Inhibition of Tacrolimus Metabolism by Cannabidiol and Its Metabolites In Vitro

Abstract

Drug interactions are major causes of interindividual variability in tacrolimus exposure and effect. Tacrolimus, a widely used drug in transplant patients, is metabolized by CYP3A4 and CYP3A5. Cannabidiol (CBD) use after transplant is common. Clinical cases suggest CBD may alter tacrolimus exposure, but the mechanism of this interaction is unknown. We hypothesize that cannabidiol will inhibit tacrolimus metabolism in vitro mainly through CYP3A5 inhibition. In pooled human liver microsomes (HLMs) and recombinant (r) CYP3A4 and CYP3A5 enzymes, tacrolimus (1 μM) metabolism was determined using substrate depletion method in the absence (control) and the presence of 10 μM CBD, 7-hydroxyCBD, and 7-carboxyCBD. Ketoconazole (1 μM) served as a positive control for the inhibition of CYP3A. Linear regression analyses were performed to obtain kinetic parameters of the depletion. Tacrolimus depletion half-life was 2.54, 0.922, and 0.351 min with pooled HLMs, rCYP3A4, and rCYP3A5, respectively. In pooled HLMs, CBD and 7-hydroxyCBD increased tacrolimus half-life by 0.8- and 2.3-fold (both p < 0.0001), respectively. In rCYP3A4, CBD, 7-hydroxyCBD, and ketoconazole prolonged tacrolimus half-life by 5.8-, 14-, and 7.7-fold, respectively. In rCYP3A5, CBD, 7-hydroxyCBD, and ketoconazole prolonged half-life by 29.3-, 19.7-, and 0.1-fold, respectively. In all experiments, 7-carboxyCBD had minimal effect on tacrolimus depletion. CBD and 7-hydroxyCBD inhibited tacrolimus metabolism in vitro. CBD showed stronger inhibition in rCYP3A5 than rCYP3A4. The demonstrated CYP3A5 selectivity of cannabidiol may contribute to the in vitro identification of CYP3A5 substrates in new drug development. Our results support the potential of a clinical drug-drug interaction between CBD and tacrolimus.

Keywords: CYP; cannabinoid; drug–drug interactions; pharmacokinetics.

FIGURE 1

Proposed inhibition of tacrolimus metabolism by cannabidiol. CBD is metabolized to 7‐OH CBD via CYP2C9 and CYP2C19, which is further metabolized to 7‐COOH CBD via both cytosolic and microsomal P450 enzymes [28, 29]. A potential 7‐CHO CBD metabolite may be appreciated between 7‐OH CBD and 7‐COOH CBD appearance. Metabolism of tacrolimus is predominantly mediated by CYP3A4 and CYP3A5 [30]. This study aims to explore the inhibition of tacrolimus metabolism by CBD and its metabolites. ALDH, aldehyde dehydrogenase; AOX, aldehyde oxidase; CBD, cannabidiol; 7‐CHO CBD, 7‐aldehydecannabidiol; 7‐COOH CBD, 7‐carboxycannabidiol; 7‐OH CBD, 7‐hydroxycannabidiol; 12‐HT, 12‐hydroxy tacrolimus; 13‐DMT, 13‐O‐desmethyl tacrolimus; 15‐DMT, 15‐O‐desmethyl tacrolimus; 31‐DMT, 31‐O‐desmethyl tacrolimus.

FIGURE 2

UHPLC chromatograms. This is an ion current versus time plot obtained from chromatography. Peaks were scaled relative to the peak with highest intensity.

FIGURE 3

Inhibition of tacrolimus depletion by CBD, 7‐OH CBD, 7‐COOH CBD, and ketoconazole with pooled HLMs. Tacrolimus (1 μM) was incubated alone and with CBD (10 μM), 7‐OH CBD (10 μM), 7‐COOH CBD (10 μM), or ketoconazole (1 μM) in the presence of 50‐donor pooled HLMs (0.5 mg/mL) and NADPH (1 mM) in phosphate buffer (0.2 M; pH 7.4) for 0, 5, 10, 15, 20, and 30 min. Tacrolimus depletion was quantified using the peak area ratio of tacrolimus to rapamycin, internal standard, and a standard curve (0 to 10 μM). The lower limit of quantification was 0.124 nM. The percentage of tacrolimus remaining was calculated and natural log‐transformed (A). Subsequently, regression analysis was performed on data in the linear range (B). The negative slope is the depletion rate constant from which half‐life was calculated. Points represent the mean ± SD (A) or mean ± SEM (B) of four technical replicates. CBD, cannabidiol; HLM, human liver microsome; 7‐COOH CBD, 7‐carboxycannabidiol; 7‐OH CBD, 7‐hydroxycannabidiol. *adjusted p < 0.05; ****adjusted p < 0.0001.

FIGURE 4

Inhibition of tacrolimus depletion by CBD, 7‐OH CBD, 7‐COOH CBD, and ketoconazole in recombinant CYP3A4 and CYP3A5 enzyme systems. Tacrolimus (1 μM) was incubated alone and with CBD (10 μM), 7‐OH CBD (10 μM), 7‐COOH CBD (10 μM), or ketoconazole (1 μM) in the presence of rCYP3A4 or rCYP3A5 (10 pmol/150 μL) and NADPH (1 mM) in phosphate buffer (0.2 M; pH 7.4) for 0, 1, 2.5, 5, 7.5, and 10 min. Tacrolimus depletion was quantified using the peak area ratio of tacrolimus to rapamycin, internal standard, and a standard curve (0–3 μM). The lower limit of quantification was 0.124 nM. For the incubation with rCYP3A4, the natural log‐transformed percentage tacrolimus remaining was plotted (A), and linear regression analysis was performed (B). For the incubation with rCYP3A5, the natural log‐transformed percentage tacrolimus remaining was plotted (C), and linear regression analysis was performed (D). Points represent the mean ± SD (A, C) or mean ± SEM (B, D) of two technical replicates. CBD, cannabidiol; rCYP, recombinant cytochrome P450; 7‐COOH CBD, 7‐carboxycannabidiol; 7‐OH CBD, 7‐hydroxycannabidiol. *adjusted p < 0.05; ****adjusted p < 0.0001.