Predicting the Potential for Cannabinoids to Precipitate Pharmacokinetic Drug Interactions via Reversible Inhibition or Inactivation of Major Cytochromes P450

Abstract

Cannabis is used for both recreational and medicinal purposes. The most abundant constituents are the cannabinoids - cannabidiol (CBD, nonpsychoactive) and (-)-trans-Δ⁹-tetrahydrocannabinol (THC, psychoactive). Both have been reported to reversibly inhibit or inactivate cytochrome P450 (CYPs) enzymes. However, the low aqueous solubility, microsomal protein binding, and nonspecific binding to labware were not considered, potentially leading to an underestimation of CYPs inhibition potency. Therefore, the binding-corrected reversible (IC_50,u) and irreversible (K _I,u ) inhibition potency of each cannabinoid toward major CYPs were determined. The fraction unbound of CBD and THC in the incubation mixture was 0.12 ± 0.04 and 0.05 ± 0.02, respectively. The IC_50,u for CBD toward CYP1A2, 2C9, 2C19, 2D6, and 3A was 0.45 ± 0.17, 0.17 ± 0.03, 0.30 ± 0.06, 0.95 ± 0.50, and 0.38 ± 0.11 µM, respectively; the IC_50,u for THC was 0.06 ± 0.02, 0.012 ± 0.001, 0.57 ± 0.22, 1.28 ± 0.25, and 1.30 ± 0.34 µM, respectively. Only CBD showed time-dependent inactivation (TDI) of CYP1A2, 2C19, and CYP3A, with inactivation efficiencies (k _inact/K _I,u) of 0.70 ± 0.34, 0.11 ± 0.06, and 0.14 ± 0.04 minutes^-1 µM^-1, respectively. A combined (reversible inhibition and TDI) mechanistic static model populated with these data predicted a moderate to strong pharmacokinetic interaction risk between orally administered CBD and drugs extensively metabolized by CYP1A2/2C9/2C19/2D6/3A and between orally administered THC and drugs extensively metabolized by CYP1A2/2C9/3A. These predictions will be extended to a dynamic model using physiologically based pharmacokinetic modeling and simulation and verified with a well-designed clinical cannabinoid-drug interaction study. SIGNIFICANCE STATEMENT: This study is the first to consider the impact of limited aqueous solubility, nonspecific binding to labware, or extensive binding to incubation protein shown by cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) on their true cytochrome P450 inhibitory potency. A combined mechanistic static model predicted a moderate to strong pharmacokinetic interaction risk between orally administered CBD and drugs extensively metabolized by CYP1A2, 2C9, 2C19, 2D6, or 3A and between orally administered THC and drugs extensively metabolized by CYP1A2, 2C9, or 3A.

Fig. 1.

Chemical structures of CBD, THC, 11-OH-THC, and COOH-THC.

Fig. 2.

Concentration-dependent reversible inhibition of CYP activity in HLMs by CBD. The order of CBD inhibition potency was CYP2C9 > 2C19 ≈ 3A ≈ 1A2 > 2D6 (Table 2). Pooled HLMs (0.1 mg/ml protein) were incubated (37°C, 15 minutes) with a CYP cocktail consisting of phenacetin (CYP1A2; 50 μM), diclofenac (CYP2C9; 5 μM), omeprazole (CYP2C19; 10 μM), dextromethorphan (CYP2D6; 5 μM), and testosterone (CYP3A; 10 μM) and varying concentrations of CBD (0.003–100 µM) or vehicle (0.2% v/v DMSO). Data represent mean ± S.D. of three independent experiments, each conducted in duplicate. Solid lines represent model fit to the data.

Fig. 3.

Concentration-dependent reversible inhibition of CYP activity in HLMs by THC. The order of THC inhibition potency was CYP2C9 > 1A2 > 2C19 > 2D6 ≈ 3A (Table 2). THC was a more potent inhibitor of CYP2C9 and 1A2 than CBD, but it was a less potent inhibitor of CYP3A (Table 2). Pooled HLMs (0.1 mg/ml protein) were incubated (37°C, 10 minutes) with a CYP cocktail consisting of phenacetin (CYP1A2; 50 μM), diclofenac (CYP2C9; 5 μM), omeprazole (CYP2C19; 10 μM), dextromethorphan (CYP2D6; 5 μM), and testosterone (CYP3A; 10 μM) and varying concentrations of THC (0.003–100 µM) or vehicle (0.2% v/v DMSO). Data represent mean ± S.D. of three independent experiments, each conducted in duplicate. Solid lines represent model fit to the data.

Fig. 4.

TDI of CYPs by CBD, THC, 11-OH-THC, and COOH-THC. When the cannabinoids were preincubated with HLMs only, CBD showed TDI of CYP1A2, 2C19, and 3A. Pooled HLMs (0.5 mg/ml protein) were preincubated with NADPH regenerating system, cannabinoid (THC, CBD, 11-OH-THC or COOH-THC; 10 μM), or vehicle (0.2% v/v DMSO) at 37°C for 0, 10, 20, or 30 minutes. Then, an aliquot (10 μl) of this mixture was incubated with NADPH regenerating system and a CYP substrate cocktail consisting of phenacetin (CYP1A2; 50 μM), diclofenac (CYP2C9; 5 μM), omeprazole (CYP2C19; 20 μM), dextromethorphan (CYP2D6; 5 μM), and testosterone (CYP3A; 20 μM) for 15 minutes. Data represent percent of activity in the vehicle-treated control group (0 minutes) that was not subjected to preincubation and are shown as means ± S.D. for three independent experiments. *P < 0.05, significantly different from the vehicle-treated control group (two-way analysis of variance test).

Fig. 5.

TDI of CYP1A2, 2C19, and 3A by various concentrations of CBD. The order of CBD inactivation potency was CYP1A2 > 2C19 ≈ CYP3A (Table 3). (A) Pooled HLMs (0.5 mg/ml protein) were preincubated with NADPH regenerating system, CBD (0.5, 1, 2.5, 5, 10, 20, 40, or 60 μM), or vehicle (0.2% v/v DMSO) at 37°C for 0, 4, 8, 12, or 16 minutes. Then, an aliquot (10 μl) of this mixture was incubated with NADPH regenerating system and a CYP substrate cocktail consisting of phenacetin (CYP1A2; 50 μM), diclofenac (CYP2C9; 5 μM), omeprazole (CYP2C19; 20 μM), dextromethorphan (CYP2D6; 5 μM), and testosterone (CYP3A; 20 μM) for 15 minutes. Data represent percent of activity in the vehicle-treated control group (0 minutes) that was not subjected to preincubation and are shown as means ± S.D. for four independent experiments. (B) Nonlinear regression model fits of the k_obs data to estimate k_inact and K_I.