Kazuhito Watanabe

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

AIMSnIn 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.nnnMAIN METHODSnDiltiazem N-demethylase activity of recombinant CYP3A4, CYP3A5, CYP3A7, and human liver microsomes (HLMs) in the presence of cannabinoids was determined.nnnKEY FINDINGSnAmong 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.nnnSIGNIFICANCEnThis 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.

Cannabidiolic Acid as a Selective Cyclooxygenase-2 Inhibitory Component in Cannabis

In the present study it was revealed that cannabidiolic acid (CBDA) selectively inhibited cyclooxygenase (COX)-2 activity with an IC50 value (50% inhibition concentration) around 2 μM, having 9-fold higher selectivity than COX-1 inhibition. In contrast, Δ9-tetrahydrocannabinolic acid (Δ9-THCA) was a much less potent inhibitor of COX-2 (IC50 > 100 μM). Nonsteroidal anti-inflammatory drugs containing a carboxyl group in their chemical structures such as salicylic acid are known to inhibit nonselectively both COX-1 and COX-2. CBDA and Δ9-THCA have a salicylic acid moiety in their structures. Thus, the structural requirements for the CBDA-mediated COX-2 inhibition were next studied. There is a structural difference between CBDA and Δ9-THCA; phenolic hydroxyl groups of CBDA are freed from the ring formation with the terpene moiety, although Δ9-THCA has dibenzopyran ring structure. It was assumed that the whole structure of CBDA is important for COX-2 selective inhibition because β-resorcylic acid itself did not inhibit COX-2 activity. Methylation of the carboxylic acid moiety of CBDA led to disappearance of COX-2 selectivity. Thus, it was suggested that the carboxylic acid moiety in CBDA is a key determinant for the inhibition. Furthermore, the crude extract of cannabis containing mainly CBDA was shown to have a selective inhibitory effect on COX-2. Taken together, these lines of evidence in this study suggest that naturally occurring CBDA in cannabis is a selective inhibitor for COX-2.

Cannabidiol-2′,6′-Dimethyl Ether, a Cannabidiol Derivative, Is a Highly Potent and Selective 15-Lipoxygenase Inhibitor

The inhibitory effect of nordihydroguaiaretic acid (NDGA) (a nonselective lipoxygenase (LOX) inhibitor)-mediated 15-LOX inhibition has been reported to be affected by modification of its catechol ring, such as methylation of the hydroxyl group. Cannabidiol (CBD), one of the major components of marijuana, is known to inhibit LOX activity. Based on the phenomenon observed in NDGA, we investigated whether or not methylation of CBD affects its inhibitory potential against 15-LOX, because CBD contains a resorcinol ring, which is an isomer of catechol. Although CBD inhibited 15-LOX activity with an IC50 value (50% inhibition concentration) of 2.56 μM, its monomethylated and dimethylated derivatives, CBD-2′-monomethyl ether and CBD-2′,6′-dimethyl ether (CBDD), inhibited 15-LOX activity more strongly than CBD. The number of methyl groups in the resorcinol moiety of CBD (as a prototype) appears to be a key determinant for potency and selectivity in inhibition of 15-LOX. The IC50 value of 15-LOX inhibition by CBDD is 0.28 μM, and the inhibition selectivity for 15-LOX (i.e., the 5-LOX/15-LOX ratio of IC50 values) is more than 700. Among LOX isoforms, 15-LOX is known to be able to oxygenate cholesterol esters in the low-density lipoprotein (LDL) particle (i.e., the formation of oxidized LDL). Thus, 15-LOX is suggested to be involved in development of atherosclerosis, and CBDD may be a useful prototype for producing medicines for atherosclerosis.

Modulation of Δ9-tetrahydrocannabinol-induced MCF-7 breast cancer cell growth by cyclooxygenase and aromatase

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), a major constituent of marijuana, has been shown to stimulate the growth of MCF-7 breast cancer cells through cannabinoid receptor-independent signaling [Takeda, S., Yamaori, S., Motoya, E., Matsunaga, T., Kimura, T., Yamamoto, I., Watanabe, K., 2008. Delta(9)-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling. Toxicology 245, 141-146]. Although the growth of MCF-7 cells is known to be stimulated by 17beta-estradiol (E(2)), the interaction of Delta(9)-THC and E(2) in MCF-7 cell growth is not fully clarified so far. In the present study, by using E(2)-sensitive MCF-7 cells that have expressed cyclooxygenase-2 (COX-2) and cytochrome P450 19 (aromatase), we studied whether or not COX-2 and aromatase are involved in Delta(9)-THC-mediated MCF-7 cell proliferation. It was shown that Delta(9)-THC-induced MCF-7 cell growth was inhibited by COX-2 inhibitors and was stimulated by arachidonic acid (a COX substrate). However, the growth of MCF-7 cells induced by Delta(9)-THC was not stimulated by PGE(2), and the expression of aromatase was not affected by COX-2 inhibitors, arachidonic acid, and PGE(2), suggesting that there is a disconnection between COX-2 (PGE(2)) and aromatase in Delta(9)-THC-mediated MCF-7 cell proliferation. On the other hand, Delta(9)-THC-induced MCF-7 cell growth was elevated by two kinds of aromatase inhibitors. Taken together with the evidence that Delta(9)-THC-induced MCF-7 cell proliferation was interfered with testosterone (an aromatase substrate) and exogenously provided E(2), it is suggested that (1) the growth stimulatory effects of Delta(9)-THC are mediated by the product(s) of COX-2 except for PGE(2), (2) the action of Delta(9)-THC is modulated by E(2), and (3) COX-2 and aromatase are individually engaged in the proliferation of MCF-7 cells induced by Delta(9)-THC.

Δ9-tetrahydrocannabinol and its major metabolite Δ9-tetrahydrocannabinol-11-oic acid as 15-lipoxygenase inhibitors.

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein, a major causal factor for atherosclerosis. Δ(9)-Tetrahydrocannabinol (Δ(9)-THC), a major component of marijuana, has suggested to suppress atherosclerosis. Although Δ(9)-THC seems to be attractive for the prevention of atherosclerosis, there is no information about whether or not 15-LOX isoform can be inhibited by Δ(9)-THC. In the present study, Δ(9)-THC was found to be a direct inhibitor for 15-LOX with an IC(50) (50% inhibition concentration) value of 2.42 μM. Furthermore, Δ(9)-THC-11-oic acid, a major and nonpsychoactive metabolite of Δ(9) -THC, but not another Δ(9)-THC metabolite 11-OH-Δ(9)-THC (psychoactive), was revealed to inhibit 15-LOX. Taken together, it is suggested that Δ(9) -THC can abrogate atherosclerosis via direct inhibition of 15-LOX, and that Δ(9)-THC-11-oic acid is shown to be an active metabolite of Δ(9) -THC in this case.

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.

Cannabidiol induces expression of human cytochrome P450 1A1 that is possibly mediated through aryl hydrocarbon receptor signaling in HepG2 cells.

AIMSnWe herein investigated the inducibility of cytochrome P450 1A1 (CYP1A1) by Δ(9)-tetrahydrocannabinol, cannabidiol (CBD), and cannabinol, three major phytocannabinoids, using human hepatoma HepG2 cells.nnnMAIN METHODSnThe expression of CYP1A1 and the aryl hydrocarbon receptor (AhR) was measured by a quantitative real-time polymerase chain reaction and/or Western blotting.nnnKEY FINDINGSnΔ(9)-Tetrahydrocannabinol and CBD concentration-dependently induced the expression of CYP1A1 mRNA, whereas cannabinol showed little or no induction. Among the phytocannabinoids tested, CBD was the most potent inducer of CYP1A1 expression. The induction of CYP1A1 expression by CBD was significantly attenuated by the knockdown of AhR expression with AhR small interfering RNAs. The role of protein tyrosine kinases (PTKs) in the CBD-mediated induction of CYP1A1 was then examined using herbimycin A, a PTK inhibitor. The upregulation of CYP1A1 by CBD was significantly suppressed by herbimycin A as was the induction by omeprazole but not 3-methylcholanthrene. The inducibility of CYP1A1 by CBD-related compounds was examined to clarify the structural requirements for CBD-mediated CYP1A1 induction. Olivetol, which corresponds to the pentylresorcinol moiety of CBD, significantly induced the expression of CYP1A1, whereas d-limonene, CBD-2-monomethyl ether, and CBD-2,6-dimethyl ether did not.nnnSIGNIFICANCEnThese results showed that CBD may have induced human CYP1A1 expression through the activation of PTK-dependent AhR signaling, in which two phenolic hydroxyl groups in the pentylresorcinol moiety of CBD may play structurally important roles.

Characterization of the structural determinants required for potent mechanism-based inhibition of human cytochrome P450 1A1 by cannabidiol.

We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2-monomethyl ether (CBDM) and CBD-2,6-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact=0.215min(-1)), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353min(-1), respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1.

Human brain microsomes: their abilities to metabolize tetrahydrocannabinols and cannabinol

In spite of the psychedelic action of Δ9-tetrahydrocannabinol (Δ9-THC) in the brain, no report on its metabolism by human brain microsomes has been published. In this study, the metabolism of Δ8-THC, Δ9-THC and cannabinol (CBN) was studied using human brain microsomes. The metabolites formed were analyzed by gas chromatography–mass spectrometry after trimethylsilylation. The three cannabinoids were biotransformed to two main metabolites by human brain microsomes. Δ8- and Δ9-THCs were mainly oxidized at the allylic positions. The main metabolites of Δ8-THC were 7α-hydroxy- and 11-hydroxy-Δ8-THCs, whereas those of Δ9-THC were 8α-hydroxy- and 11-hydroxy-Δ9-THCs. CBN was metabolized to 8-hydroxy- and 11-hydroxy-CBNs. Although the primary metabolic pathways of the THCs and CBN in brain microsomes are different from those in liver microsomes for other mammalian species, those in human brain microsomes were similar to those in human liver microsomes.