Cyp3a4 Is a Major Isoform Responsible for Oxidation of 7-hydroxy-d- Tetrahydrocannabinol to 7-oxo-d-tetrahydrocannabinol in Human Liver Microsomes

The human liver enzyme microsomal alcohol oxygenase was able to oxidize both 7aand 7b-hydroxy-D-tetrahydrocannabinol (7aand 7b-hydroxy-D-THC) to 7-oxo-D-THC. The oxidative activity was determined by using a panel of 12 individual cDNA-expressed human cytochrome P450s (CYPs) (1A1, 1A2, 2A6, 2B6, 2C8, 2C9Arg, 2C9-Cys, 2C19, 2D6-Met, 2D6-Val, 2E1 and 3A4). Among the CYP isoforms examined, CYP3A4 showed the highest activity for both of substrates. The metabolism of 7aand 7b-hydroxy-D-THC to 7-oxo-D-THC was also detected for CYPs 1A1 (4.8% of CYP3A4), 1A2 (4.7%), 2A6 (2.3%), 2C8 (16.6%), and 2C9-Cys (5.4%), and CYPs 1A1 (0.4%), 2C8 (1.3%), 2C9-Arg (4.3%), and 2C9-Cys (0.9%), respectively. The 7aand 7b-hydroxy-D-THC microsomal alcohol oxygenase activities in human liver were significantly inhibited by addition of 100 mM troleandomycin, 1 mM ketoconazole, and anti-CYP3A antibody, although these activities were not inhibited by 1 mM 7,8-benzoflavone and 50 mM sulfaphenazole. When the substrates were incubated with the CYP3A4-expressed microsomes under oxygen-18 gas phase, atmospheric oxygen was incorporated into 35% of 7-oxo-D-THC formed from 7a-OH-DTHC, but only 12% of 7-oxo-D-THC formed from 7b-OH-D-THC. These results indicate that CYP3A4 is a major isoform responsible for the oxidation of 7aand 7b-hydroxy-D-THC to 7-oxo-D-THC in liver microsomes of humans, although the oxidation mechanisms for 7aand 7b-hydroxy-D-THC might be different. Tetrahydrocannabinol (THC) , a psychotropic component of Cannabis sativa L. (marijuana), is mainly hydroxylated at allylic position at C-7 (for D-THC), C-8 (for D-THC), and C-11 (both of Dand D-THC) together with oxidation at the pentyl side chain in various animals, including humans (Harvey and Paton, 1986). At present, over 80 metabolites have been identified from THC (Harvey and Paton, 1986). Cytochrome P450 (CYP) has been suggested to play a major role in the oxidation of THC in humans (Halldin et al., 1982; Yamamoto et al., 1983, 1984). The metabolic reaction is complicated and the isoforms responsible for particular metabolic reaction have not been completely elucidated. In some cases, the effects of metabolites are greater than those of THC (Yamamoto and Yoshimura, 1982; Yamamoto, 1986) and these metabolites have been suggested to contribute to the psychoactivity of the parent compound. The content of D-THC in marijuana of Mexican origin is 10% of the total THC (Hively et al., 1966), but it is possible that it is actually formed by isomerization (Turner et al., 1980). There have been many reports concerning metabolism of D-THC because it shows comparable pharmacological activity to D-THC and is chemically more stable. Furthermore, D-THC and its derivatives have been used for the study of medicinal chemistry and pharmacology (Mechoulam et al., 1999; Waser and Martin, 1999; Zurier et al., 1999). We have shown that 7-oxo-D-THC formed from 7-hydroxy-D-THC is one of the active metabolites (Narimatsu et al., 1984). Bornheim et al. (1992) have reported that antibody raised against mouse hepatic CYP3A inhibited the formation of most of the 8-hydroxy-D-THC and 8-oxoD-THC formation from D-THC by human liver microsomes. However, the contribution of the human CYP3A isoform to the formation of 8-oxo-D-THC has not been directly demonstrated. D-THC, not 8-hydroxy-D-THC, was used for substrate although 8-oxo-D-THC was biotransformed from 8-hydroxylated metabolites by further enzymatic oxidation. Its is well known that secondary alcohols such as hydroxysteroids and xenobiotics are oxidized to the corresponding ketones by dehydrogenases in microsomes and cytosol (Maser and Bannenberg, 1994; Furster et al., 1996; Yamano et al., 1997). We have found, however, that the 7-oxo-D-THC was biotransformed from 7aand 7b-hydroxy-D-THC by the liver microsomal enzyme microsomal alcohol oxygenase (MALCO) (Narimatsu et al., 1988). We have recently purified two CYP isoforms, named P450GPF-B and P450 MDX-B, that are the major enzymes responsible for the formation of 7-oxo-D-THC in liver microsomes of guinea pig (Matsunaga et al., 1997) and mouse (Matsunaga et al., 1998), respectively. These enzymes are estimated to be CYP3A isoforms from NH2-terminal This work was partially supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, and by the Special Research Fund of Hokuriku University. 1 Abbreviations used are: THC, tetrahydrocannabinol; CYP, cytochrome P450; MALCO, microsomal alcohol oxygenase; GC/MS, gas chromatography/mass