Inhibitory effects of D 9 -tetrahydrocannabinol (D 9 -THC), cannabidiol (CBD), and cannabinol (CBN) on the catalytic activities of human recombinant cytochrome P450 (CYP) 2A6 and CYP2B6 were investigated. D 9 -THC, CBD, and CBN noncompetitively inhibited coumarin 7-hydroxylase activity of recombinant CYP2A6 with the apparent K i values of 28.9, 55.0, and 39.8 lM, respectively. On the other hand, D 9 -THC, CBD, and CBN inhibited 7-benzoxyresorufin O-debenzylase activity of recombinant CYP2B6 in a mixed fashion with the K i values of 2.81, 0.694, and 2.55 lM, respectively. Because the inhibition of CYP2B6 by CBD was the most potent, investigation was conducted to determine which moiety of the CBD structure was responsible for the inhibition. Olivetol and d-limonene, the partial structure of CBD, inhibited the CYP2B6 activity to some extent. Inhibitory effects of CBD-2 0 -monomethyl ether and CBD-2 0 ,6 0 -dimethyl ether attenuated with the number of methylations on the phenolic hydroxyl groups in the resorcinol moiety of CBD. Cannabidivarin, a CBD analogue having a propyl side chain, inhibited the CYP2B6 activity less potently than CBD possessing a pentyl side chain. Therefore, both structures of pentylresorcinol and terpene moieties of CBD were suggested to play important roles in the CYP2B6 inhibition. D 9 -THC, CBD, and CBN showed metabolismdependent inhibition for CYP2A6 but not for CYP2B6. Furthermore, D 9 -THC and CBN were characterized as mechanism-based inhibitors for CYP2A6. The k inact and K I values of D 9 -THC were 0.0169 min -1 and 0.862 lM, respectively; the k inact and K I values of CBN were 0.00909 min -1 and 1.01 lM, respectively. These results indicated that D 9 -THC, CBD, and CBN showed differential inhibition against CYP2A6 and CYP2B6.

-An analysis of mRNA levels of 39 Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a was performed using a real-time reverse transcriptase-polymerase chain reaction. Relative expression levels were quantified by normalized β-actin levels, and compared to the mRNA expression level of the cannabinoid receptor (CB1R), which is abundantly expressed in the mouse brain. Mean 2^-ddCts of CB1R in mouse brain, C-1300N18, and NB2a cells were 1.043, 1.003, and 1.005, respectively. Among Cyp mRNAs in the mouse brain, Cyp1b1 mRNA was the most abundantly expressed (2^-ddCt = 0.310), followed by Cyp46a1 mRNA (0.246). The other Cyp mRNAs moderately expressed (0.011 ~ 0.117) were Cyp1a1, 1a2, 2b10, 2c29, 2c50, 2d9, 2d10, 2d12, 2d22, 2d26, 3a11, 3a41, 4f14, 4f15, 4f16, and 4x1. On the other hand, 10 out of 39 Cyp mRNAs (Cyp 2b9, 2b13, 2b19, 2c37, 2c38, 2c55, 3a44, 4a12, 4a14, and 4f18) were not detectable (2^-ddCt < 0.001). In the neuroblastoma cell lines, C-1300N18 and NB2a, Cyp1b1 mRNA was also the most abundant and preferentially expressed, and relative expression levels to CB1R were 4.674 and 5.084, respectively. Thirteen other Cyp mRNAs (Cyp1a1, 1a2, 2a5, 2b10, 2c44, 2c50, 2c55, 2d10, 2d22, 3a11, 4f13, 4f15, and 4f16) were detected in the neuroblastoma cell lines, whereas 17 Cyp mRNAs (Cyp2c29, 2c37, 2c39, 2c40, 2d12, 2d34, 2e1, 3a16, 3a25, 3a41, 3a44, 4a10, 4a12, 4a14, 4f14, 4x1, and 46a1) were not under the current conditions. The pattern of Cyp mRNA expression was similar for both neuroblastoma cell lines. The present results provide fundamental and useful information on the significance of particular Cyp enzymes in the mouse brain and neuroblastoma cell lines, C-1300N18 and NB2a, which may be valuable tools for investigations on the neural expression and function of Cyp1b1.