1 enzyme based on V max /K m , increasing the rate of JWH-018 metabolism and allowed for a much more rapid elimination. These results suggest that genetic polymorphisms of P450 enzymes result in the production of varying levels of biologically active JWH-018 metabolites in some individuals, offering a mechanistic explanation for the diverse clinical toxicity often observed following JWH-018 abuse. [ABSTRACT FROM AUTHOR]

New designer drugs such as K2, Spice, and "bath salts" present a formidable challenge for law enforcement and public health officials. The following report summarizes a three-year study of 1320 law enforcement cases involving over 3000 products described as vegetable material, powders, capsules, tablets, blotter paper, or drug paraphernalia. All items were seized in Arkansas from January 2010 through December 2012 and submitted to the Arkansas State Crime Laboratory for analysis. The geographical distribution of these seizures co-localized in areas with higher population, colleges, and universities. Validated forensic testing procedures confirmed the presence of 26 synthetic cannabinoids, 12 designer stimulants, and 5 hallucinogenic-like drugs regulated by the Synthetic Drug Prevention Act of 2012 and other state statutes. Analysis of paraphernalia suggests that these drugs are commonly used concomitantly with other drugs of abuse including marijuana, MDMA, and methamphetamine. Exact designer drug compositions were unpredictable and often formulated with multiple agents, but overall, the synthetic cannabinoids were significantly more prevalent than all the other designer drugs detected. The synthetic cannabinoids JWH-018, AM2201, JWH-122, JWH-210, and XLR11 were most commonly detected in green vegetable material and powder products. The designer stimulants methylenediox- ypyrovalerone (MDPV), 3,4-methylenedioxy-N-methylcathinone (methylone), and a-methylamino- valerophenone (pentedrone) were commonly detected in tablets, capsules, and powders. Hallucinogenic drugs were rarely detected, but generally found on blotter paper products. Emerging designer drug products remain a significant problem and continued surveillance is needed to protect public health. [ABSTRACT FROM AUTHOR]

AKB48 and its fluorinated derivative 5F-AKB48 are synthetic cannabinoids (SCs) which have caused hospitalizations and deaths in human users. Abuse of SCs is dangerous because users may mistake them for natural cannabis, which is generally considered to be unlikely to elicit adverse effects. The present studies were designed to investigate the in vitro oxidative metabolism of 5F-AKB48 by human microsomal fractions from different organs and sexes as well as recombinant human cytochrome P450s (P450s). Mass spectrometry data tentatively provides evidence for the existence of mono-, di-, and trihydroxylated metabolites in a successive metabolism. Experiments utilizing P450s revealed that the most active enzymes (CYP2D6, CYP2J2, CYP3A4, and CYP3A5) effectively produced mono- and dihydroxylated metabolites, while CYP3A4/5 also produced significant amounts of the trihydroxylated metabolite. Moreover, although the affinity and potency of Phase I metabolite 4OH-5F-AKB48 is reduced when compared to that of the parent drug, this metabolite nevertheless retains similar high affinity for CB1 receptors, and greater efficacy for G protein activation, when compared to THC. Finally, 5F-AKB48 produced time- and dose-dependent cannabimimetic effects in mice which were more potent, but shorter acting, than those of Δ9-THC, and were attenuated by prior treatment with the CB1 antagonist rimonabant. Based on our data, we hypothesize that while many cases of toxicity result from genetic mutations, which can lead to a decrease or even absence of activity for Phase I drug-metabolizing enzymes, other P450s could potentially increase their role in the metabolism of these SCs. Because many metabolites of SCs remain biologically active, they could contribute to the deleterious effects of these substances. [ABSTRACT FROM AUTHOR]