Identification and Characterization of Cannabichromene's Major Metabolite Following Incubation with Human Liver Microsomes

Abstract

Cannabichromene (CBC) is a minor cannabinoid within the array of over 120 cannabinoids identified in the Cannabis sativa plant. While CBC does not comprise a significant portion of whole plant material, it is available to the public in a purified and highly concentrated form. As minor cannabinoids become more popular due to their potential therapeutic properties, it becomes crucial to elucidate their metabolism in humans. Therefore, the goal of this was study to identify the major CBC phase I-oxidized metabolite generated in vitro following incubation with human liver microsomes. The novel metabolite structure was identified as 2'-hydroxycannabicitran using gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Following the identification, in silico molecular modeling experiments were conducted and predicted 2'-hydroxycannabicitran to fit in the orthosteric site of both the CB₁ and CB₂ receptors. When tested in vitro utilizing a competitive binding assay, the metabolite did not show significant binding to either the CB₁ or CB₂ receptors. Further work necessitates the determination of potential activity of CBC and the here-identified phase I metabolite in other non-cannabinoid receptors.

Keywords: 2′-hydroxycannabicitran; CB1 receptor; CB2 receptor; binding; cannabichromene; cannabicitran; gas chromatography–mass spectrometry; human drug metabolism; human liver microsomes; molecular docking; nuclear magnetic resonance spectroscopy; phase I metabolism.

Figure 1

Structures and numbering of (A) CBC, (B) cannabicitran (CBT-C), and the main oxidized CBC metabolite identified in the present study, (C) 2′-hydroxycannabicitran. In this figure and throughout the present manuscript, CBC, CBT-C, and 2′-hydroxycannabicitran are numbered according to a terpenoid system of numbering [36,37]. The theoretical exact masses and chemical formulas are shown below each respective structure.

Figure 2

Upscaled metabolite generation following CBC incubation with HLMs analyzed via LC-MS/MS. The figure shows overlaid extracted ion chromatograms recorded on a Sciex API4000 MS/MS system (Supplementary Materials Section S1.2). The blue tracer represents CBC ([M+H]⁺, m/z = 315.5, primary peak retention time at 17.9 min), the red tracer represents +16 Da from CBC ([M+H]⁺, m/z = 331.5, indicating addition of one oxygen, primary peak retention time at 14.9 min), and the dark green tracer represents +32 Da from CBC ([M+H]⁺, m/z = 347.5 indicating addition of two oxygens, primary peak retention time at 8.8 min). The neon green peak at 21.0 min was present in the background controls and is not considered a metabolite. Additional extracted ion chromatogram tracers and analytical details are available in the Supplementary Materials (Section S1.2). As shown, HLMs generated one major metabolite, which we later identified to be 2′-hydroxycannabicitran. Based on this result, we decided to focus here on said major metabolite, its structural identification, and its potential interaction with the CB₁ and CB₂ receptors. No attempt was made during the present study to structurally identify any of the other minor metabolites.

Figure 3

(A) Staggered overlay of GC-MS/MS total ion chromatogram (TIC, black tracer) of CBC incubated with HLMs, extracted ion chromatogram of the fragment m/z = 303.2 ((i), green tracer), extracted ion chromatogram of the fragment m/z = 391.2 ((ii), red tracer), and extracted ion chromatogram of the fragment m/z = 319.1 ((iii), blue tracer). Extracted fragments were selected based upon expected major fragments of metabolites following the formation of the chromenyl ion. Intensities of the extracted fragments were multiplied by a factor of 10 for ease of visibility. Major fragments are shown due to the considerable fragmentation of the molecular ion following ionization, as expected from previous reports [32,33]. Mass spectra of CBC and the major oxidized metabolite 2′-hydroxycannabicitran are shown in (B,C), respectively. Proposed structures for possible chromenyl ions are shown in (D(i–iii)). Importantly, these structures are representative possibilities and an identical modification elsewhere on the molecule resulting in the same chemical formula would be correct.

Figure 4

¹H NMR of 2′-hydroxycannabicitran. Solvent peaks and impurities are labeled accordingly. There was poor resolution between H-8′b and H-4′a as well as H-4″, H-9′, and H-3″; therefore, all subsets were integrated as a group.

Figure 5

Concentration–response curves of tested ligands in radioligand binding assays at (A) CB₁R and (B) CB₂R. Data shown represent the means ± S.E.M. of four to ten experiments conducted in duplicate.