Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes

doi:10.1016/j.lfs.2011.05.018

Life Sciences

Volume 89, Issues 5–6, 1 August 2011, Pages 165-170

https://doi.org/10.1016/j.lfs.2011.05.018 Get rights and content

Abstract

Aims

Cannabidiol (CBD), one of the major constituents in marijuana, has been shown to be extensively metabolized by experimental animals and humans. However, human hepatic enzymes responsible for the CBD metabolism remain to be elucidated. In this study, we examined in vitro metabolism of CBD with human liver microsomes (HLMs) to clarify cytochrome P450 (CYP) isoforms involved in the CBD oxidations.

Main methods

Oxidations of CBD in HLMs and recombinant human CYP enzymes were analyzed by gas chromatography/mass spectrometry.

Key findings

CBD was metabolized by pooled HLMs to eight monohydroxylated metabolites (6α-OH-, 6β-OH-, 7-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs). Among these metabolites, 6α-OH-, 6β-OH-, 7-OH-, and 4″-OH-CBDs were the major ones as estimated from the relative abundance of m/z 478, which was a predominant fragment ion of trimethylsilyl derivatives of the metabolites. Seven of 14 recombinant human CYP enzymes examined (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) were capable of metabolizing CBD. The correlations between CYP isoform-specific activities and CBD oxidative activities in 16 individual HLMs indicated that 6β-OH- and 4″-OH-CBDs were mainly formed by CYP3A4, which was supported by inhibition studies using ketoconazole and an anti-CYP3A4 antibody. The correlation and inhibition studies also showed that CBD 6α-hydroxylation was mainly catalyzed by CYP3A4 and CYP2C19, whereas CBD 7-hydroxylation was predominantly catalyzed by CYP2C19.

Significance

This study indicated that CBD was extensively metabolized by HLMs. These results suggest that CYP3A4 and CYP2C19 may be major isoforms responsible for 6α-, 6β-, 7-, and/or 4″-hydroxylations of CBD in HLMs.

Introduction

Cannabidiol (CBD), one of the major constituents in marijuana (Fig. 1), is not psychoactive but has several pharmacological effects such as drug-induced sleep prolongation, antiepileptic, anxiolytic, and antiemetic actions (Mechoulam et al., 2002). Some of these pharmacological effects may be of therapeutic importance. Recently, Sativex^®, a marijuana extract containing CBD, has been clinically used for the symptomatic relief for neuropathic pain and spasticity in multiple sclerosis (Collin et al., 2010, Kavia et al., 2010, Selvarajah et al., 2010, Wade et al., 2010).

CBD is extensively metabolized by experimental animals and humans (Huestis, 2005). Martin et al. (1976) reported that CBD was oxidized by a 10,000 ×g supernatant from rat livers to form 7-hydroxy-CBD (7-OH-CBD) as a major metabolite together with seven other metabolites; 6α-OH-, 6β-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs. A comparative study using liver microsomes from seven experimental animals has shown that CBD is metabolized to these eight metabolites and 10-OH-CBD, and the metabolic profiles of CBD are considerably different among the animal species (Harvey and Brown, 1990). In an in vivo metabolism study in mice, 22 metabolites including CBD-7-oic acids and glucuronide conjugates were characterized in the liver following intraperitoneal administration of CBD (Martin et al., 1977). In addition, 33 metabolites were identified in urine from a dystonic patient treated chronically with CBD (Harvey and Mechoulam, 1990). From these findings, the main metabolic pathway of CBD in humans is suggested to be oxidation of C-atom at the 7-position followed by further hydroxylation in the pentyl side chain and the terpene moiety (Harvey and Mechoulam, 1990). The formation of these hydroxylated metabolites of CBD is thought to be catalyzed by cytochrome P450 (CYP), because it has been previously reported that a purified mouse CYP2C enzyme metabolizes CBD to form 6″-OH-, 7-OH-, and 4″-OH-CBDs (Bornheim and Correia, 1991). However, overall metabolism of CBD with human liver microsomes (HLMs) and CYP isoforms responsible for CBD oxidations in human livers remain unclear.

In the present study, we investigated in vitro metabolism of CBD with HLMs. We report herein that CBD is metabolized by HLMs to produce four major metabolites, 6α-OH-, 6β-OH-, 7-OH-, and 4″-OH-CBDs together with four minor metabolites 1″-OH-, 2″-OH-, 3″-OH-, and 5″-OH-CBDs. Furthermore, our study suggests that CYP3A4 and CYP2C19 may play important roles in the 6α-, 6β-, 7-, and 4″-hydroxylations of CBD in HLMs.

Section snippets

Materials

CBD was isolated from cannabis leaves using the method of Aramaki et al. (1968). 6α-OH-CBD, 6β-OH-CBD, and 5′-nor-Δ⁸-tetrahydrocannabinol-4′-oic acid methyl ester (5′-nor-Δ⁸-THC-4′-oic acid methyl ester) were prepared by the previous methods (Lander et al., 1976, Ohlsson et al., 1979). The purities of these cannabinoids were determined to be at least 98% by gas chromatography (GC). NADP and glucose 6-phosphate were purchased from Böehringer-Mannheim GmbH (Darmstadt, Germany). Glucose

Metabolism of CBD by pooled HLMs

Fig. 2 shows representative mass chromatograms of the TMS derivatives of CBD metabolites formed with pooled HLMs. HLMs produced eight monohydroxylated metabolites of CBD, 6α-OH-, 6β-OH-, 7-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs, in the presence of NADPH (Fig. 2). The typical fragment ions of these eight metabolites were in good agreement with a previous report (Martin et al., 1976). CBD metabolites formed with HLMs were analyzed by GC/MS after trimethylsilylation. All of

Discussion

In the present study, we demonstrated that CBD is extensively metabolized by HLMs. Under the conditions of this study, eight monohydroxylated metabolites of CBD were detected in the incubation with HLMs. Based on the relative abundance of these metabolites formed, it is assumed that 6α-, 6β-, 7-, and 4″-hydroxylations are predominant metabolic pathways of CBD in HLMs. Harvey and Mechoulam (1990) have previously reported that the main metabolic pathway of CBD in humans may be 7-hydroxylation

Conclusions

The present study conclusively demonstrated that CBD was extensively metabolized by HLMs. Our results suggest that CYP3A4 and CYP2C19 may play pivotal roles in the formation of 6α-OH-, 6β-OH-, 7-OH-, and 4″-OH-CBDs in HLMs (Fig. 5).

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (Grant Number 20590127) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the ‘Academic Frontier’ Project for Private Universities (Grant Number 05F016) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2005–2009).

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Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes

Abstract

Aims

Main methods

Key findings

Significance

Introduction

Section snippets

Materials

Metabolism of CBD by pooled HLMs

Discussion

Conclusions

Conflict of interest statement

Acknowledgments

Arch Biochem Biophys

Biochem Pharmacol

Life Sci

Forensic chemical study on marihuana I. A detection method of the principal constituents by thin-layer and gas chromatographies

Chem Pharm Bull

Zur identifikation potentieller metabolite von cannabis-inhaltstoffen: kernresonanz- und massenspektroskopische untersuchungen an seitenkettenhydroxylierten cannabinoiden

Helv Chim Acta

Purification and characterization of the major hepatic cannabinoid hydroxylase in the mouse: a possible member of the cytochrome P-450IIC subfamily

Mol Pharmacol

Anticonvulsant activity of four oxygenated cannabidiol derivatives

Res Commun Chem Pathol Pharmacol

A double-blind, randomized, placebo-controlled, parallel-group study of Sativex, in subjects with symptoms of spasticity due to multiple sclerosis

Neurol Res

In vitro metabolism of cannabidiol in seven common laboratory mammals

Res Commun Subst Abuse

Metabolites of cannabidiol identified in human urine

Xenobiotica