Determination of IC50 and Ki Values.

For those cannabinoids/THC metabolites that exhibited a >50% decrease in relative P450 activity for any given probe substrate at ≤10 µM, IC₅₀ determinations were performed in both microsomes from HEK293 CYP450-overexpressing cell lines (described above) and in HLMs using multiple concentrations (ranging between 0.5 and 100 µM) of cannabinoid/metabolite in incubations as described above, with all determinations performed for three independent experiments with three replicates per experiment.

To identify possible metabolism-dependent inhibition of the potential enzymes, IC₅₀ shift studies (Parkinson et al., 2011) were performed using HEK-overexpressing cell lines. Reactions containing the test cannabinoid or THC metabolite (inhibitor) but no probe substrate were incubated with or without NADPH at 37°C for 30 minutes. After this 30-minute preincubation, probe substrate was added to the incubation mixture and incubated at 37°C for up to 30 minutes (Supplemental Table 1). Peak areas corresponding to the probe metabolite were determined, and the percentage of relative activity was calculated by comparing the peak area in incubations containing the inhibitors to incubations containing only the vehicle control, as described below. The differences between the IC₅₀ values obtained with or without the NADPH preincubation period were compared, and the fold-IC₅₀ shift was determined. The cannabinoid/THC metabolite with a fold-IC₅₀ shift of ≥1.5 was considered a time-dependent inhibitor as recommended by the FDA guidelines.

IC₅₀ data were used as a guide to generate appropriate probe substrate and test inhibitor concentrations for the determination of the K_i values for each isoform. The P450 enzyme-specific probe substrate concentrations used were 1–25 μM phenacetin for CYP1A2, 12.5–100 μM bupropion for CYP2B6, 2.5–30 μM diclofenac for CYP2C9, 0.5–5 μM omeprazole for CYP2C19, 1–10 μM dextromethorphan for CYP2D6, 12.5–100 μM chlorzoxazone for CYP2E1, and 1–25 μM midazolam for CYP3A4.

Data were exported and analyzed using an Excel spreadsheet (Microsoft). The amount of metabolite formed at each concentration relative to the control (percent relative activity) of specific enzyme in the presence and absence of probe inhibitor or test compound was calculated as:

% Relative activity = (Peak area with inhibitor/Peak area without inhibitor) × 100%

Percent Inhibition = [(Peak area without inhibitor − peak area with inhibitor)/peak area without inhibitor] × 100

The IC₅₀ values were calculated by plotting the percent inhibition of P450 enzyme activities versus the log concentration of the test inhibitors using GraphPad Prism 7.04 software (GraphPad Software Inc., San Diego, CA).

To calculate K_i values, inhibition data were fit to different models of enzyme inhibition (competitive, uncompetitive, or noncompetitive) by nonlinear least-squares regression analysis using the GraphPad Prism 7.04 software. K_i values were calculated with the use of nonlinear regression according to the equations:

where I is compound concentration, K_i is the inhibition constant, S is the substrate concentration, and K_m is the substrate concentration at half of the V_max of the reaction (Cornish-Bowden, 1974).