Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes
Introduction
Tetrahydrocannabinols (THCs) and cannabinol (CBN), which are the major constituents of marijuana, are known to be extensively metabolized by experimental animals and humans (Harvey, 1984). Cytochrome P450 (CYP) is mainly involved in the primary metabolism of the cannabinoids in hepatic microsomes (Yamamoto et al., 1995). Our previous studies demonstrated that the major CYP enzymes involved in the hepatic metabolism of THCs in mice and male rats are CYP2C29 (Watanabe et al., 1993) and CYP2C11 (Narimatsu et al., 1990), respectively. The metabolic reaction of THCs is complicated and over 40 metabolites have been identified in humans in vivo [Agurell et al., 1986]. Many metabolites of CBN have also been identified in humans in vivo (Agurell et al., 1986). However, relatively limited information is available for in vitro metabolism of these cannabinoids in humans (Halldin et al., 1982, Yamamoto et al., 1983), in particular CYP enzymes involved in the metabolism of THCs and CBN are not fully elucidated. Bornheim et al. (1992) suggested in a study with immunoinhibition and purified enzyme that CYP2C9 was mainly responsible for the 11-hydroxylation of Δ9-THC in human hepatic microsomes. Our previous study suggested that a member of the CYP2C subfamily is primarily responsible for the 11-hydroxylation of Δ8-THC, Δ9-THC, and CBN in human hepatic microsomes from an elderly woman, although the enzyme involved in the reaction was not entirely specified (Watanabe et al., 1995). The 11-hydroxylation of THCs and CBN has been shown to be the metabolic activation pathway of both cannabinoids (Watanabe et al., 1980; Yamamoto et al., 1987, Yamamoto et al., 2003). Recently, Bland et al. (2005) reported the kinetic nature and pharmacogenetics of CYP2C9 enzymes for the metabolic interaction of Δ9-THC and phenytoin. The present paper describes the CYP enzymes responsible for the major metabolism of THCs and CBN in human hepatic microsomes.
Section snippets
Chemicals
Δ9-THC and CBN were isolated from cannabis leaves by the methods of Aramaki et al. (1968). Δ8-THC was prepared by acidic isomerization of Δ9-THC as described by Gaoni and Mechoulam (1966). 7α-Hydroxy-Δ8-THC (Mechoulam et al., 1972), 7β-hydroxy-Δ8-THC (Mechoulam et al., 1972), 11-hydroxy-Δ8-THC (Inayama et al., 1974), 8β-hydroxy-Δ9-THC (Pitt et al., 1975), 9α,10α-epoxyhexahydrocannabinol (9α,10α-EHHC) (Narimatsu et al., 1983), 11-hydroxy-Δ9-THC (Pitt et al., 1975), 8-hydroxy-CBN (Inayama et al.,
Metabolism of cannabinoids by human hepatic microsomes
The most predominant metabolites of THCs and CBN formed by pooled human hepatic microsomes were 11-hydroxy metabolites. The catalytic activities (nmol/min/mg protein) for the 11-hydroxylation of pooled microsomes for Δ8-THC, Δ9-THC, and CBN were 0.492, 0.515, and 0.448, respectively. Δ8-THC, Δ9-THC, and CBN were also biotransformed to 7α-hydroxy-Δ8-THC (0.355 nmol/min/mg protein), 8β-hydroxy-Δ9-THC (0.344 nmol/min/mg protein), and 8-hydroxy-CBN (0.039 nmol/min/mg protein), respectively, as the
Discussion
Δ8-THC was mainly converted to 7α-hydroxy-Δ8-THC and 11-hydroxy-Δ8-THC by human hepatic microsomes. Δ9-THC and CBN were also primarily oxidized at the 8- and 11-positions by human hepatic microsomes. The 11-hydroxylation of three cannabinoids was highly inhibited by sulfaphenazole, which has been confirmed as a potent inhibitor of CYP2C enzymes, suggesting that CYP2C enzymes are involved in the 11-hydroxylation of the cannabinoids in human hepatic microsomes as indicated in the case of Δ9-THC
Acknowledgement
A part of this work was supported by the Academic Frontier Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan (2005–2009).
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