5 research outputs found
Monohydroxylated metabolites of the K2 synthetic cannabinoid JWH-073 retain intermediate to high cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist to partial agonist activity
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 (Kb~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
Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids
Tetrahydrocannabinol (Δ9-THC), the primary psychoactive
ingredient in marijuana, is subject to cytochrome P450 oxidation and
subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many
studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible
for these cytochrome P450-dependent oxidations, but little work has been done
to characterize phase II metabolic pathways. In this study, we test the
hypothesis that there are specific human UGTs responsible for classic
cannabinoid metabolism. The activities of 12 human recombinant UGTs toward
classic cannabinoids [cannabinol (CBN), cannabidiol (CBD),
(–)-Δ8-THC, (–)-Δ9-THC,
(±)-11-hydroxy-Δ9-THC (THC-OH), and
(–)-11-nor-9-carboxy-Δ9-THC (THC-COOH)] were evaluated
using high-performance liquid chromatography-tandem mass spectrometry and
labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7
toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate
sufficient activity for further characterization of steady-state kinetics. CBN
was the most recognized substrate as evidenced by activities from hepatic
UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect
the introduction of an aromatic ring to Δ9-THC, leading to
favorable π stacking with phenylalanines in the UGT active site. Likewise,
oxidation of Δ9-THC to THC-OH results in UGT1A9 and UGT1A10
activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly
leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a
substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of
THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes
play a critical role in the metabolic clearance of cannabinoids. Taken
together, glucuronidation of cannabinoids depends on upstream processing
including enzymes such as CYP2C9 and CYP3A4
Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids
ABSTRACT: Tetrahydrocannabinol (⌬ 9 -THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UG