An Update on Synthetic Cannabinoid Research
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Synthetic cannabinoid receptor agonists (SCRAs) are a class of psychoactive substances (NPSs) that exhibit high affinity binding to the cannabinoid CB1 and CB2 receptors and are used as an alternative to the natural cannabinoid (-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC). SCRAs are marketed under brand names such as “K2” and “Spice” and are popular drugs of abuse among male teenagers and young adults. Typically, these products contain a mixture of SCRAs that are sprayed on dried plant material and marketed to suggest a similarity to cannabis. In addition to their desirable euphoric and relaxation effects, SCRAs can cause severe toxicity, including seizures, respiratory depression, cardiac arrhythmias, stroke and psychosis.
One of the first SCRAs to appear on the illicit drug market was JWH-018, which was synthesized in the 1990s by Dr. John W. Huffman and colleagues at Clemson University. Since the appearance of JWH-018, SCRAs have grown in number and evolved in chemical diversity to evade forensic detection and government drug enforcement scheduling. Over 200 SCRAs, many produced in southeast Asia, are currently monitored by the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime.
In general, SCRAs consist of four chemical components: a tail, a core, a linker, and a linked group (Figure 1). A large number of SCRAs, including JWH-018 and AMB-FUBINACA (Figure 1), contain an indole or indazole core moiety with a carboxamide linker. The introduction of the indazole core/carboxamide linker was found to increase the potency of the SCRAs at CB1 and CB2 receptors. This high potency is consistent with the “zombie outbreak” that occurred with AMB-FUBINACA in which dozens of users experienced strong intoxicating effects of the cannabinoid.
Emergence of new SCRAs
Historically, most countries passed regulations controlling the use of individual SCRAs based on chemical structure. A major landmark was set by the Chinese government in July 2021 with the imposition of a class-wide ban on SCRAs. According to the legislation, restricted substances are defined by seven core structures found in existing SCRAs and include indole carboxamides and indazole carboxamides. As a result, new SCRAs (Figure 1) began to emerge after July 2021 that circumvented the seven-core structural definition. In 2022, Customs and Border Protection officers seized a shipment of the SCRAs BZO-4en-POXIZID and ADB-FUBIATA entering into the United States. OXIZID class SCRAs (BZO-4en-POXIZID, 5-fluoro BZO-POXIZID, etc.) contain an oxoindole core moiety and a hydrazide linker, making them exempt from the Chinese class-wide ban. ADB-FUBIACA differs from ADB-FUBICA, a controlled substance, by the addition of a methylene group between the indole core and the amido group. SCRAs such MDMB-5Br-INACA (no tail group), CUMYL-CHSINACA (cyclohexane sulfonyl tail), CH-PIATA (indole-3-acetamide core/linker), A-PONASA (sulfonamide linker group) and others are being synthesized that evade the restriction.
A novel mechanism of SCRA action
Abuse of SCRAs is associated with side effects including strokes, anxiety, psychosis, and seizures that are more severe than those from ingestion of Δ9-THC. In addition, SCRAs are known to cause hypertension, myocardial infarctions, arrhythmias, and other cardiovascular disorders. The mechanism of these side effects is not fully understood, but likely involves interactions beyond SCRA agonism at the CB1 and CB2 receptors. For example, some SCRAs have “off target” effects by binding to transient receptor potential (TRP) channels causing cell membrane potential depolarization and cellular Ca2+ influx. Now a new study published in the FASEB Journal reports that SCRAs inhibit the enzyme monoamine oxidase A and may thus elevate body levels of the neurotransmitters dopamine and epinephrine.
The monoamine oxidase (MAO) enzymes MAO-A and MAO-B catalyze the oxidation of monoamines including norepinephrine, epinephrine, dopamine, and serotonin. MAO inhibitors (MAOIs) such as isocarboxazid and phenelzine have been used to treat depression, while others (selegiline) are employed in Parkinson’s disease. Treatment with MAO inhibitors is linked to severe cardiovascular side effects when taken along with foods and beverages containing the amino acid tyramine. Normally, tyramine is broken down by the MAO enzymes, but in the presence of MAOIs the increased blood levels of tyramine can produce a hypertensive crisis.
In their FASEB Journal paper, Christopher Pudney and colleagues used a combination of in silico docking and in vitro enzyme kinetic experiments to examine the effects of SCRAs on MAO-A and MAO-B. SCRAs were found to selectively inhibit the MAO-A enzyme. SCRAs containing an aromatic ring (5-fluoro PB-22 & AM-2201) in the linked group position were more potent than SCRAs with other linked groups (5-fluoro ADB & 5-fluoro MDMB PICA). In contrast, changing the core moiety from an indole to indazole group was without effect. Since high, micromolar concentrations of the compounds were required for MAO-A inhibition, it will be important to examine SCRA-mediated changes in MAO activity and blood pressure in an in vivo model.
Urine detection of OXIZID SCRAs
SCRAs undergo extensive metabolism in the body with little or none of the original drug (parent drug) being found in the urine. For this reason, consumption of SCRAs is detected through the measurement of urinary metabolites (or biomarkers) that result from the liver metabolism of the compounds. SCRAs, like many drugs, undergo hepatic microsomal phase 1 metabolism via the cytochrome P450 (CYP450) enzymes. Unfortunately, biomarkers for new classes of SCRAs, such as the OXIZIDs, have not been available, something that hampers the ability of forensic scientists to identify OXIZID drug abuse.
In a report published in Clinical Chemistry in 2022, Dr. Eric Chan and his research team at the University of Singapore identified the urinary biomarkers for BZO-HEXOXIZID, BZO-POXIZID, 5-flouro-BZO-POXIZID and BZO-CHMOXIZID. The investigators used a two-pronged approach in measuring the OXIZID metabolites. First, the SCRAs were incubated in vitro with both human microsomes and recombinant CYP450 enzymes and a panel of metabolic biomarkers obtained using mass spectrometry. Second, the in vitro results were confirmed by measuring the biomarkers in urine samples from drug users who had consumed the SCRAs. As an example, in vitro metabolism of 5-fluoro-BZO-POXIZID by CYP3A4 and CYP3A5 resulted in the formation of several hydroxylated metabolites (Figure 2) with the 5-hydroxypentyl metabolite being the most abundant. As corroboration, these biomarkers were found to be present in urine samples obtained from anonymous drug users of 5-flouro-BZO-POXIZID. Thus, these biomarkers can now be applied by drug enforcement agencies to monitor the abuse and spread of OXIZID SCRAs. This methodology could be applied to screen for other emerging SCRAs.