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Aims: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development., Main Methods: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively., Key Findings: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [ 3 H]WIN-55,212-2, but not CB1/CB2R radioligand [ 3 H]CP-55,940. Homologous receptor binding demonstrated that [ 3 H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [ 3 H]WIN-55,212-2, whereas two ligands enhanced [ 3 H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [ 3 H]WIN-55,212-2 displacement exhibited high nanomolar affinity (K i ) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity., Significance: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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)-1 H -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.

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 K m value of 38 µM and a V max 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.

K2 or Spice products are emerging drugs of abuse that contain synthetic cannabinoids (SCBs). Although assumed by many teens and first time drug users to be a "safe" and "legal" alternative to marijuana, many recent reports indicate that SCBs present in K2 produce toxicity not associated with the primary psychoactive component of marijuana, ∆(9)-tetrahydrocannabinol (Δ(9)-THC). This mini-review will summarize recent evidence that use of K2 products poses greater health risks relative to marijuana, and suggest that distinct pharmacological properties and metabolism of SCBs relative to Δ(9)-THC may contribute to the observed toxicity. Studies reviewed will indicate that in contrast to partial agonist properties of Δ(9)-THC typically observed in vitro, SCBs in K2 products act as full cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) agonists in both cellular assays and animal studies. Furthermore, unlike Δ(9)-THC metabolism, several SCB metabolites retain high affinity for, and exhibit a range of intrinsic activities at, CB1 and CB2Rs. Finally, several reports indicate that although quasi-legal SCBs initially evaded detection and legal consequences, these presumed "advantages" have been limited by new legislation and development of product and human testing capabilities. Collectively, evidence reported in this mini-review suggests that K2 products are neither safe nor legal alternatives to marijuana. Instead, enhanced toxicity of K2 products relative to marijuana, perhaps resulting from the combined actions of a complex mixture of different SCBs present and their active metabolites that retain high affinity for CB1 and CB2Rs, highlights the inherent danger that may accompany use of these substances., (© 2013.)

"K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 μM) and ANF (0.1 - 5.0 μM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB(1)) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (K(m) = 0.81-7.3 μM) and low to high reaction velocities (V(max) = 0.0053-2.7 nmol of product · min(-1) · nmol protein(-1)). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of "K2" parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.

K2 and several similar purported "incense products" spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3-M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist (K(b)∼40nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad (e.g., locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice. In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products., (Copyright © 2012 Elsevier Inc. All rights reserved.)

K2, a synthetic cannabinoid (SC), is an emerging drug of abuse touted as "legal marijuana" and marketed to young teens and first-time drug users. Symptoms associated with K2 use include extreme agitation, syncope, tachycardia, and visual and auditory hallucinations. One major challenge to clinicians is the lack of clinical, pharmacological, and metabolic information for the detection and characterization of K2 and its metabolites in human samples. Information on the metabolic pathway of SCs is very limited. However, previous reports have shown the metabolites of these compounds are excreted primarily as glucuronic acid conjugates. Based on this information, this study evaluates nine human recombinant uridine diphosphate-glucuronosyltransferase (UGT) isoforms and human liver and intestinal microsomes for their ability to glucuronidate hydroxylated metabolites of 1-naphthalenyl-1(1-pentyl-1H-indol-3-yl)-methanone (JWH-018) and (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073), the two most common SCs found in K2 products. Conjugates were identified and characterized using liquid chromatography/tandem mass spectrometry, whereas kinetic parameters were quantified using high-performance liquid chromatography-UV-visible methods. UGT1A1, UGT1A3, UGT1A9, UGT1A10, and UGT2B7 were shown to be the major enzymes involved, showing relatively high affinity with K(m) ranging from 12 to 18 μM for some hydroxylated K2s. These UGTs also exhibited a high metabolic capacity for these compounds, which indicates that K2 metabolites may be rapidly glucuronidated and eliminated from the body. Studies of K2 metabolites will help future development and validation of a specific assay for K2 and its metabolites and will allow researchers to fully explore their pharmacological actions.

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ9-THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ9-THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo , producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ9-THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana. [ABSTRACT FROM AUTHOR]

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs. [ABSTRACT FROM AUTHOR]

Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23-year-old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over-the-counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH-073 found in smoke residue, no other substances were detected. Psychiatric complications including self-induced, lethal trauma can occur after the use of SC products. [ABSTRACT FROM AUTHOR]

Recently, hydroxylated metabolites of JWH-018, a syntheticcannabinoidfound in many K2/Spice preparations, have been shown to retain affinityand activity for cannabinoid type 1 receptors (CB1Rs). The activityof glucuronidated metabolites of JWH-018 is not known; hence, thisstudy investigated the affinity and activity of a major metabolite,JWH-018-N-(5-hydroxypentyl) β-d-glucuronide(018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (Ki= 922 nM), has no effect on G-protein activity, butantagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylationby cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferasesproduces a metabolite, 018-gluc, which possesses antagonistic activityat CB1Rs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Tamoxifen produces cytotoxicity via estrogen-receptor (ER) independent mechanisms. [•] Tamoxifen binds to CB1 and CB2 cannabinoid receptors and acts as an inverse agonist. [•] CB1 and CB2 receptors are novel molecular targets for Tamoxifen. [•] ER-independent effects for Tamoxifen may be mediated via CB1 and/or CB2 receptors. [Copyright &y& Elsevier]

Designer synthetic cannabinoids like JWH-018 and AM2201 have unique clinical toxicity. Cytochrome-P450-mediated metabolism of each leads to the generation of pharmacologically active (ω)- and (ω-1)- monohydroxyl metabolites that retain high affinity for cannabinoid type-1 receptors, exhibit Δ9-THC-like effects in rodents, and are conjugated with glucuronic acid prior to excretion in human urine. Previous studies have not measured the contribution of the specific (ω-1)-monohydroxyl enantiomers in human metabolism and toxicity. This study uses a chiral liquid chromatography-tandem mass spectroscopy approach (LC-MS/MS) to quantify each specific enantiomer and other nonchiral, human metabolites of JWH-018 and AM2201 in human urine. The accuracy (average % RE = 18.6) and reproducibility (average CV = 15.8%) of the method resulted in low-level quantification (average LLQ = 0.99 ng/mL) of each metabolite. Comparisons with a previously validated nonchiral method showed strong correlation between the two approaches (average r2 = 0.89). Pilot data from human urine samples demonstrate enantiospecific excretion patterns. The (S)-isomer of the JWH-018-(ω-1)-monohydroxyl metabolite was predominantly excreted (>87%) in human urine as the glucuronic acid conjugate, whereas the relative abundance of the corresponding AM2201-(ω-1)-metabolite was low (<5%) and did not demonstrate enantiospecificity (approximate 50:50 ratio of each enantiomer). The new chiral method provides a comprehensive, targeted metabolomic approach for studying the human metabolism of JWH-018 and AM2201. Preliminary evaluations of specific enantiomeric contributions support the use of this approach in future studies designed to understand the pharmacokinetic properties of JWH-018 and/or AM2201. [ABSTRACT FROM AUTHOR]

"K2/SPICE" products are commonly laced with aminoalkylindole synthetic cannabinoids (i.e., JWH-018 and JWH-073) and are touted as "legal" marijuana substitutes. Here we validate a liquid chromatography- tandem mass spectrometry (LC-MS/MS) method for measuring urinary concentrations of JWH-018, JWH-073, and several potential metabolites of each. The analytical procedure has high capacity for sample throughput and does not require solid phase or liquid extraction. Evaluation of human urine specimens collected after the subjects reportedly administered JWH-018 or a mixture of JWH-018 and JWH-073 provides preliminary evidence of clinical utility. Two subjects that consumed JWH-018 primarily excreted glucuronidated conjugates of 5-(3-(1-naphthoyl)-1H-indol-1-yl)-pentanoic acid (>30 ng/mL) and (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalene-1-yl)-methanone (>50 ng/mL). Interestingly, oxidized metabolites of both JWH-018 and JWH-073 were detected in these specimens, suggesting either metabolic demethylation of JWH-018 to JWH-073 or a nonreported, previous JWH-073 exposure. Metabolic profiles generated from a subject who consumed a mixture of JWH-018 and JWH-073 were similar to profiles generated from subjects who presumably consumed JWH-018 exclusively. Oxidized metabolites of JWH-018 and JWH-073 were of the same pattern, but JWH-018 metabolites were excreted at lower concentrations. These results begin clinically validating the LC-MS/MS assay for detecting and quantifying aminoalkylindole metabolites. Full validation awaits further testing. [ABSTRACT FROM AUTHOR]