Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

Abstract

Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes).

Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Figures

Fig. 1.

t-Cinnamic aldehyde (cinnamaldehyde).

Fig. 2.

(A) A representative binding spectra of purified rCYP2A6 with increasing concentrations of t-CA. (B) A representative curve generated from fitting changes in absorbance (A386 nm–A418 nm) to a one-site ligand binding model as a function of t-CA concentration.

Fig. 3.

(A) Inactivation of coumarin hydroxylase activity as a function of t-CA concentration, preincubation period, and NADPH in a recombinant CYP2A6 system (●, buffer; ▪, NADPH; ▴, 10 µM t-CA; ▾, 20 µM t-CA; ♦, 40 µM t-CA; ○, 80 µM t-CA; □, 120 µM t-CA). Data points are the mean of three individual experiments conducted on separate days (N = 3 for each point). The percentage of control activity was determined by comparing the activity to the average activity of samples with CYP2A6 that did not contain NADPH or t-CA. (B) Determination of kinact and KI by fitting kobs (min−1) to eq. 1.

Fig. 4.

(A) Inactivation of coumarin hydroxylase activity as a function of t-CA concentration, preincubation period, and NADPH in human liver microsomes (●, buffer; ▪, NADPH; ▴, 10 µM t-CA; ▾, 20 µM t-CA; ♦, 40 µM t-CA; ○, 80 µM t-CA; □, 120 µM t-CA). Each point is the mean of three individual experiments conducted on separate days (N = 3 for each point). The percentage of control activity was determined by comparing the activity to the average activity of samples with human liver microsomes that did not contain NADPH or t-CA. (B) Determination of kinact and KI by fitting kobs (min−1) to eq. 1.

Fig. 5.

Effect of t-CA and NADPH on CYP2A6 CO-binding spectra: 2A6 with t-CA (red line), 2A6 with NADPH (blue line), and 2A6 with t-CA and NADPH (green line).

Fig. 6.

Potential mechanisms for reactions of t-CA with CYP2A6. I. Direct reactions by conjugate addition with a cysteine residue of CYP2A6 (A) and reaction with a lysine residue (B) to form an imine (Schiff base). II. Reactions involving bioactivation of t-CA to form an uncharacterized reactive metabolite that reacts with and inactivates CYP2A6.