We have recently reported the potent direct inhibition of human CYP1A1 activity by CBD.8) The competitive inhibition of CYP1A1 by CBD suggested that the phytocannabinoid exerts the inhibitory effect at the active site of CYP1A1.8) This is not surprising because human CYP1A1 is capable of metabolizing CBD.29) On the other hand, it has been shown that CBD is also a mechanism-based inhibitor of CYP1A1.8) However, it is suggested that the contribution of CBD as a mechanism-based inhibitor may be of small importance to CYP1A1 inhibition observed in the present study coincubated CBD with a substrate because the preincubation of a mechanism-based inhibitor with an alternative substrate in the presence of NADPH attenuates inactivation.30,31) In this study, we examined the inhibitory effects of nine CBD-related compounds on CYP1A1 activity to clarify the structural requirements for potent direct CYP1A1 inhibition by CBD under the condition of a short incubation period to minimize the contribution of CBD-related compounds to metabolism-dependent inhibition.
The partial inhibition of CYP1A1 by olivetol but not d-limonene suggests that the pentylresorcinol structure in CBD is essential for inhibition although the whole structure of CBD is required for the overall inhibition of CYP1A1 activity. The structure of the resorcinol moiety in CBD contains two free phenolic hydroxyl groups and a phenyl ring, which are likely to interact with amino acids within the active site of CYP1A1. Inhibition studies with CBDM and CBDD indicate that both free phenolic hydroxyl groups in CBD are required for potent CYP1A1 inhibition. Furthermore, our molecular modeling supports the importance of two free phenolic hydroxyl groups in CYP1A1 inhibition. We have recently reported that CYP2B6, CYP2D6, CYP3A4, and CYP3A5 are more potently inhibited by CBD than by Δ9-THC and cannabinol.9–11) The structural requirements for the inhibitory effects of CBD on these CYP enzymes indicate that both free phenolic hydroxyl groups in the phytocannabinoid contribute to these inhibitions. A similar observation was seen in the inhibitory effects of CBD on CYP-mediated drug oxidations in mouse liver microsomes.22) Thus, these findings suggest that these two free phenolic hydroxyl groups of CBD may be key functional groups common to the inhibition of many CYP enzymes by CBD.
It should be noted that CBDD preserves some degree of the ability to inhibit CYP1A1 activity even though both phenolic hydroxyl groups are methylated. This result reveals that the mechanism underlying CBD-mediated CYP1A1 inhibition cannot be explained only by the presence of two free phenolic hydroxyl groups of the phytocannabinoid. The molecular modeling of CYP1A1 with CBD suggested a possible stacking interaction between the phenyl ring of the resorcinol moiety in CBD and Phe-224 in CYP1A1. The importance of the Phe residue in ligand binding has been shown by previous studies. Lewis et al.32) reported that Ala substitution for Phe-224 in CYP1A1 decreases the catalytic efficiency (Vmax/Km) of EROD activity to approximately one third that of wild-type CYP1A1. For CYP1A2, it has been reported that all mutant enzymes to substitute Ile, Thr, or Tyr residues for Phe-226, which corresponds to Phe-224 in CYP1A1, show considerably reduced EROD activity.33) A crystal structure of CYP1A2 bound to α-naphthoflavone, a representative CYP1 inhibitor, shows that Phe-226 as well as Phe-125 forms a stacking interaction with this inhibitor.34) Phe-224 in CYP1A1 is highly conserved among the CYP1 family.35) Thus, it is conceivable that this residue may interact with the phenyl ring of the resorcinol moiety in CBD. In addition, CBD was accommodated in a torsional conformation of the cyclohexenyl ring of the terpene moiety relative to the phenyl ring of the resorcinol moiety within the active site of CYP1A1 (Fig. 7). This finding is compatible with the results of inhibition studies with Δ9-THC, CBE, and CBDM suggesting that the torsional structure of CBD may be preferentially recognized by CYP1A1. It is surmised that such a torsional conformation of CBD is stabilized by two hydrogen bonds in the CYP1A1 active site, as well as the stacking interaction between the phenyl rings of CBD and Phe-224 in CYP1A1.
There are several studies on the inhibition of human CYP1A1 by naturally occurring compounds containing the resorcinol moiety in their structures.30,36–38) Interestingly, the inhibition of human CYP1A1 by oxyresveratrol having two resorcinol moieties (i.e., 2,4,3′,5′-tetrahydroxystilbene) has been shown to be markedly potentiated by the methylation of all four phenolic hydroxyl groups in oxyresveratrol.36,37) This is in contrast to the result obtained in this study. The presence of free phenolic hydroxyl groups in oxyresveratrol is unlikely to be important in the strong inhibition of human CYP1A1. The role of free phenolic hydroxyl groups for CYP1A1 inhibition may be specific for CBD.
In addition, we focused on a role of the pentyl side chain of CBD in CYP1A1 inhibition because it has been shown that human CYP1A1 is capable of oxidizing the carbon atom at the 1″-position of the pentyl side chain in CBD.29) The side-chain shortening of CBD and olivetol attenuated their inhibitory potencies against CYP1A1 activity. Interestingly, orcinol preserves some degree of the ability to inhibit CYP1A1 activity, although the inhibition by orcinol has not been observed with the other CYP enzymes examined.11,13) The lack of the side chain in the pentylresorcinol structure caused a loss in CYP1A1 inhibition. When the carbon atom at the 1″-position of the pentyl group in CBD comes close to the heme iron, the distal portion of the side chain in CBD may interact with the proposed hydrophobic region within the active site of CYP1A1.32) These results suggest that the pentyl side chain of CBD also plays an important role in potent CYP1A1 inhibition.
CYP1A1 is known to be responsible for the metabolism of several clinically used drugs.20,21) It has been previously reported that granisetron 7-hydroxylation, a major metabolic pathway, is almost exclusively catalyzed by CYP1A1 in human liver microsomes.20) The potent inhibition of CYP1A1 by CBD might influence the pharmacokinetics of granisetron. At present, however, the roles of CYP1A1 and CBD-mediated inhibition in drug clearance are limited because of a limited number of drugs metabolized by CYP1A1 and very low constitutive expression of CYP1A1 in the liver. On the other hand, CYP1A1 is suggested to induce genotoxicity and caricinogenicity by the bioactivation of procarcinogens, such as benzo[a]pyrene and other polycyclic aromatic hydrocarbons.39) Therefore, CBD and its structurally related compounds which potently inhibit CYP1A1 activity would be expected as a lead compound in anticancer chemotherapy.
In conclusion, our inhibition study indicated that two free phenolic hydroxyl groups and the pentyl side chain in the pentylresorcinol moiety of CBD play important roles in direct CYP1A1 inhibition although the whole structure of CBD is essential for potent inhibition. Our molecular modeling supported some results of the inhibition study, i.e., the importance of these two free phenolic hydroxyl groups in CYP1A1 inhibition and the torsional conformation of CBD within the CYP1A1 active site. Furthermore, this modeling speculated a possible stacking interaction between the phenyl ring of CBD and Phe-224 in CYP1A1. This study will provide useful information to understand the precise mechanisms underlying the potent inhibition of human CYP1A1 by CBD.