Natural Products Inhibition of Cytochrome P450 2B6 Activity and Methadone Metabolism

Abstract

Methadone is cleared predominately by hepatic cytochrome P450 (CYP) 2B6-catalyzed metabolism to inactive metabolites. CYP2B6 also catalyzes the metabolism of several other drugs. Methadone and CYP2B6 are susceptible to pharmacokinetic drug-drug interactions. Use of natural products such as herbals and other botanicals is substantial and growing, and concomitant use of prescription medicines and non-prescription herbals is common and may result in interactions, often precipitated by CYP inhibition. Little is known about herbal product effects on CYP2B6 activity, and CYP2B6-catalyzed methadone metabolism. We screened a family of natural product compounds used in traditional medicines, herbal teas, and synthetic analogs of compounds found in plants, including kavalactones, flavokavains, chalcones and gambogic acid, for inhibition of expressed CYP2B6 activity and specifically inhibition of CYP2B6-mediated methadone metabolism. An initial screen evaluated inhibition of CYP2B6-catalyzed 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation. Hits were further evaluated for inhibition of racemic methadone metabolism, including mechanism of inhibition and kinetic constants. In order of decreasing potency, the most effective inhibitors of methadone metabolism were dihydromethysticin (competitive, K _i 0.074 µM), gambogic acid (noncompetitive, K _i 6 µM), and 2,2'-dihydroxychalcone (noncompetitive, K _i 16 µM). Molecular modeling of CYP2B6-methadone and inhibitor binding showed substrate and inhibitor binding position and orientation and their interactions with CYP2B6 residues. These results show that CYP2B6 and CYP2B6-catalyzed methadone metabolism are inhibited by certain natural products, at concentrations which may be clinically relevant. SIGNIFICANCE STATEMENT: This investigation identified several natural product constituents which inhibit in vitro human recombinant CYP2B6 and CYP2B6-catalyzed N-demethylation of the opioid methadone. The most potent inhibitors (K _i) were dihydromethysticin (0.074 µM), gambogic acid (6 µM) and 2,2'-dihydroxychalcone (16 µM). Molecular modeling of ligand interactions with CYP2B6 found that dihydromethysticin and 2,2'-dihydroxychalcone bound at the active site, while gambogic acid interacted with an allosteric site on the CYP2B6 surface. Natural product constituents may inhibit CYP2B6 and methadone metabolism at clinically relevant concentrations.

Graphical abstract

Fig. 1.

Structures of kavalactones (1–6), gambogic acid (7), euphorbiasteroid (8), daphnoretin (9), and cardiotonic steroids (10–13).

Fig. 2.

Concentration-dependent inhibition of 7-ETFMC O-deethylation by twelve compounds that passed the initial ETFMC assay screening. Substrate concentration was fixed (50 µM 7-ETFMC). Results are the mean ± S.D. of triplicate determinations. Solid lines represent predicted concentrations based on parameters obtained by nonlinear regression analysis of measured concentrations using the four-parameter logistic nonlinear regression model as described in the Methods section.

Fig. 3.

Inhibition of racemic methadone demethylation by seven compounds identified as inhibitory in the coumarin assay screen and yangonin (3). (A) dihydromethysticin, methysticin; (B) gambogic acid, dihydrokavain; (C) 2,2'-dihydroxychalcone, desmethoxyyangonin; (D) yangonin. Substrate concentration was fixed at 2 µM racemic methadone. Results are the mean ± S.D. of triplicate determinations. Solid lines represent predicted concentrations based on parameters obtained by nonlinear regression analysis of measured concentrations using the four-parameter logistic nonlinear regression model as described in the Methods section.

Fig. 4.

Inhibition of racemic methadone demethylation by (A) dihydromethysticin, (B) gambogic acid, (C) methysticin, (D) dihydrokavain, (E) desmethoxyyangonin, and (F) 2,2’-dihydroxychalcone. Results are the mean ± S.D. of triplicate determinations. Lines are predicted concentrations based on kinetic parameters obtained by nonlinear regression analysis of measured concentrations using the models of competitive inhibition (dihydromethysticin, dihydrokavain), non-competitive inhibition (gambogic acid, desmethoxyyangonin), and non-competitive inhibition with substrate inhibition (methysticin, 2,2’-dihydroxychalcone).

Fig. 5.

Binding poses of (A) R-methadone and (B) S-methadone at the substrate binding pocket of CYP2B6. The backbone of methadone is orange. The heme is purple. The CYP residues interacting with methadone are dark cyan.

Fig. 6.

Binding poses of clopidogrel at the substrate binding pocket of CYP2B6 in two orientations: (A) ‘thiophene down’ orientation and (B) ‘chlorophenyl down’ orientation. The backbone of clopidogrel is orange. The heme is purple. The CYP2B6 residues interacting with clopidogrel are dark cyan.

Fig. 7.

Binding poses of (A) dihydromethysticin and (B) 2,2’-dihydroxychalcone at the CYP2B6 substrate binding pocket. The backbone of the inhibitor ligand is orange. The heme is purple. The CYP2B6 residues interacting with the inhibitors are dark cyan.

Fig. 8.

Docking of gambogic acid into an alternative binding site of CYP2B6 in the groove between helix C and helix H. (A) Details of the interactions with surrounding residues (The backbone of gambogic acid is shown in blue). (B) Gambogic acid (backbone in cyan) shown as ball and stick on protein surface. (C) Gambogic acid (cyan) shown as spheres on the protein surface.