Biologic Materials, Chemicals, and Reagents

Pooled human liver microsomes (HLMs) (mixed gender; pool of 200 donors) and human plasma from a single healthy male donor were purchased from SEKISUI Xenotech, LLC (Kansas City, KS) and Bloodworks (Seattle, WA), respectively. THC, CBD, omeprazole, 5-hydroxy omeprazole, testosterone, 6β-hydroxy testosterone, dextromethorphan, dextrorphan, phenacetin, acetaminophen, diclofenac, and 4’-hydroxy diclofenac were purchased from Cayman Chemical (Ann Arbor, MI). 7-COOH CBD and 7-OH CBD were procured from BDG Synthesis (Lower Hutt, New Zealand). Methanolic solutions of 11-COOH THC, 11-OH THC, deuterated THC (THC-d3), and deuterated CBD (CBD-d3) were purchased from Cerilliant (Round Rock, TX). Acetonitrile, bovine serum albumin (BSA), and formic acid were bought from Fisher Scientific (Hampton, NH). Coumarin, 7-hydroxycoumarin, bupropion, 4-hydroxy bupropion, amodiaquine, N-desethylamodiaquine, tolbutamide, D-glucose 6-phosphate, β-nicotinamide adenine dinucleotide phosphate (NADP+), and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s phosphate-buffered saline was procured from Thermo Scientific (Asheville, NC). Micro ultracentrifuge polycarbonate tubes were purchased from Beckman Coulter (Brea, CA), and ultralow-binding microcentrifuge tubes were purchased from Genesee Scientific (EI Cajon, CA). All other chemicals and experimental reagents were obtained from reputable commercial sources.

Reversible Inhibition of P450s by Cannabinoids

As per our previous study (Bansal et al., 2020), low-binding microcentrifuge tubes were used to reduce the nonspecific binding and adsorption of cannabinoids to labware, and we included BSA (0.2%) in all incubations to increase cannabinoid solubility and reduce nonspecific binding.

The inhibition of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A by each cannabinoid was simultaneously evaluated using a previously validated P450 cocktail assay (Dinger et al., 2014). A metabolite of each probe substrate selectively formed by a single P450 was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (described below). The probe substrate/metabolite pair and probe substrate concentration were as follows: phenacetin (K_m = 1.7–152 μM)/acetaminophen and 50 μM (CYP1A2); coumarin (K_m = 0.3–2.3 μM)/7-hydroxycoumarin and 2 μM (CYP2A6); bupropion (K_m = 67–168 μM)/4-hydroxy-bupropion and 5 μM (CYP2B6); amodiaquine (K_m = 1.9–2.4 μM)/N-desethylamodiaquine and 2 μM (CYP2C8); diclofenac (K_m = 3.4–52 μM)/4’-hydroxydiclofenac and 5 μM (CYP2C9); omeprazole (K_m = 17–26 μM)/5-hydroxyomeprazole and 10 μM (CYP2C19); dextromethorphan (K_m = 0.44–8.5 μM)/dextrorphan and 5 μM (CYP2D6); and testosterone (K_m = 52–94 μM)/6β-hydroxytestosterone and 10 μM (CYP3A) (Dinger et al., 2014; Spaggiari et al., 2014; Dahlinger et al., 2016). The probe substrates showed minimal interactions with each other (data not shown).

As an initial screen, each cannabinoid (10 μM) was added to an incubation mixture (200 μl) containing potassium phosphate buffer (100 mM; pH 7.4), HLMs (0.1 mg/ml), P450 cocktail, and BSA (0.2%). Stock solutions of probe substrates and cannabinoids were prepared in 100% DMSO. The final concentration of DMSO (v/v) in the incubation mixture was 0.2–0.5%. Each incubation mixture was prewarmed at 37°C with constant stirring for 10 minutes, and then the reaction was initiated with a NADPH regenerating system (1.3 mM NADP+, 3.3 mM D-glucose 6-phosphate, 3.3 mM magnesium chloride, and 0.4 units/ml glucose-6-phosphate dehydrogenase). After 15 minutes, the reaction was terminated with 200 μl ice-cold acetonitrile containing tolbutamide (internal standard; 25 nM final concentration) to precipitate the proteins. After vortexing, each sample was centrifuged at 18,000 g for 10 minutes. Then, an aliquot of the supernatant (50 μl) was stored at −20°C until analysis by LC-MS/MS (described below). Three independent experiments were conducted, each in duplicate.

Because 10 μM is much greater than circulating cannabinoid unbound plasma concentrations observed when cannabis or CBD products are used for medicinal or recreational purposes, IC₅₀ values were determined only for those cannabinoids that likely inhibit P450s (in the screening assay) at pharmacologically relevant concentrations. Therefore, varying concentrations of CBD (0.003–100 μM), 7-OH CBD (0.003–50 μM), THC (0.003–100 μM), or 11-OH THC (0.01–100 μM) were used to determine their P450-inhibitory potencies. P450 inhibition assays were conducted as described above. Three independent experiments were conducted, each in duplicate. IC₅₀ was determined by nonlinear least-squares regression analysis (GraphPad Prism 6.01, GraphPad Software Inc., San Diego, CA) using the following equation: where [I] represents the inhibitor concentration, A₀ and A_max represent minimum and maximum % P450 activity, respectively, and Hill Slope describes the steepness of the curve. The F-test and the Akaike information criterion were used as measures of goodness of fit. Residual plots were used to decide among standard, Poisson (1/y), or relative (1/y²) weighting.

Time-Dependent Inhibition of P450s by Cannabinoids

The TDI potential of each cannabinoid was screened at 10 μM. The previously reported TDI (Bansal et al., 2020) of CYP1A2, 2C19, and 3A by CBD (10 μM) was repeated as a positive control. Each primary incubation mixture (200 μl) consisted of HLMs (0.5 mg/ml protein), potassium phosphate buffer (100 mM; pH 7.4), BSA (0.2%), and cannabinoid. Blank DMSO (0.5% v/v) was used in place of cannabinoid as a negative control. The mixture was prewarmed at 37°C with constant stirring for 5 minutes, and then the reaction was initiated with the NADPH regenerating system and incubated at 37°C for 0 or 30 minutes. An aliquot (5 μl) of the primary incubation mixture was transferred to a prewarmed (37°C) secondary incubation mixture (195 μl) containing potassium phosphate buffer (pH 7.4), P450 cocktail (50 μM phenacetin, 2 μM coumarin, 5 μM bupropion, 2 μM amodiaquine, 5 μM diclofenac, 20 μM omeprazole, 5 μM dextromethorphan, and 20 μM testosterone), and the NADPH regenerating system. After 15 minutes, the reaction was terminated with 200 μl ice-cold acetonitrile containing tolbutamide (internal standard; 25 nM final concentration) to precipitate the proteins. Reaction mixtures were processed and analyzed as described for the reversible inhibition experiments. Three or four independent experiments were conducted, each singly.

Based on the screening results, cannabinoids that were likely to demonstrate TDI for P450s at pharmacologically relevant concentrations were studied in detail to determine the TDI kinetics. Briefly, varying concentrations of 7-OH CBD (1, 5, 10, 15, 20, 25, 30, 40, or 50 μM) or blank DMSO (0.5% v/v) were preincubated with HLMs (0.5 mg/ml protein) and BSA (0.2%) for 0, 4, 8, 12, or 16 minutes. The assay proceeded as described above. Four independent experiments were conducted, each in singlicate. The observed first-order rate constants for inactivation (k_obs) were estimated as described previously (Bansal et al., 2020). The half-maximal inactivation concentration (K_I) and maximal inactivation rate constant (k_inact) were estimated by nonlinear least-squares regression analysis (GraphPad Prism 6.01, GraphPad Software Inc.) of the k_obs versus inactivator concentration ([I]) data using the following equation:

Cannabinoid Plasma Protein Binding

The high nonspecific binding of cannabinoids to plastic and adsorption to filters or membranes preclude the use of ultrafiltration or equilibrium dialysis to determine their fraction unbound in plasma. Therefore, as previously described, the ultracentrifugation method and diluted plasma were used to measure the extent of plasma protein binding of the cannabinoids (Patilea-Vrana and Unadkat, 2019). Briefly, CBD (0.05, 5, or 50 μM), 7-OH CBD (0.25 or 1 μM), or THC (0.5 or 5 μM) was added to low-binding tubes and incubated with diluted plasma (10-fold dilution with Dulbecco’s phosphate buffer saline) (total volume = 200 μl) for 20 minutes at 37°C with shaking to facilitate nonspecific and protein binding. Two sets of these samples were prepared: one set (180 μl/sample) was subjected to ultracentrifugation at 37°C for 90 minutes, and the other (control) set (180 μl/sample) was incubated in the ultracentrifuge tubes at 37°C for 90 minutes without centrifugation. Samples undergoing ultracentrifugation were centrifuged at 435,000 g for 90 minutes at 37°C using the Sorval Discovery M150 SE centrifuge and S100-AT3 rotor (Thermo Scientific). The middle (aqueous) layer that was collected from the sample underwent centrifugation to determine unbound cannabinoid concentration. The total (bound and unbound) concentration of cannabinoid was quantified in the control incubations. Samples were analyzed using LC-MS/MS (described below). Because both sets of samples were prepared in the low-binding tubes and transferred into the ultracentrifuge tubes, the same degree of nonspecific binding was assumed for both sample types. The fraction unbound in undiluted plasma (f_u,p) was estimated using the following equation (Schuhmacher et al., 2000), where f_u,d represents the fraction unbound in the diluted plasma and DF represents the dilution factor (0.1):

Cannabinoid Protein Binding in the Incubation Mixtures

Although we and Nasrin and colleagues (2021) have previously determined f_u,inc of CBD and THC by the tube adsorption method (Bansal et al., 2020), the ultracentrifugation method was used to determine f_u,inc of CBD, 7-OH CBD, and THC. This method was used because the tube adsorption method requires determination of the partition ratio of unbound cannabinoid to the walls of the tube in protein-free aqueous buffer. Given the limited solubility of the cannabinoids in protein-free buffer, the ultracentrifugation method avoids this problem. Briefly, CBD (0.1, 1, or 5 μM), 7-OH CBD (1 or 10 μM), or THC (0.5 or 5 μM) was added to low-binding tubes and incubated with HLMs (0.1 or 0.5 mg/ml) in 0.1 mM potassium phosphate buffer (total volume, 200 μl) containing BSA (0.2%) for 15 minutes at 37°C with shaking to facilitate nonspecific and protein binding. Reaction mixtures were processed and analyzed as described for f_u,p determination. The f_u,inc of each tested cannabinoid was used to compute their corresponding unbound IC₅₀ and K_I values. Because 11-OH THC and 7-OH CBD have similar lipophilicity and have a similar chemical structure (ChemDraw Ultra, CambridgeSoft Corporation, Cambridge, MA), the f_u,inc for 11-OH THC was assumed to be same as that for 7-OH CBD.

LC-MS/MS Analysis

Each P450-mediated metabolite was quantified using an ultrahigh performance liquid chromatography (UPLC)-MS/MS system which consisted of an ACQUITY UPLC system (Waters, Milford, MA) coupled with a SCIEX 6500 QTRAP mass spectrometer (SCIEX, Framingham, MA) equipped with an electrospray ion source. Chromatography was performed on an ACQUITY UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 μm) which was maintained at 45°C and was preceded by an ACQUITY UPLC BEH C₁₈ VanGuard pre-column (2.1 × 5 mm, 1.7 μm). The autosampler compartment were set at 4°C to prevent the evaporation of the samples. The sample injection volume was 10 μl. Water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid were used as the mobile phase A and B, respectively, with a flow rate of 0.4 ml/min. The gradient conditions were optimized over 4.5 minutes as follows: 5% B at 0.0–0.3 minutes, linear increase from 5% to 60% B at 0.3–3.0 minutes and from 60% to 90% B at 3.0–4.0 minutes, linear decrease from 90% to 5% B at 4.0–4.2 minutes, and 5% B at 4.2–4.5 minutes. The quantification was performed in the positive multiple reaction monitoring mode after optimizing the compound- and ion source-dependent parameters. Acetaminophen, 7-hydroxycoumarin, 4-hydroxy bupropion, N-desethylamodiaquine, 4’-hydroxy diclofenac, 5-hydroxy omeprazole, dextrorphan, 6β-hydroxy testosterone, and tolbutamide were quantified using the following mass transitions of mass-to-charge ratio: 152.0 → 110.0, 162.9 → 106.9, 256.2 → 238.1, 328.2 → 283.1, 312.0 → 231.0, 362.0 → 214.0, 258.1 → 157.0, 305.3 → 269.1, and 271.3 → 155.0, respectively. The optimized spray voltage, ion source temperature, curtain gas, ion source gas 1, and ion source gas 2 were 5500 V, 600°C, 30 psi, 50 psi, and 50 psi, respectively.

CBD, 7-OH CBD, CBD-d3 (internal standard for CBD and 7-OH CBD assay), THC, and THC-d3 (internal standard for THC assay) were quantified using the same LC-MS/MS system, column, and solvents as mentioned above. The flow rate was optimized to be 0.3 ml/min. The gradient conditions were optimized over 4.5 minutes as follows: 10% B at 0.0–0.5 minutes, linear increase from 10% to 95% B at 0.5–2.0 minutes, 95% B at 2.0–4.0 minutes, linear decrease from 95% to 10% B at 4.0–4.1 minutes, and 10% B at 4.1–4.5 minutes. CBD (or THC), 7-OH CBD, and CBD-d3, (or THC-d3) were quantified using the following mass transitions of mass-to-charge ratio: 315.2 → 193.3, 331.2 → 313.1, and 318.3 → 196.3, respectively. The optimized spray voltage, ion source temperature, curtain gas, ion source gas 1, and ion source gas 2 were 4000 V, 300°C, 30 psi, 50 psi, and 50 psi, respectively. After method development, the LC-MS/MS methods were validated. The calibration curves were linear over the calibrator concentration range used (r² > 0.97) and the accuracy and precision of the quality control samples (low, middle, and high end of the calibration curves) were within 14% of the nominal concentrations and CV% of <12.5%, respectively.

Prediction of the Potential of the Cannabinoids to Precipitate Pharmacokinetic Drug Interactions

Unbound reversible inhibition (IC_50,u) and inactivation (K_I,u and k_inact) kinetics of CBD, 7-OH CBD, and 11-COOH CBD or THC, 11-OH THC, and 11-COOH THC were incorporated into a previously described mechanistic static model (Fahmi et al., 2008; Cheong et al., 2017) to predict the magnitude of drug-drug interaction (eq. 4). Assuming competitive inhibition, the k_i,u of the cannabinoids was assumed to be equal to IC_50,u when the P450 inhibition experiments were performed at probe concentration <K_m (CYP2B6, 2C19, and 3A) or half the IC_50,u when the P450 inhibition experiments were performed at probe concentration ≅ K_m (CYP1A2, 2C8, 2C9, and 2D6) (Cheng and Prusoff, 1973). AUCR (the magnitude of drug-drug interaction) represents the ratio of area under the plasma concentration-time curve of the object (probe) drug in the presence (AUC’_PO) of the cannabinoid P450 inactivator/inhibitor to that of the probe drug AUC in the absence of the inactivator/inhibitor. where A is the term that describes TDI of a P450 enzyme in the liver:

B is the term that describes reversible inhibition of a P450 enzyme in the liver:

X is the term that describes TDI of a P450 enzyme in the intestine:

Y is the term that describes reversible inhibition of a P450 enzyme in the intestine: and n is the number of inactivators/inhibitors (parent and metabolites) present. [I]_H and [I]_G are the inhibitor/inactivator concentrations in the liver and intestine, respectively. k_deg,H and k_deg,G represent the degradation rates of the P450 in the liver and intestine, respectively. The fraction of the object drug metabolized by a given P450 is represented by f_m, and the fraction of the object drug escaping intestinal metabolism is represented by F_G. The f_m and F_G values of object drugs and k_deg values of P450 enzymes are provided in Supplemental Table 1.

 A variation of the four different models (Models A–D) described by Tseng et al. (2021) was used to predict the magnitude of a pharmacokinetic cannabinoid-drug interaction in the liver or intestine after a single (THC) or multiple (CBD) doses of oral or inhaled cannabinoid (liver only) administration (Bansal et al., 2020). AUCR values were predicted using the estimated [I]_H and [I]_G concentrations (Supplemental Table 2). [I]_H was estimated to be either the unbound maximum hepatic inlet concentration (C_{max,hepatic inlet,u}, Model A), the unbound maximum systemic concentration (C_max,sys,u, Model B) or, the unbound average systemic concentration (C_ave,sys,u, Models C and D) (Tseng et al., 2021). [I]_G of the cannabinoids, due to their poor aqueous solubility, was estimated as their maximum intestinal fluid solubility (Models A–C) (Bansal et al., 2020) or the unbound average portal vein concentration (C_ave,portal,u, Model D) to predict any interaction in the intestine (Tseng et al., 2021).

To predict drug interactions precipitated by the cannabinoid metabolites 7-OH CBD, 7-COOH CBD, 11-OH THC, or 11-COOH THC, the C_max,sys,u and C_ave,sys,u of each was set equal to [I]_H. The doses (and justification) of CBD and THC to predict in vivo hepatic and intestinal P450-mediated drug interactions were the same as described previously (Bansal et al., 2020). Briefly, the average low, average high, and maximum doses of CBD or THC used for recreational or medicinal purposes were estimated from those reported in clinical trials, case reports, social media, cannabinoid vendor websites, and newspapers (Bansal et al., 2020). The oral doses for CBD were 70 mg, 700 mg, and 2,000 mg and for THC were 20 mg, 130 mg, and 160 mg. The inhaled doses of THC were 25 mg, 75 mg, and 100 mg.