Investigation of phase II metabolism of 11-hydroxy-Δ-9-tetrahydrocannabinol and metabolite verification by chemical synthesis of 11-hydroxy-Δ-9-tetrahydrocannabinol-glucuronide

Abstract

(−)-Δ-9-tetrahydrocannabinol ((−)-Δ-9-THC) is the main psychoactive constituent in cannabis. During phase I metabolism, it is metabolized to (−)-11-hydroxy-Δ-9-tetrahydrocannabinol ((−)-11-OH-Δ-9-THC), which is psychoactive, and to (−)-11-nor-9-carboxy-Δ-9-tetrahydrocannabinol ((−)-Δ-9-THC-COOH), which is psychoinactive. It is glucuronidated during phase II metabolism. The biotransformation of (−)-Δ-9-tetrahydrocannabinol-glucuronide ((−)-Δ-9-THC-Glc) and (−)-11-nor-9-carboxy-Δ-9-tetrahydrocannabinol-glucuronide ((−)-Δ-9-THC-COOH-Glc) is well understood, which is mainly due to the availability of commercial reference standards. Since such a standardized reference is not yet available for (−)-11-hydroxy-Δ-9-tetrahydrocannabinol-glucuronide ((−)-11-OH-Δ-9-THC-Glc), its biotransformation is harder to study and the nature of the glucuronide bonding—alcoholic and/or phenolic—remains unclear. Consequently, the aim of this study was to investigate the biotransformation of (−)-11-OH-Δ-9-THC-Glc in vitro as well as in vivo and to identify the glucuronide by chemically synthesis of a reference standard. For in vitro analysis, pooled human S9 liver fraction was incubated with (−)-Δ-9-THC. Resulting metabolites were detected by high-performance liquid chromatography system coupled to a high-resolution mass spectrometer (HPLC-HRMS) with heated electrospray ionization (HESI) in positive and negative full scan mode. Five different chromatographic peaks of OH-Δ-9-THC-Glc have been detected in HESI positive and negative mode, respectively. The experiment set up according to Wen et al. indicates the two main metabolites being an alcoholic and a phenolic glucuronide metabolite. In vivo analysis of urine (n = 10) and serum (n = 10) samples from cannabis users confirmed these two main metabolites. Thus, OH-Δ-9-THC is glucuronidated at either the phenolic or the alcoholic hydroxy group. A double glucuronidation was not observed. The alcoholic (−)-11-OH-Δ-9-THC-Glc was successfully chemically synthesized and identified the main alcoholic glucuronide in vitro and in vivo. (−)-11-OH-Δ-9-THC-Glc is the first reference standard for direct identification and quantification. This enables future research to answer the question whether phenolic or alcoholic glucuronidation forms the predominant way of metabolism.

Insights into the human metabolism of hexahydrocannabinol by non-targeted liquid chromatography–high-resolution tandem mass spectrometry

“…Drawing parallels with the human metabolism of THC and CBD, it was anticipated that metabolic transformation reactions would involve Position 11 as well as the pentyl side chain ( 6–8 ). Additionally, it was expected that both the parent drug and its oxidized forms might undergo glucuronidation reactions ( 9 ). Leveraging the available LC–MS-MS data, these hypotheses were verified, enabling insights into the biotransformation pathways of HHC in humans.…”

“…Another observed metabolism route for CBD includes oxidation of the pentyl side chain ( 7 , 8 ). Phase II metabolism primarily involves glucuronidation, with parent drugs and their oxidized forms as precursors ( 9 ).…”

The Intoxication Equivalency of 11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) Relative to Δ9-tetrahydrocannabinol (THC)

“…The wide availability of cannabis necessitates warning against its misuse (Carvalho and Evans-Gilbert, 2019) as there have been numerous case reports of acute cannabis intoxication resulting in seizures, ataxia, lethargy, tremors, and even comas among children (Emoto and Kallail, 2020). Cannabis or THC is becoming increasingly important as a therapeutic agent (Hassenberg et al, 2020). Although THC is considered the principal psychoactive agent of the cannabis plant (Dumbraveanu et al, 2023), and 11-OH-THC is the primary metabolite of THC, there is limited data on the pharmacology of 11-OH-THC.…”

Cannabis Pharmacogenomics: A Path to Personalized Medicine

“…Data on the association of UGT1A9 polymorphism with the metabolism of cannabinoids are limited. A recent study demonstrated significantly lower 11-OH-THC concentrations of homozygote carriers of the derived alleles in UGT1A9 440/331 compared with homozygote carriers of the ancestral alleles [ 244 , 245 ].…”