Synthetic Cannabinoid JWH-018 Research Articles

Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases

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.

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

BackgroundK2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ9-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).Methods/Principal FindingsJWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [3H]CP-55,940, and then for CB1R intrinsic efficacy using an [35S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting Ki values that were lower than or equivalent to Δ9-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ9-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ9-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Conclusions/SignificanceUnlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ9-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.

Structure characterisation of urinary metabolites of the cannabimimetic JWH-018 using chemically synthesised reference material for the support of LC-MS/MS-based drug testing

As recently reported, the synthetic cannabinoid JWH-018 is the subject of extensive phase I and II metabolic reactions in vivo. Since these studies were based on LC-MS/MS and/or GC-MS identification and characterisation of analytes, the explicit structural assignment of the metabolites was only of preliminary nature, if possible at all. Here, we report the chemical synthesis of five potential in vivo metabolites of JWH-018 derivatives featuring an alkylcarboxy (M1), a terminal alkylhydroxy (M2), a 5-indolehydroxy (M3), an N-dealkylated 5-indolehydroxy (M4) and a 2'-naphthylhydroxy (5) analogue, respectively, and their characterisation by nuclear magnetic resonance spectroscopy. The collision-induced dissociation (CID) patterns of the protonated compounds were studied by high-resolution/high-accuracy tandem mass spectrometry (MS( n )) applying an LTQ Orbitrap with direct infusion and electrospray ionisation of target analytes. An unusual dissociation behaviour including a reversible ion-molecule reaction between a naphthalene cation (m/z 127) and water in the gas phase of the MS was shown to be responsible for nominal neutral losses of 10u in the course of the CID pathway. LC-MS/MS-supported comparison of synthesised reference standards with an authentic urine sample using an API 4000 QTrap mass spectrometer identified the synthetic JWH-018 analogues M1-M4 as true in vivo metabolites, presuming a chromatographic separation of potentially present regioisomeric analogues. Existing doping control methods were expanded and validated according to international guidelines in order to allow for the detection of the carboxy and the alkylhydroxy metabolites, respectively, as urinary markers for the illegal intake of the synthetic cannabinoid JWH-018. Both metabolites were quantified in authentic doping control urine samples that had been suspicious of JWH-018 abuse after routine screening procedures, and a stable isotope-labelled (13)C(8)-(15)N-carboxy metabolite was synthesised for future analytical applications.

Chromatography–mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures

The Russian Federation prohibited the distribution of herbal mixtures with synthetic aminoalkylindoles JWH-018 and JWH-073, agonist cannabinoid receptors, on January 22, 2010. The lack or low content of their native compounds in urine requires detailed identification of their metabolites, which are excreted with urine and are present in blood. Using gas and liquid chromatography–mass spectrometry, we identified a series of metabolites in urine samples from humans and rats that were products of the following reactions: (a) mono- and dihydroxylation of the parent compounds with hydroxyl groups located at aromatic and aliphatic residues, (b) carboxylation, (c) N-dealkylation and (d) N-dealkylation and hydroxylation. The prevailing urinary metabolites in humans are monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms are found in rats. Twenty-six samples of herbal smoking mixtures with JWH-018, purchased in Russia, were analysed.

Synthetic cannabinoid JWH-018 and psychosis: An explorative study

Background Aroma, Spice, K2 and Dream are examples of a class of new and increasingly popular recreational drugs. Ostensibly branded “herbal incense”, they have been intentionally adulterated with synthetic cannabinoids such as JWH-018 in order to confer on them cannabimimetic psychoactive properties while circumventing drug legislation. JWH-018 is a potent cannabinoid receptor agonist. Little is known about its pharmacology and toxicology in humans. This is the first research considering the effects of JWH-018 on a psychiatric population and exploring the relationship between JWH-018 and psychotic symptoms. Method This paper presents the results of semi-structured interviews regarding the use and effects of JWH-018 in 15 patients with serious mental illness in a New Zealand forensic and rehabilitative service. Results All 15 subjects were familiar with a locally available JWH-018 containing product called “Aroma” and 86% reported having used it. They credited the product's potent psychoactivity, legality, ready availability and non-detection in drug testing as reasons for its popularity, with most reporting it had replaced cannabis as their drug of choice. Most patients had assumed the product was “natural” and “safe”. Anxiety and psychotic symptoms were common after use, with 69% of users experiencing or exhibiting symptoms consistent with psychotic relapse after smoking JWH-018. Although psychological side effects were common, no one reported becoming physically unwell after using JWH-018. Three subjects described developing some tolerance to the product, but no one reported withdrawal symptoms. Conclusion It seems likely that JWH-018 can precipitate psychosis in vulnerable individuals. People with risk factors for psychosis should be counseled against using synthetic cannabinoids.

Screening for the synthetic cannabinoid JWH‐018 and its major metabolites in human doping controls

Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine.

JOHN W. HUFFMAN

IN DECEMBER 2008, John W. Huffman, a professor of organic chemistry at Clemson University, received an e-mail that illustrates how pure scientific research can be exploited in unintended ways. The e-mail, from a blogger in Germany, alerted Huffman to the fact that the synthetic cannabinoid JWH-018 had been detected as an ingredient in some herbal blends marketed over the Internet as a legal substitute for marijuana. Huffman knows JWH-018 well. “We made the stuff in 1995,” he says. “I had an undergraduate student working under the supervision of a very capable postdoc, and this was just one of the things we made.” Their research was published in 1998 in the Journal of Pharmacology & Experimental Therapeutics ( 1998, 285, 995). JWH-018 is the 18th compound that Huffman’s research group synthesized in a series of more than 470 analogs and metabolites of ▵ 9 -tetrahydrocannabinol (THC), the active component of marijuana. Huffman created these compounds to ...