Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

Life Sciences 80 (2007) 1415 – 1419 www.elsevier.com/locate/lifescie

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes Kazuhito Watanabe a,⁎, Satoshi Yamaori a , Tatsuya Funahashi a , Toshiyuki Kimura a , Ikuo Yamamoto b a

Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan b Department of Hygienic Chemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-machi, Nobeoka 882-8508, Japan Received 9 August 2006; accepted 27 December 2006

Abstract In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7α-position for Δ8-THC and the 8βposition for Δ9-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7α-(Δ8-THC), 8β-(Δ9-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Δ8-THC (7.60 nmol/min/nmol CYP), Δ9-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7α-(5.34 nmol/min/nmol CYP) and 7β-hydroxylation (1.39 nmol/min/nmol CYP) of Δ8-THC, the 8β-hydroxylation (6.10 nmol/min/nmol CYP) and 9α,10α-epoxidation (1.71 nmol/min/nmol CYP) of Δ9-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7α- and 7β-hydroxylation of Δ8-THC, and the 9α,10α-epoxidation of Δ9-THC. © 2007 Elsevier Inc. All rights reserved. Keywords: Tetrahydrocannabinol; Cannabinol; CYP2C9; CYP3A4; Human hepatic microsomes; Oxidative metabolism

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 ⁎ Corresponding author. Tel.: +81 76 229 6220; fax: +81 76 229 6221. E-mail address: [email protected] (K. Watanabe). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.12.032

[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 11hydroxylation 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, Δ9THC, 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

1416

K. Watanabe et al. / Life Sciences 80 (2007) 1415–1419

et al., 1987, 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. Materials and methods 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), 11hydroxy-Δ8-THC (Inayama et al., 1974), 8β-hydroxy-Δ9-THC (Pitt et al., 1975), 9α,10α-epoxyhexahydrocannabinol (9α,10αEHHC) (Narimatsu et al., 1983), 11-hydroxy-Δ9-THC (Pitt et al., 1975), 8-hydroxy-CBN (Inayama et al., 1974), 11-hydroxy-CBN (Yamamoto et al., 1987), and 5′-nor-Δ8-THC-4′-oic acid methyl ester (Ohlsson et al., 1979) were prepared according to the previous methods. Purities of these cannabinoids prepared were checked to be more than 98% by gas chromatography (GC). GC analyses were conducted by the methods previously described, except that the detector temperature was 280 °C and flow rate of carrier gas was 50 mL/min (Watanabe et al., 1986). NADPH was purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan); NADP and glucose 6-phosphate were from Boehringer-Mannheim GmbH (Darmstadt, Germany); glucose 6-phosphate dehydrogenase (type V, EC 1.1.1.49) and sulfaphenazole were from Sigma Chemical Co. (St. Louis, MO, USA); bis-trimethylsilyltrifluoroacetamide, trimethylsilylimidazole, trimethylchlorosilane, and 7,8-benzoflavone were from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan); ketoconazole and other chemicals were from Wako Pure Chemicals (Osaka, Japan).

ester (1 μg) as the internal standard. After the evaporation of the organic solvent, the residue was trimethylsilylated as described previously and subjected to GC-MS (Narimatsu et al., 1990, 1991; Watanabe et al., 1995). GC-MS conditions; 1) a JEOL JMS 06 gas chromatograph coupled with a JEOL JMS DX-300 GCG mass spectrometer and a JEOL DA mass data system: column, 2% OV-17 on Chromosorb W (60–80 mesh, 3 mm × 1.5 m); column temperature, 260 °C; ionization, 70 eV; ionizing current, 300 μA; carrier gas, He 40 mL/min; 2) a Shimadzu GCMSQP2010: column, DB-1 (0.25 mm × 30 m); column temperature, 50 °C (1 min), 25 °C/min (6 min), 10 °C/min (10 min), 300 °C (5 min hold); ion source temperature, 250 °C, interface temperature 280 °C, ionization, 70 eV; emission current, 60 μA; carrier gas, He 2.04 mL/min. Under these conditions, the retention times (min, condition-1; condition-2) of TMS derivatives of cannabinoid metabolites were as follows; 7αhydroxy-Δ8-THC (1.93, 14.53), 7β-hydroxy-Δ8-THC (2.47, 15.17), 11-hydroxy-Δ8-THC (3.67, 15.68), 8β-hydroxy-Δ9THC (1.73, 15.32), 9α,10α-EHHC (2.07, 13.76), 11-hydroxyΔ9-THC (3.07, 15.45), 8-hydroxy-CBN (3.90, 16.08), 11hydroxy-CBN (4.16, 16.18), and 5′-nor-Δ8-THC-4′-oic acid methyl ester (3.60, 14.95). For the study on inhibitory effects of CYP-selective inhibitors on cannabinoid metabolism, the incubations were carried out under the same manner described above with 7,8benzoflavone (10 μM) (Baldwin et al., 1995), ketoconazole (10 μM) (Baldwin et al., 1995), and sulfaphenazole (50 μM) (Ono et al., 1996). Other method Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Results

Enzyme source

Metabolism of cannabinoids by human hepatic microsomes

Pooled human hepatic microsomes were purchased from Xenotech (Lenexa, KS, USA) and human CYPs (1A1, 1A2, 2A6, 2B6, 2C8, 2C9-Arg, 2C9-Cys, 2C19, 2D6-Met, 2D6-Val, 2E1 and 3A4) containing microsomes prepared from human Blymphoblastoid cells expressed cDNA were obtained from Gentest (Woburn, MA, USA).

The most predominant metabolites of THCs and CBN formed by pooled human hepatic microsomes were 11-hydroxy Table 1 Effects of various CYP inhibitors on metabolic formation of cannabinoid metabolites formed with human hepatic microsomes Relative activity (% of control)

Cannabinoid metabolism Δ -THC, Δ -THC or CBN (final conc. 64 μM) was added to a mixture of human hepatic microsomes (10 μg protein) or the microsomes from human B lymphoblastoid cells (10 to 50 pmol CYP), NADPH (1 mM) or an NADPH-generating system (NADP 0.5 mM, glucose 6-phosphate 10 mM, 1 unit of glucose 6-phosphate dehydrogenase, magnesium chloride 10 mM), and 100 mM of potassium phosphate buffer (pH 7.4) to make a final volume of 0.5 mL. The mixtures were incubated at 37 °C for 20 min and then extracted with 4 mL of ethyl acetate after addition of 0.5 mL of 1 M KH2PO4 and 5′-nor-Δ8-THC-4′-oic acid methyl 8

Δ9-THC

Δ8-THC

9

Control Ketoconazole Sulfaphenazole 7,8-Benzoflavone

CBN

7α-OH

11-OH

8β-OH

11-OH

8-OH

11-OH

100 4 72 124

100 74 38 92

100 9 73 125

100 69 13 99

100 36 94 185

100 85 11 93

The data are means of duplicate incubations. The concentrations of inhibitors used are 10 μM for ketoconazole and 7,8benzoflavone, and 50 μM for sulfaphenazole. 7α-OH: 7α-hydroxylation of Δ8-THC; 7β-OH: 7β-hydroxylation of Δ8-THC; 8β-OH: 8β-hydroxylation of Δ9-THC; 8-OH: 8-hydroxylation of CBN; 11-OH: 11-hydroxylation.

K. Watanabe et al. / Life Sciences 80 (2007) 1415–1419

1417

metabolites. The catalytic activities (nmol/min/mg protein) for the 11-hydroxylation of pooled microsomes for Δ8-THC, Δ9THC, and CBN were 0.492, 0.515, and 0.448, respectively. Δ8-THC, Δ9-THC, and CBN were also biotransformed to 7αhydroxy-Δ8-THC (0.355 nmol/min/mg protein), 8β-hydroxyΔ9-THC (0.344 nmol/min/mg protein), and 8-hydroxy-CBN (0.039 nmol/min/mg protein), respectively, as the second abundant metabolites by human hepatic microsomes. Inhibition by CYP specific inhibitors Ketoconazole, sulfaphenazole, and 7,8-benzoflavone are known to be selective inhibitors of human CYP3A, CYP2C, and CYP1A, respectively (Baldwin et al., 1995; Ono et al., 1996). We used these form-selective inhibitors, and determined their inhibitory effects on the metabolism of cannabinoids by human hepatic microsomes (Table 1). The formation of 11hydroxy metabolites from three cannabinoids was inhibited by 62–89%, when the cannabinoids were incubated with sulfaphenazole (50 μM). Ketoconazole (10 μM) inhibited the formation of 7α-hydroxy-Δ8-THC, 8β-hydroxy-Δ9-THC and 8-hydroxy-CBN from the corresponding cannabinoids by 96, 91, and 64%, respectively. 7,8-Benzoflavone (10 μM) had little effect on the formation of these metabolites from three cannabinoids. Catalytic activities of human CYPs expressed in human B lymphoblastoid cells Table 2 summarizes the catalytic activities for three cannabinoids of human CYPs expressed in microsomes of human B lymphoblastoid cells. Human CYP2C9-Arg expressed in the microsomes exhibited a high 11-hydroxylation activity Table 2 Catalytic activities for cannabinoids of human CYPs expressed in microsomes of human B lymphoblastoid cells Catalytic activity (nmol/min/nmol CYP) 11-Hydroxylation

CYP1A1 CYP1A2 CYP2A6 CYP2B6 CYP2C8 CYP2C9-Arg CYP2C9-Cys CYP2C19 CYP2D6-Met CYP2D6-Val CYP2E1 CYP3A4

CYP3A4

Δ8-THC

Δ9-THC

CBN

b0.01 b0.01 b0.01 b0.01 b0.01 7.60 10.2 0.41 0.04 b0.01 b0.01 b0.01

b0.01 b0.01 b0.01 b0.01 b0.01 19.2 3.65 0.22 0.01 b0.01 b0.01 b0.01

b0.01 b0.01 b0.01 b0.01 b0.01 6.62 0.64 0.23 b0.01 b0.01 b0.01 b0.01

7α-OH

7β-OH

8β-OH

EHHC

8-OH

5.34

1.39

6.10

1.71

1.45

The data are means of duplicate incubations. 7α-OH: 7α-hydroxylation of Δ8-THC; 7β-OH: 7β-hydroxylation of Δ8-THC; 8β-OH: 8β-hydroxylation of Δ9-THC; EHHC: 9α,10α-epoxidation of Δ9THC; 8-OH: 8-hydroxylation of CBN.

Fig. 1. Catalytic sites of human CYPs on cannabinoid structure 7α-OH: 7αhydroxylation of Δ8-THC; 7β-OH: 7β-hydroxylation of Δ8-THC; 8β-OH: 8βhydroxylation of Δ9-THC; 8-OH, 8-hydroxylation of CBN; 9α,10α-EHHC: 9α,10α-epoxidation of Δ9-THC.

for THCs and CBN (6.62–19.2 nmol/min/nmol CYP). CYP2C9-Cys also efficiently catalyzed the 11-hydroxylation of the cannabinoids (0.64–10.2 nmol/min/nmol CYP). Expressed CYP2C19 had little activity (b0.5 nmol/min/nmol CYP) for the formation of 11-hydroxy metabolites from the cannabinoids. Expressed human CYP3A4 catalyzed the 7α(5.34 nmol/min/nmol CYP) and 7β-(1.39 nmol/min/nmol CYP) hydroxylation of Δ 8 -THC, and the 8β-hydroxylation (6.10 nmol/min/nmol CYP) and 9α,10α-epoxidation (1.71 nmol/min/nmol CYP) of Δ9-THC. Expressed CYP3A4 also catalyzed the formation of 8-hydroxy-CBN, which has been reported as a new metabolite of CBN by human hepatic microsomes (Watanabe et al., 2006), although CYP3A4 did not catalyze the 11-hydroxylation of three cannabinoids at a detectable level. No other CYPs expressed in the microsomes of human B lymphoblastoid cells catalyzed efficiently the 8-(or 7-) and 11-hydroxylations of these cannabinoids. Discussion Δ8 -THC was mainly converted to 7α-hydroxy-Δ 8 -THC and 11-hydroxy-Δ 8 -THC by human hepatic microsomes. Δ9 -THC and CBN were also primarily oxidized at the 8- and 11positions 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 Δ 9-THC by Bornheim et al. (1992) and Bland et al. (2005), and that of Δ8 -THC by Watanabe et al. (1995). In the present study, the 11hydroxylation of Δ8 -THC and Δ 9 -THC was also inhibited by ketoconazole, a typical inhibitor of CYP3A enzymes to some extent (26–31% at 10 μM). Ketoconazole has been reported to inhibit CYP2C-mediated tolbutamide hydroxylation at a higher concentration (Newton et al., 1995). Recently, Bland et al. (2005) also reported that ketoconazole slightly inhibited CYP2C9-mediated 11-hydroxy-Δ9 -THC formation at higher concentrations (N10 μM). These findings indicate that the

1418

K. Watanabe et al. / Life Sciences 80 (2007) 1415–1419

ketoconazole inhibition observed in the present study is due to a nonselective interaction of the inhibitor with CYP2C9 at a relatively higher concentration at 10 μM. Expressed CYP2C9 but not CYP2C8 and CYP2C19 efficiently catalyzed the 11hydroxylation of Δ8 -THC, Δ9 -THC, and CBN. These results indicate that CYP2C9 is a major enzyme responsible for the 11hydroxylation of the cannabinoids in human hepatic microsomes as reported previously in the case of Δ 9 -THC (Bornheim et al., 1992; Bland et al., 2005). The other major metabolic sites for three cannabinoids by human hepatic microsomes were at the 7α-position for Δ8-THC, and at the 8(β)-position for Δ9-THC and CBN. Ketoconazole inhibited most of these reactions by human hepatic microsomes. Even at a higher concentration of 10 μM, however, 7,8benzoflavone failed to inhibit the 7α-hydroxylation of Δ8-THC and the 8-hydroxylation of Δ9-THC and CBN, but rather stimulated these reactions. The precise mechanism for the stimulation of cannabinoids metabolism by the inhibitor is not clear at present, although some metabolic reactions catalyzed by CYP3A enzymes are known to be stimulated by 7,8-benzoflavone as an effector (Schwab et al., 1988; Emoto et al., 2001). We also reported that 7,8-benzoflavone stimulated CYP3Amediated metabolism of 7β-hydroxy-Δ8-THC by monkey hepatic microsomes (Funahashi et al., 2005). In addition, expressed CYP3A4 catalyzed the 8(β)-hydroxylation of Δ9THC and CBN, and the 7α-hydroxylation of Δ8-THC. The expressed CYP3A4 was also able to catalyze the 9α,10αepoxidation of Δ9-THC and the 7β-hydroxylation of Δ8-THC. The former is known to be an active metabolite of Δ9-THC (Narimatsu et al., 1983). These results suggest that CYP3A4 is a major enzyme responsible for the 7α- and 7β-hydroxylation of Δ8-THC, and the 9α,10α-epoxidation of Δ9-THC in human hepatic microsomes. Metabolism by CYP2C9 and CYP3A4 accounts for most of the primary metabolites of the three cannabinoids formed, indicating that two CYP enzymes are major enzymes involved in the metabolism of Δ8-THC, Δ9-THC, and CBN by human hepatic microsomes (Fig. 1). 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). References Agurell, S., Halldin, M., Lindgren, J.-E., Ohlsson, A., Widman, M., Gillespie, H., Hollister, L., 1986. Pharmacokinetics and metabolism of Δ1-tetrahydrocannabinol and other cannabinoids with emphasis on man. Pharmacological Reviews 38, 21–43. Aramaki, H., Tomiyasu, N., Yoshimura, H., Tsukamoto, H., 1968. Forensic chemical study on marihuana. I. A detection method of the principal constituents by thin-layer and gas chromatographies. Chemical and Pharmaceutical Bulletin 16, 822–826. Baldwin, S.J., Bloomer, J.C., Smith, G.J., Ayrton, A.D., Clarke, S.E., Chenery, R.J., 1995. Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9. Xenobiotica 25, 261–270.

Bland, T.M., Haining, R.L., Tracy, T.S., Callery, P.S., 2005. CYP2C-catalyzed delta(9)-tetrahydrocannabinol metabolism: kinetics, pharmacogenetics and interaction with phenytoin. Biochemical Pharmacology 70, 1096–1103. Bornheim, L.M., Lasker, J.M., Raucy, J.L., 1992. Human hepatic microsomal metabolism of Δ1-tetrahydrocannabinol. Drug Metabolism and Disposition 20, 241–246. Emoto, C., Yamazaki, H., Iketani, H., Yamasaki, S., Satoh, T., Shimizu, R., Suzuki, S., Shimada, N., Nakajima, M., Yokoi, T., 2001. Cooperativity of αnaphthoflavone in cytochrome P4503A-dependent drug oxidation activities in hepatic microsomes from mouse and human. Xenobiotica 31, 265–275. Funahashi, T., Tanaka, Y., Yamaori, S., Kimura, T., Matsunaga, T., Ohmori, S., Kageyama, T., Yamamoto, I., Watanabe, K., 2005. Stimulatory effects of testosterone and progesterone on the NADH- and NADPH-dependent oxidation of 7β-hydroxy-Δ8-tetrahydrocannabinol to 7-oxo-Δ8-tetrahydrocannabinol in monkey liver microsomes. Drug Metabolism and Pharmacokinetics 20, 358–367. Gaoni, Y., Mechoulam, R., 1966. Hashish-VII, the isomerization of cannabidiol to tetrahydrocannabinols. Tetrahedron 22, 1481–1488. Halldin, M., Widman, M., Bahr, C.V., Lindgren, J.-E., Martin, B.R., 1982. Identification of in vitro metabolites of Δ1-tetrahydrocannabinol formed by human liver. Drug Metabolism and Disposition 10, 297–301. Harvey, D.J., 1984. Chemistry, metabolism, and pharmacokinetics of the cannabinoids. In: Nahas, G.G. (Ed.), Marihuana in Science and Medicine. Raven Press, New York, pp. 37–107. Inayama, S., Sawa, A., Hosoya, E., 1974. The oxidation of Δ1- and Δ6tetrahydrocannabinol with selenium dioxide. Chemical and Pharmaceutical Bulletin 22, 1519–1525. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Mechoulam, R., Varconi, H., Ben-Zvi, Z., Edery, H., Grunfeld, Y., 1972. Synthesis and biological activity of five tetrahydrocannabinol metabolites. Journal of American Chemical Society 94, 7930–7931. Narimatsu, S., Yamamoto, I., Watanabe, K., Yoshimura, H., 1983. 9α,10αEpoxyhexahydrocannabinol formation from Δ9-tetrahydrocannabinol by liver microsomes of phenobarbital-treated mice and its pharmacological activities in mice. Journal of Pharmacobio-Dynamics 6, 558–564. Narimatsu, S., Watanabe, K., Matsunaga, T., Yamamoto, I., Imaoka, S., Funae, Y., Yoshimura, H., 1990. Cytochrome P-450 isozyme in metabolic activation of Δ9-tetrahydrocannabinol by rat liver microsomes. Drug Metabolism and Disposition 18, 943–948. Narimatsu, S., Watanabe, K., Yamamoto, I., Yoshimura, H., 1991. Sex difference in the oxidative metabolism of Δ9-tetrahydrocannabinol in the rat. Biochemical Pharmacology 41, 1187–1194. Newton, D.J., Wang, R.W., Lu, A.Y.H., 1995. Cytochrome P450 inhibitors, evaluation of specificities in the in vitro metabolism of therapeutic agents by human liver microsomes. Drug Metabolism and Disposition 23, 154–158. Ohlsson, A., Agurell, S., Leander, K., Dahmen, J., Edery, H., Porath, G., Levy, S., Mechoulam, R., 1979. Synthesis and psychotropic activity of side chain hydroxylated Δ6-tetrahydrocannabinol metabolite. Acta Pharmaceutica Suecica 16, 21–33. Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F.J., Tsutsui, M., 1996. Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26, 681–693. Pitt, C.G., Fowler, M.S., Sathe, S., Srivastava, S.C., Williams, D.L., 1975. Synthesis of metabolites of Δ9-tetrahydrocannabinol. Journal of American Chemical Society 97, 3798–3802. Schwab, G.E., Raucy, J.L., Johnson, E.F., 1988. Modulation of rabbit and human hepatic cytochrome P-450-catalyzed steroid hydroxylations by α-naphthoflavone. Molecular Pharmacology 33, 493–499. Watanabe, K., Yamamoto, I., Oguri, K., Yoshimura, H., 1980. Comparison in mice of pharmacological effects of Δ8-tetrahydrocannabinol and its metabolites oxidized at 11-position. European Journal of Pharmacology 63, 1–6. Watanabe, K., Arai, M., Narimatsu, S., Yamamoto, I., Yoshimura, H., 1986. Effect of repeated administration of 11-hydroxy-Δ8-tetrahydrocannabinol, an active metabolite of Δ8-tetrahydrocannabinol, on the hepatic microsomal drug-metabolizing enzyme system of mice. Biochemical Pharmacology 35, 1861–1865.

K. Watanabe et al. / Life Sciences 80 (2007) 1415–1419 Watanabe, K., Narimatsu, S., Matsunaga, T., Yamamoto, I., Yoshimura, H., 1993. A cytochrome P450 isozyme having aldehyde oxygenase activity plays a major role in metabolizing cannabinoids by mouse hepatic microsomes. Biochemical Pharmacology 46, 405–411. Watanabe, K., Matsunaga, T., Yamamoto, I., Funae, Y., Yoshimura, H., 1995. Involvement of CYP2C in the metabolism of cannabinoids by human hepatic microsomes from an old woman. Biological and Pharmaceutical Bulletin 18, 1138–1141. Watanabe, K., Yamaori, S., Funahashi, T., Kimura, T., Yamamoto, I., 2006. 8Hydroxycannabinol, a new metabolite of cannabinol formed by human hepatic microsomes. Forensic Toxicology 24, 80–82. Yamamoto, I., Narimatsu, S., Watanabe, K., Shimonishi, T., Yoshimura, H., Nagano, T., 1983. Human liver microsomal oxidation of Δ8-tetrahydrocannabinol. Chemical and Pharmaceutical Bulletin 31, 1784–1787.

1419

Yamamoto, I., Watanabe, K., Kuzuoka, K., Narimatsu, S., Yoshimura, H., 1987. The pharmacological activity of cannabinol and its major metabolite, 11hydroxycannabinol. Chemical and Pharmaceutical Bulletin 35, 2144–2147. Yamamoto, I., Watanabe, K., Narimatsu, S., Yoshimura, H., 1995. Recent advances in the metabolism of cannabinoids. International Journal of Biochemistry and Cell Biology 27, 741–746. Yamamoto, I., Watanabe, K., Matsunaga, T., Kimura, T., Funahashi, T., Yoshimura, H., 2003. Pharmacology and toxicology of marijuana — on the metabolic activation of cannabinoids and its mechanism. Journal of Toxicology. Toxin Reviews 22, 577–589.