Satoshi Yamaori

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes.

Inhibitory effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K(i) value (0.155microM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K(i)=0.0790 and 0.148microM, respectively) compared with CYP1A1 (K(i)=0.541microM). Delta(9)-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K(i)=2.47-7.54microM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Delta(9)-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k(inact)/K(I) value (540l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.

Potent inhibition of human cytochrome P450 3A isoforms by cannabidiol: Role of phenolic hydroxyl groups in the resorcinol moiety

AIMS In this study, we examined the inhibitory effects of Δ(9)-tetrahydrocannabinol (Δ(9)-THC), cannabidiol (CBD), and cannabinol (CBN), the three major cannabinoids, on the activity of human cytochrome P450 (CYP) 3A enzymes. Furthermore, we investigated the kinetics and structural requirement for the inhibitory effect of CBD on the CYP3A activity. MAIN METHODS Diltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined. KEY FINDINGS Among the three major cannabinoids, CBD most potently inhibited CYP3A4 and CYP3A5 (IC(50)=11.7 and 1.65 μM, respectively). The IC(50) values of Δ(9)-THC and CBN for CYP3A4 and CYP3A5 were higher than 35 μM. For CYP3A7, Δ(9)-THC, CBD, and CBN inhibited the activity to a similar extent (IC(50)=23-31 μM). CBD competitively inhibited the activity of CYP3A4, CYP3A5, and HLMs (K(i)=1.00, 0.195, and 6.14 μM, respectively). On the other hand, CBD inhibited the CYP3A7 activity in a mixed manner (K(i)=12.3 μM). Olivetol partially inhibited all the CYP3A isoforms tested, whereas d-limonene showed lack of inhibition. The lesser inhibitory effects of monomethyl and dimethyl ethers of CBD indicated that the ability of CYP3A inhibition by the cannabinoid attenuated with the number of methylation on the phenolic hydroxyl groups in the resorcinol moiety. SIGNIFICANCE This study indicated that CBD most potently inhibited catalytic activity of human CYP3A enzymes, especially CYP3A4 and CYP3A5. These results suggest that two phenolic hydroxyl groups in the resorcinol moiety of CBD may play an important role in the CYP3A inhibition.

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

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.

Ethnic differences between Japanese and Caucasians in the expression levels of mRNAs for CYP3A4, CYP3A5 and CYP3A7 : lack of co-regulation of the expression of CYP3A in Japanese livers

Using a newly developed real-time reverse transcriptase-polymerase chain reaction method, mRNAs were quantitated for CYP3A4, CYP3A5 and CYP3A7 in adult livers from 24 Japanese and 24 Caucasian subjects to elucidate the potential ethnic differences in the expression levels of human cytochrome P450 (CYP) 3As. The expression level of CYP3A4 mRNA in Japanese livers (n = 24) was approximately three times higher than that in Caucasian livers (n = 24, p < 0.001). The mean level of CYP3A5 mRNA was approximately twice higher in Japanese (n = 9) than in Caucasians (n = 5) heterozygous for the CYP3A5 *1 allele (p = 0.057). The CYP3A7 mRNA level was twice higher in Japanese (n = 24) than in Caucasians (n = 22) carrying the CYP3A7 *1A/ *1A genotype (p = 0.042). The level of CYP3A4 mRNA did not correlate with those of CYP3A5 (r = 0.044, n = 24) or CYP3A7 (r = 0.21, n = 24) mRNAs in Japanese livers in contrast to co-regulatory expression of CYP3A4, CYP3A5 and CYP3A7 in Caucasian livers. The results indicate that there are ethnic differences in the expression levels of adult liver CYP3A mRNAs between Japanese and Caucasians, and that the mechanism(s) regulating the hepatic CYP3A expression may be different between these ethnic groups.

Cannabidiol, a major phytocannabinoid, as a potent atypical inhibitor for CYP2D6.

Δ9-Tetrahydrocannabinol, cannabidiol (CBD), and cannabinol are the three major cannabinoids contained in marijuana, which are devoid of nitrogen atoms in their structures. In this study, we investigated the inhibitory effects of the major phytocannabinoids on the catalytic activity of human CYP2D6. These major cannabinoids inhibited the 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) and dextromethorphan O-demethylase activities of recombinant CYP2D6 and pooled human liver microsomes in a concentration-dependent manner (IC50 = 4.01–24.9 μM), indicating the strongest inhibitory potency of CBD. However, these cannabinoids showed no or weak metabolism-dependent inhibition. CBD competitively inhibited the CYP2D6 activities with the apparent Ki values of 1.16 to 2.69 μM. To clarify the structural requirement for CBD-mediated CYP2D6 inhibition, effects of CBD-related compounds on the AMMC O-demethylase activity of recombinant CYP2D6 were examined. Olivetol (IC50 = 7.21 μM) inhibited CYP2D6 activity as potently as CBD did (IC50 = 6.52 μM), whereas d-limonene did not show any inhibitory effect. Pentylbenzene failed to inhibit CYP2D6 activity. Furthermore, neither monomethyl nor dimethyl ethers of CBD inhibited the activity. Cannabidivarin having a propyl side chain inhibited CYP2D6 activity; its inhibitory effect (IC50 = 10.2 μM) was less potent than that of CBD. On the other hand, orcinol and resorcinol showed lack of inhibition. The inhibitory effect of CBD on CYP2D6 activity was more potent than those of 16 compounds without nitrogen atoms tested, such as progesterone. These results indicated that CBD caused potent direct CYP2D6 inhibition, in which two phenolic hydroxyl groups and the pentyl side chain of CBD may play important roles.

Implication of intestinal VDR deficiency in inflammatory bowel disease.

BACKGROUND To investigate the function of the intestinal Vdr gene in inflammatory bowel disease (IBD), in conjunction with the discovery of possible metabolic markers for IBD using intestine-specific Vdr knockout mice. METHODS Vdr(ΔIEpC) mice were generated, phenotyped and treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis. Colitis was diagnosed by evaluating clinical symptoms and intestinal histopathology. Gene expression analysis was carried out. In addition, metabolic markers of IBD were explored by metabolomics. RESULTS Vdr(ΔIEpC) mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. In the acute model which was treated with 3% DSS for six days, Vdr(ΔIEpC) mice showed a high score of IBD symptoms; enlarged mucosal layer and damaged muscularis layer. In the recovery experiment model, where mice were treated with 3% DSS for four days and water for three days, Vdr(ΔIEpC) mice showed a high score of IBD symptoms; severe damage of mucosal layer and increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in Vdr(ΔIEpC) mice. CONCLUSIONS Disruption of the intestinal Vdr gene showed phenotypical changes that may exacerbate IBD. These results suggest that VDR may play an important role in IBD. GENERAL SIGNIFICANCE VDR function has been implicated in IBD. This is of value for understanding the etiology of IBD and for development of diagnostic biomarkers for IBD.

Δ9-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling

We recently reported that Delta(9)-tetrahydrocannabinol (Delta(9)-THC) has the ability to stimulate the proliferation of human breast carcinoma MCF-7 cells. However, the mechanism of action remains to be clarified. The present study focused on the relationship between receptor expression and the effects of Delta(9)-THC on cell proliferation. RT-PCR analysis demonstrated that there was no detectable expression of CB receptors in MCF-7 cells. In accordance with this, no effects of cannabinoid 1/2 (CB1/2) receptor antagonists and pertussis toxin on cell proliferation were observed. Although MCF-7 cell proliferation is suggested to be suppressed by Delta(9)-THC in the presence of CB receptors, it was revealed that Delta(9)-THC could exert upregulation of living cells in the absence of the receptors. Interestingly, Delta(9)-THC upregulated human epithelial growth factor receptor type 2 (HER2) expression, which is known to be a predictive factor of human breast cancer and is able to stimulate cancer cells as well as MCF-7 cells. Actinomycin D-treatment interfered with the upregulation of HER2 and cell proliferation by cannabinoid. Taken together, these studies suggest that, in the absence of CB receptors, Delta(9)-THC can stimulate the proliferation of MCF-7 cells by modulating, at least in part, HER2 transcription.

Conversion of cannabidiol to Δ9-tetrahydrocannabinol and related cannabinoids in artificial gastric juice, and their pharmacological effects in mice

Cannabidiol (CBD), a nonpsychoactive cannabinoid, was found to be converted to 9α-hydroxyhexahydrocannabinol (9α-OH-HHC) and 8-hydroxy-iso-hexahydrocannabinol (8-OH-iso-HHC) together with Δ9-tetrahydrocannabinol (Δ9-THC), a psychoactive cannabinoid, and cannabinol in artificial gastric juice. These cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS) by comparison with the spectral data of the authentic compounds. Pharmacological effects of 9α-OH-HHC and 8-OH-iso-HHC in mice were examined using catalepsy, hypothermia, pentobarbital-induced sleep prolongation, and antinociception against acetic acid-induced writhing as indices. The ED50 values (effective dose producing a 50% reduction of control; mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso-HHC for the cataleptogenic effect were 8.0 and 30.4, respectively. 8-OH-iso-HHC (10 mg/kg, i.v.) produced a significant hypothermia from 15 to 90 min after administration, although 9α-OH-HHC failed to induce such an effect at the same dose. However, both HHCs (10 mg/kg, i.v.) significantly prolonged pentobarbital-induced sleeping time by 1.8 to 8.0 times as compared with the control solution with 1% Tween 80-saline. The ED50 values (mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso-HHC for the antinociceptive effect were 14.1 and 39.4, respectively. The present study demonstrated that CBD can be converted to Δ9-THC and its related cannabinoids, 9α-OH-HHC and 8-OH-iso-HHC, in artificial gastric juice, and that these HHCs show Δ9-THC-like effects in mice, although their pharmacological effects were less potent than those of Δ9-THC.

Δ8-Tetrahydrocannabinol induces cytotoxicity in macrophage J774-1 cells: Involvement of cannabinoid receptor 2 and p38 MAPK

Tetrahydrocannabinol (THC), a psychoactive component of marijuana, is known to exert cytotoxicity in immune cells. In the present study, we examined the cytotoxicity of Δ⁸-THC in mouse macrophage J774-1 cells and a possible involvement of cannabinoid receptors and stress-responsive mitogen-activated protein kinases (MAPKs) in the cytotoxic process. J774-1 cells were treated with Δ⁸-THC (0-20 μM) for up to 6 h. As measured by the MTT and LDH assays, Δ⁸-THC induced cell death of J774-1 cells in a concentration- and/or exposure time-dependent manner. Δ⁸-THC-induced cell damage was associated with vacuole formation, cell swelling, chromatin condensation, and nuclear fragmentation. The cytotoxic effect of Δ⁸-THC was significantly prevented by a caspase-1 inhibitor Ac-YVAD-cmk but not a caspase-3 inhibitor z-DEVD-fmk. The pretreatment with SR144528, a CB₂ receptor-selective antagonist, effectively suppressed Δ⁸-THC-induced cytotoxicity in J774-1 cells, which exclusively expressed CB₂ receptors as indicated by real-time polymerase chain reaction analysis. In contrast, AM251, a CB₁ receptor-selective antagonist, did not affect the cytotoxicity. Pertussis toxin and α-tocopherol significantly attenuated Δ⁸-THC-induced cytotoxicity suggesting that G(i/o) protein coupling signal transduction and oxidative stress are responsible for the cytotoxicity. Δ⁸-THC stimulated the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in J774-1 cells, which were effectively antagonized by the pretreatment with SR144528. In addition, SB203580, a p38 MARK inhibitor, significantly attenuated the cytotoxic effect of Δ⁸-THC, whereas SP600125, a JNK inhibitor, significantly enhanced the cytotoxicity. These results suggest that the cytotoxicity of Δ⁸-THC to J774-1 cells is exerted mediated through the CB₂ receptor followed by the activation of p38 MAPK.

8-Hydroxycannabinol: a new metabolite of cannabinol formed by human hepatic microsomes

Metabolism of cannabinol (CBN) was studied in vitro using hepatic microsomes from human livers. The metabolites formed were analyzed by thinlayer chromatography (TLC) and identified by gas chromatography-mass spectrometry as their trimethylsilyl derivatives. 11-Hydroxy-CBN, the major metabolite, was detected together with a smaller amount of another mono-hydroxylated metabolite. The minor metabolite was identified as 8-hydroxy-CBN, after comparing its Rf value by TLC, retention time by GC, and the mass spectrum with those of the authentic compound. 8-Hydroxy-CBN was confirmed to be a new metabolite of CBN formed by human hepatic microsomes.