Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole S

Home , Cinnamaldehyde

Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/02/05/dmd.115.067942.DC1

1521-009X/44/4/534–543$25.00 http://dx.doi.org/10.1124/dmd.115.067942 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:534–543, April 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole s

Jeannine Chan, Tyler Oshiro, Sarah Thomas, Allyson Higa, Stephen Black, Aleksandar Todorovic, Fawzy Elbarbry, and John P. Harrelson

Chemistry Department, Pacific University Oregon, Forest Grove, Oregon (J.C., T.O., A.H., S.B.); and School of Pharmacy, Pacific University Oregon, Hillsboro, Oregon (S.T., A.H., S.B., A.T., F.E., J.P.H.)

Received October 16, 2015; accepted February 4, 2016

ABSTRACT Downloaded from Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; of inhibition modestly and statistically significantly. The carbon cinnamal; (E)-3-phenylprop-2-enal] is common through diet and monoxide binding spectrum was dramatically diminished after expo- through the use of cinnamon powder for diabetes and to provide sure to NADPH and t-CA, suggesting degradation of the heme or flavor and scent in commercial products. We evaluated the likeli- CYP2A6 apoprotein. Using a static model and mechanism-based 21 hood of t-CA to influence metabolism by inhibition of P450 enzymes. inhibition parameters (KI = 18.0 mM; kinact = 0.056 minute ), changes

IC50 values from recombinant enzymes indicated that an interaction in the area under the concentration-time curve (AUC) for nicotine and dmd.aspetjournals.org is most probable for CYP2A6 (IC50 = 6.1 mM). t-CA was 10.5-fold letrozolewerepredictedinthepresenceoft-CA (0.1 and 1 mM). The more selective for human CYP2A6 than for CYP2E1; IC50 values for AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or source of pharmacokinetic variability for CYP2A6 substrates due to more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 mM). Inhibition of metabolism-dependent inhibition, especially in scenarios when expo- CYP2A6 by t-CA was metabolism-dependent; inhibition required sure to t-CA is elevated due to high dietary exposure, or when cinnamon NADPH and increased with time. Glutathione lessened the extent is used as a treatment of specific disease states (e.g., diabetes). at ASPET Journals on October 2, 2021

Introduction contributes to cinnamon’s flavor and aroma and is the major component Dietary and herbal agents can profoundly impact the nature of drug of cinnamon oil. Human exposure to t-CA is common due to routine use response. Consequently, drug-diet/drug-herbal interactions are an impor- in fragrances and food (Peters and Caldwell, 1994). The concentration tant consideration for the effective use of medications. Modulation of drug of t-CA in commercial cinnamon powder ranges from 8.2 to 27.5 mg metabolism by diet/herbs can significantly alter drug clearance, resulting per gram (Friedman et al., 2000). Doses from 1 to 10 g of encapsulated in toxicity or reduced efficacy. For example, grapefruit juice inhibits the cinnamon powder are used for diabetes mellitus to lower blood sugar metabolism of terfenadine, elevating concentrations that ultimately affect (Pham et al., 2007; Crawford, 2009; Akilen et al., 2010), corresponding, cardiac rhythm (Benton et al., 1996). Other examples include leafy green therefore, to exposure of 8 to 275 mg of t-CA (Friedman et al., 2000; vegetables with warfarin (O’Reilly and Rytand, 1980; Suttie et al., 1988), Kirkham et al., 2009). There are also reports of cinnamon exhibiting St. John’s wort with CYP3A substrates (Borrelli and Izzo, 2009), and antimicrobial, anti-inflammatory, lipid-lowering, anticancer, and amy- tyramine-containing foods with monoamine oxidase inhibitors (Blackwell loid plaque-reducing effects (George et al., 2013; Long et al., 2015). et al., 1967; Flockhart, 2012). The growing use of cinnamon as a complementary treatment, in Phenylpropanoids are a major class of plant secondary metabolites addition to dietary exposure, increases the likelihood of drug interac- with diverse functions (Yu and Jez, 2008). Due to their ubiquity, tions with t-CA. phenylpropanoids are a potential cause of drug-diet interactions. There Metabolism by CYP2A6 is a major clearance route for nicotine are several low-molecular-weight phenylpropanoids present in cinna- (Nakajima et al., 1996; Messina et al., 1997) and letrozole (Desta et al., mon, most notably trans-cinnamic aldehyde (t-CA) (Fig. 1), which 2009, 2011; Murai et al., 2009), an aromatase inhibitor used to treat breast cancer. CYP2A6 exhibits extensive genetic diversity (http://www. cypalleles.ki.se/cyp2a6.htm). With letrozole, CYP2A6 genetic variation contributes to as large as 12-fold differences in steady-state plasma This work was supported by the Medical Research Foundation of Oregon, the concentrations in postmenopausal breast cancer patients (Desta et al., M. J. Murdock Charitable Trust, the Pacific Research Institute for Science and Mathematics, and the Pacific University College of Health Professions and School 2011). For nicotine, variations in metabolism contribute to differences in of Pharmacy. The CYP2A6 plasmid, provided as a gift, was supported by the smoking behavior, including smoking cessation rates (Rao et al., 2000; National Institutes of Health [Grant R01 GM076343]. Ariyoshi et al., 2002; Minematsu et al., 2003; Fujieda et al., 2004; dx.doi.org/10.1124/dmd.115.067942. Iwahashi et al., 2004; Schoedel et al., 2004; Ray et al., 2009; Chenoweth s This article has supplemental material available at dmd.aspetjournals.org. et al., 2013, 2015). However, nicotine addiction is a complex disease, and

ABBREVIATIONS: AUC, area under the concentration-time curve; HPLC, high-performance liquid chromatography; MBI, mechanism-based inhibition; P450, cytochrome P450; rCYP2A6, recombinant CYP2A6; t-CA, trans-cinnamic aldehyde.

534 CYP2A6 Inhibition by Cinnamic Aldehyde 535

1 mM 7-ethoxyresorufin (CYP1A2), 2.5 mM 7-ethoxy-4-trifluoromethyl coumarin (CYP2B6), 75 mM 7-methoxy-4-trifluoromethyl coumarin (CYP2C9), 25 mM 3-cyano-7-ethoxycoumarin (CYP2C19), 1.5 mM 3-[2-(N,N-diethyl-N- methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D6), and 50 mM 7-benzyloxy-trifluoromethylcoumarin (CYP3A4). t-CA stocks were prepared in acetonitrile in accordance with the manufacturer’s directions. The final concentration of acetonitrile in assays was 0.05%. Positive control inhibitors were 1-aminobenzotriazole (CYP1A2), tranylcypromine (CYP2A6, CYP2B6, Fig. 1. t-Cinnamic aldehyde (cinnamaldehyde). and CYP2C19), sulfaphenazole (CYP2C9), quinidine (CYP2D6), and ketoconazole (CYP3A4). Assays were conducted in 96-well plates, and the fluorescent intensity was measured using a Synergy2 microplate reader (BioTek, Winooski, genetics alone does not fully explain interindividual differences in VT). Each experiment was conducted in triplicate on at least three different days $ smoking addiction/cessation (Berrettini and Doyle, 2012; Bergen et al., (i.e., at least three trials; N 9). The mean IC50 was determined from taking the 2013). Similarly, there are unaccounted factors that contribute to the antilog of the mean of the logIC50 values for the trials. The mean of the logIC50 noteworthy variability in letrozole pharmacokinetics (Desta et al., 2011). values from the trials was analyzed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA) to generate the 95% confidence intervals displayed in A potential contributor to the variation in metabolism and drug response Table 1. The logIC50 value for CYP2A6 was compared with each logIC50 is modulation of CYP2A6 activity by dietary and herbal substances. value for the other P450 isoforms using a t test (two-tailed; unequal variance) However, unlike drugs, which undergo thorough analysis for risk of to evaluate the probability that differences in logIC50 values were due to drug-drug interactions (Food and Drug Administration, 2012), most coincidence. Downloaded from herbal or dietary agents have not undergone such an intense level of CYP2E1 activity was measured using the formation of 6-hydroxychlorzoxazone scrutiny. Thus, there is a substantial knowledge gap regarding the risk of as previously reported, with modification (Peter et al., 1990; Elbarbry et al., drug-herb and drug-diet interactions (Brantley et al., 2014a,b). 2007). Incubation mixtures consisted of 60 mM chlorzoxazone, 0.1 mM CYP2E1 Given the abundant chemical diversity in herbs and the human diet, Supersomes with cytochrome b5, and 50 mM potassium phosphate buffer m there are still many unknowns about the extent to which herbs/food (pH 7.4) in a final volume of 150 l. After a preincubation period of 3 minutes at 37C, the reaction was started by addition of NADPH (final concentration =

affect CYP2A6 activity. Previously, isoflavones (Nakajima et al., dmd.aspetjournals.org 1 mM) and incubated at 37C for 9 minutes in a shaking water bath. The reaction 2006), menthol (MacDougall et al., 2003; Kramlinger et al., 2012), was terminated by addition of trichloroacetic acid (6 ml), followed by the menthofuran (Khojasteh-Bakht et al., 1998), isothiocyanates (Nakajima addition of 7-hydroxycoumarin (internal standard). The supernatant was et al., 2001; von Weymarn et al., 2007), and several others (Di et al., analyzed by high-performance liquid chromatography (HPLC), as described 2009) were studied and found to inhibit CYP2A6. Based on the later. Each experiment was conducted in duplicate over four different days structural and biophysical studies of CYP2A6, low-molecular-weight (N = 8). Standard curves for 6-hydroxychlorzoxazone were generated from phenylpropanoids, such as t-CA (Fig. 1), are reasonable candidates as serially diluted standards suspended in incubation buffer, 0.1 mM CYP2E1 CYP2A6 inhibitors, since structural evidence indicates that the active Supersomes, trichloroacetic acid, chlorzoxazone (60 mM), and NADPH (1 mM) site volume of CYP2A6 is more restricted (Yano et al., 2005) and “less that were processed similar to experimental samples, except that trichloroacetic at ASPET Journals on October 2, 2021 flexible” (Wilderman et al., 2013) than other cytochrome P450s acid was added prior to NADPH. The mean IC50 value, variability, 95% (P450s). The goals of this study were to investigate inhibition of confidence interval, and statistical comparison with the CYP2A6 IC50 value were determined by the same method as described earlier for the other P450 CYP2A6 by t-CA, evaluate selectivity in comparison with other drug- isoforms. Experiments were conducted in microcentrifuge tubes. metabolizing P450s, and assess the likelihood of drug interactions with IC50 Measurement for Inhibition of 7-Hydroxycoumarin Formation by t-CA by estimating changes in nicotine and letrozole area under the t-CA in Human Liver Microsomes. Human liver microsomes (50-donor pool; concentration-time curve (AUC) using a model that accounts for 0.2 mg/ml), coumarin (3 mM), incubation buffer (50 mM potassium phosphate; mechanism-based inhibition, variability in the fraction metabolized pH = 7.4), and t-CA (0–500 mM; dissolved in incubation buffer) were by CYP2A6, and intrinsic degradation rates of CYP2A6 (Mayhew preincubated for 5 minutes at 37C in a shaking water bath. Following the et al., 2000; Venkatakrishnan and Obach, 2007; Grimm et al., 2009; addition of NADPH (1 mM), the samples (100 ml) were heated at 37C for Mohutsky and Hall, 2014). 6minutes,terminatedwith4ml of trichloroacetic acid, mixed with a vortex mixer, and centrifuged at 11,000 rpm (11,228g) for 5 minutes in a 5415D centrifuge (Eppendorf, Hamburg, Germany). The concentration of 7-hydroxycoumarin in the Materials and Methods

Materials. CYP2A6 Supersomes (with P450 reductase and cytochrome b5), TABLE 1 human liver microsomes (50-donor pool), and P450 High Throughput Screening Inhibition of xenobiotic-metabolizing P450 isoforms by t-CA Kits were obtained from Corning Gentest (Woburn, MA). The human liver IC50 values are the mean of at least three experiments conducted on separate days. Values in microsomes included male (53%) and female (47%) donors with an age distribu- parenthesis represent the 95% confidence interval (lower limit–upper limit). tion of 19–77. Glutathione, coumarin, 7-hydroxycoumarin, methoxylamine hydro- chloride, methoxypsoralen, ketoconazole, letrozole, 1-aminobenzotriazole, Fold Difference Compared P450 Isoform IC50 N trans-cinnamic aldehyde, and chlorzoxazone were purchased from Sigma- with CYP2A6 Aldrich (St. Louis, MO). NADPH tetrasodium salt, 6-hydroxychlorzoxazone, mM and 4,49-methanol-bisbenzonitrile were purchased from EMD Chemicals 2A6 6.1*** (2.9–12.7) 15 — (San Diego, CA), U.S. Biologicals (Marblehead, MA), and Santa Cruz 1A2 106.3 (68.5–164.8) 12 17.4 Biotechnology (Dallas, TX), respectively. 2B6 107.3 (88.3–130.3) 12 17.6 Inhibition of Major Xenobiotic-Metabolizing P450s by t-CA. Fluorescent 2C9 96.2 (57.0–162.5) 9 15.8 – assays were used to evaluate the selectivity of t-CA for CYP2A6 and many of the 2C19 212.8(132.7 349.2) 9 34.9 2D6 . 2000a 9 Not determined major drug-metabolizing human P450s. Corning Gentest P450 High Throughput 2E1 64.0 (56.9–71.9) 8 10.5 Screening Kits were used to measure t-CA inhibition of CYP2A6, CYP1A2, 3A4 116.1 (51.1–263.0) 9 19.0 CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 essentially according to a $ m the manufacturer’s directions. All assays used P450 Supersomes with P450 Fifty percent inhibition was only observed at concentrations 2000 M; the IC50 for quinidine (positive control) was ,1 mM(N =9). reductase (and cytochrome b for CYP2A6, 2B6, 2C9, 2C19, and 3A4) and an 5 ***P , 0.001 in the comparison of logIC50 for CYP2A6 to logIC50 for CYP1A2, 2B6, 2C9, NADPH-regenerating system. The substrates were 3 mM coumarin (CYP2A6), 2C19, 2E1, and 3A4. 536 Chan et al. supernatant was measured by HPLC-fluorescence as described later. Stock solutions containing buffer, letrozole (0.5 mM), and NADPH (1 mM). The concentration of coumarin were prepared in dimethylsulfoxide and diluted in incubation buffer of CYP2A6 the secondary incubation was 0.1 mM. After heating at 37C for prior to experiments. The final concentration of dimethylsulfoxide in incuba- 72 minutes, the incubations were terminated with 8 ml of trichloroacetic acid, tions was 0.25% (v/v). Standard curves were generated by serial dilution of mixed with a vortex mixer, and centrifuged at 11,288g for 5 minutes. The 7-hydroxycoumarin in incubation buffer for quantification. Experiments were supernatant was analyzed by HPLC-fluorescence, as described later. Preliminary conducted in microcentrifuge tubes. experiments conducted under the same conditions as the secondary incubations NADPH- and Time-Dependent Inhibition of 7-Hydroxycoumarin showed metabolite (4,49-methanol-bisbenzonitrile) formation was linear to at Formation with Recombinant CYP2A6 and Human Liver Microsomes. least 120 minutes. The experiments were conducted in microcentrifuge tubes. CYP2A6 Supersomes (0.2 mM) or human liver microsomes (50-donor pool; For microsomal studies, human liver microsomes (50-donor pool; 7.5 mg/ml) 2 mg/ml) were preincubated for 5 minutes at 37C in incubation buffer. Select were preincubated for 5 minutes at 37C in incubation buffer (50 mM potassium samples contained t-CA (0–120 mM), NADPH (1 mM), glutathione (5 mM), and phosphate, pH = 7.4). Select samples contained t-CA (80 mM), NADPH (1 mM), methoxylamine (1 mM) or a combination thereof. Incubations were initiated by and methoxylamine (1 mM) or a combination thereof. Incubations were initiated addition of NADPH and heated at 37C. At specific time points (0, 3, 6, 9, 12, 15, by addition of NADPH and heated at 37C. Aliquots (30 mL) were removed from and 18 minutes), 20-ml aliquots were removed from the primary incubation and the primary incubation, after incubating for 18 minutes, and diluted to 300 mlina diluted to 200 ml in a secondary incubation containing buffer, coumarin (3 mM), secondary incubation containing buffer, letrozole (0.5 mM), and NADPH and NADPH (1 mM). The concentration of CYP2A6 or microsomal protein in (1 mM). The protein concentration in the secondary incubation was 0.75 mg/ml. the secondary incubation was 0.02 mM and 0.2 mg/ml, respectively. After After heating at 37C for 30 minutes, the incubations were terminated with 12 mlof heating at 37C for 5.5 minutes, the incubations were terminated with 8 mlof trichloroacetic acid, mixed with a vortex mixer, and centrifuged at 11,288g for trichloroacetic acid, mixed with a vortex mixer, and centrifuged at 11,288g for 5 minutes. The supernatant was analyzed by HPLC-fluorescence, as described

5 minutes. The supernatant was analyzed by HPLC-fluorescence, as described later. Preliminary experiments, as described earlier for the NADPH/time-dependent Downloaded from later. Preliminary experiments conducted under the same conditions as the experiment, showed metabolite formation in the secondary incubations was in the secondary incubations showed 7-hydroxycoumarin formation was linear to at linear range with respect to time and protein concentration. All experiments were least 14 and 18 minutes in recombinant and microsomal systems, respectively. conducted in microcentrifuge tubes. Product formation was also within the linear range as a function of protein HPLC Analysis of 6-Hydroxychlorzoxazone, 7-Hydroxycoumarin, and concentration for both systems. All experiments were conducted in micro- 4,49-methanol-bisbenzonitrile. The metabolites were quantified with a Prom- centrifuge tubes. The percentage of remaining activity was determined by inence HPLC (Shimadzu, Kyoto, Japan), which included the following: two comparing to control samples that did not contain NADPH and t-CA in the LC-20AD pumps, degasser, autosampler, column oven, communication bus module, dmd.aspetjournals.org primary incubation. diode array detector, and fluorescence detector. For 6-hydroxychlorzoxazone,

KI and kinact values were determined using nonlinear regression via GraphPad chromatographic separation was carried out on a reversed-phase C18 column Prism 5 and eq. 1 (Silverman, 1988): (150 4.6-mm i.d., 3.5-mm particle size; Agilent Technologies, Santa Clara, CA) that was kept at 40C based on a previously reported method (Elbarbry et al., kinact •½I 2007). 6-Hydroxychlorzoxazone, 7-hydroxycoumarin (internal standard), and kobs ¼ : ð1Þ KI þ½I chlorzoxazone were eluted under isocratic conditions at retention times of 3.5, 5, and 15 minutes, respectively, using a mobile phase composed of acetonitrile and The partition ratio for t-CA inhibition of CYP2A6 was determined from 0.25% acetic acid (20:80 v/v) with a flow rate of 1.0 ml/min. Peaks were detected at ASPET Journals on October 2, 2021 recombinant CYP2A6 (i.e., Supersomes). The percentage of remaining activity by absorbance at 287 nm (bandwidth = 4 nm); 7-hydroxycoumarin was also was graphed versus the molar ratio of t-CA to CYP2A6. The partition ratio was detected by fluorescence (excitation = 370 nm; emission = 450 nm). To quantify estimated from the intersection point of the two lines generated from linear coumarin hydroxylase activity, the supernatant (25 ml) from incubations with regression of the low and high molar ratio regions of the graph (Silverman, 1988; coumarin was injected onto a Gemini column (100 4.6 mm, 5 mm; Phenomenex, Ghanbari et al., 2006). Torrance, CA). 7-Hydroxycoumarin was eluted with 35% acetonitrile (flow rate = NADPH- and Time-Dependent Inhibition of Letrozole Metabolism in 0.6 ml/min) and detected by fluorescence. Analysis of 4,49-methanol- Human Liver Microsomes. Human liver microsomes (50-donor pool; 7.5 mg/ml) bisbenzonitrile, as an indicator of letrozole metabolism, was based on a method were preincubated in a total volume of 250 ml for 5 minutes at 37C in buffer reported previously with some modifications (Marfil et al., 1996). Supernatant (50 mM potassium phosphate, pH = 7.4). Select samples also contained NADPH (100 mL) was injected onto a Phenomenex Kinetix C18 column (100 4.6 mm, (1 mM) and t-CA (8 and 80 mM) or a combination thereof; stocks were prepared 5 mm) using a Shimadzu Prominence HPLC (10 mM potassium phosphate, pH = in incubation buffer. At 9 minutes, 30-ml aliquots were removed from the 2.1 with 25% acetonitrile; 1.3 ml/min). 4,49-Methanol-bisbenzonitrile and primary incubation, diluted to 300 ml in incubation buffer containing letrozole letrozole were detected by fluorescence (excitation = 230 nm; emission = 295 nm); (0.5 mM) and NADPH (1 mM), and incubated for an additional 30 minutes a representative HPLC trace is available (Supplemental Fig. 1). at 37C. Incubations were terminated with trichloroacetic acid (12 ml) and Spectral Binding Studies with t-CA and Purified CYP2A6. CYP2A6 was centrifuged at 11,228g for 5 minutes. The supernatant was removed and expressed and purified as previously reported, with some slight modifications transferred to a vial for HPLC analysis. 4,49-Methanol-bisbenzonitrile formation (Stephens et al., 2012). Spectral ligand binding assays were conducted using was linear for at least 90 minutes as a function of time, and linear up to 1 mg/ml as modified recombinant human CYP2A6, which had an N-terminal trans- a function of microsomal protein concentration in the secondary incubation. membrane sequence truncation (Δ2–23) and a C-terminal His4-tag. The Standard curves for 4,49-methanol-bisbenzonitrile were generated from serially recombinant protein was expressed from pKK2A6dH (gift from Emily Scott, diluted standards suspended in incubation buffer, human liver microsomes University of Kansas, Lawrence, KS) in TOPP3 or DH5a Escherichia coli (0.75 mg/ml), trichloroacetic acid (12 ml), letrozole (0.5 mM), and NADPH with a 48- or 72-hour induction time in d-amino levulinic acid–supplemented (1 mM) that were processed similar to experimental samples, except that Terrific Broth media. The protein was purified using Ni affinity chromatog- trichloroacetic acid was added prior to NADPH. The percentage of remaining raphy, followed by carboxymethyl sepharose cation-exchange chromatogra- activity was determined by comparing to control samples that did not contain phy (Stephens et al., 2012). P450 content was determined from the carbon NADPH and t-CA in the primary incubation. monoxide difference spectrum (Omura and Sato, 1962, 1964; Guengerich Effect of Methoxylamine on NADPH-Dependent Inhibition of Letrozole et al., 2009). Metabolism with Recombinant CYP2A6 and Human Liver Microsomes. For the ligand binding titrations, samples of 1 mM purified CYP2A6, in CYP2A6 Supersomes (0.5 mM) were preincubated for 5 minutes at 37Cin 50 mM potassium phosphate buffer (pH 7.4), were titrated with aliquots of t-CA incubation buffer (50 mM potassium phosphate, pH = 7.4). Select samples dissolved in 50 mM potassium phosphate buffer. Difference spectra were contained t-CA (80 mM), NADPH (1 mM), and methoxylamine (1 mM) or a recorded on a Shimadzu UV-1601 spectrophotometer, and the difference in combination thereof. Incubations were initiated by addition of NADPH and absorbance between the minimum and maximum, ΔA, was measured. Equilib- heated at 37C. Aliquots (40 mL) were removed from the primary incubation, rium dissociation constants were determined from nonlinear least-squares fits to after incubating for 18 minutes, and diluted to 200 ml in a secondary incubation the equation for one-site specific ligand binding using GraphPad Prism 5. CYP2A6 Inhibition by Cinnamic Aldehyde 537

CYP2A6 Carbon Monoxide Binding Spectra in the Presence of t-CA were based on a pharmacogenetic study that evaluated the fm of high (fm = 0.77) and NADPH. CO binding assays were conducted using modified recombinant and intermediate (fm = 0.60) metabolizers, which was consistent with previous human CYP2A6, which had an N-terminal transmembrane sequence truncation observations that 70–80% of a nicotine dose is metabolized to cotinine

(Δ2–23) and a C-terminal His4-tag, as described earlier. Rat P450 reductase was (Benowitz and Jacob, 1994), with CYP2A6 responsible for 90% of this pathway. expressed and purified as previously reported (Shen et al., 1989). CYP2A6 and For letrozole, a high fm of 0.80 was selected, based on reports that at least 85% of rat P450 reductase were combined in a 1:4 ratio (final sample concentrations a letrozole dose is eliminated by P450-mediated metabolism, with CYP2A6 0.4 and 1.6 mM, respectively) and left at room temperature for 10 minutes. being the enzyme responsible for approximately 93% of metabolism at

Following the addition of buffer (50 mM potassium phosphate buffer, pH 7.4), therapeutic letrozole concentrations. The low fm of 0.31 was selected based on NADPH (1 mM) and/or t-CA (80 mM) samples were incubated for 18 minutes at in vitro studies of letrozole metabolism involving microsomes with reduced 37C, and then CO-binding spectra were acquired using established methods CYP2A6 activity (Sioufi et al., 1997; Murai et al., 2009; Desta et al., 2011). (Omura and Sato, 1962, 1964; Guengerich et al., 2009). Based on our literature searches, the systemic t-CA concentration in human Prediction of t-CA–Mediated Changes in Nicotine and Letrozole AUC. blood following oral administration has yet to be reported. To estimate the blood To estimate the potential for interactions between t-CA and CYP2A6 drug substrates, a concentrations of t-CA that could be expected for humans upon oral dosing, a model (eq. 2) was selected that takes into account mechanism-based inhibition (MBI) body surface area normalization method (Food and Drug Administration, 2005; parameters (KI and kinact), the fraction of the dose metabolized by CYP2A6 (fm), and Reagan-Shaw et al., 2008) was used. The method utilizes an animal dose and theinvivodegradationrateofCYP2A6(kdeg) (Mayhew et al., 2000; Venkatakrishnan accounts for differences in body weight and body surface area between species to and Obach, 2007; Grimm et al., 2009; Mohutsky and Hall, 2014). AUCi and AUC estimate a human dose. A 250-mg/kg dose of t-CA in rats was used for the refer to the AUC in the presence and absence of inhibitor, respectively: estimation; this dose resulted in blood concentrations on the order of 1 mg/ml ( 7.6 mM) in rats (Yuan et al., 1992).

AUC 1 Downloaded from i ¼ 2 3 : ð2Þ Statistical Analysis. The t test (paired; two-tailed distribution) was used to AUC evaluate the probability that differences between mean values were due to 6 fm;CYP 7 þ 2 4 •½ 5 1 fm;CYP coincidence. þ kinact I 1 • þ½ kdeg ðKI I Þ

Nicotine and letrozole were selected because CYP2A6 metabolism has been Results reported as a primary route of elimination for both. The magnitude of kdeg can Inhibition of Major Xenobiotic-Metabolizing P450s by t-CA. dmd.aspetjournals.org vary; low and high values from the literature were used to estimate the potential The selectivity of t-CA was evaluated by measuring inhibition of the variation in t-CA–induced AUC changes. The in vivo half-life (t1/2) for CYP2A6 ranges from 19 to 37 hours (Renwick et al., 2000; Yang et al., 2008). Values of kdeg were determined from the relationship kdeg = 0.693/t1/2, generating kdeg 21 values of 0.00031–0.00061 minute . High and low values for fm were selected to account for genetic variability in CYP2A6 activity. For nicotine, the fm values at ASPET Journals on October 2, 2021

Fig. 3. (A) Inactivation of coumarin hydroxylase activity as a function of t-CA concentration, preincubation period, and NADPH in a recombinant CYP2A6 system (d, buffer; j, NADPH; m,10mM t-CA; .,20mM t-CA; ♦,40mM t-CA; s, 80 mM t-CA; u, 120 mM t-CA). Data points are the mean of three individual Fig. 2. (A) A representative binding spectra of purified rCYP2A6 with increasing experiments conducted on separate days (N = 3 for each point). The percentage of concentrations of t-CA. (B) A representative curve generated from fitting changes in control activity was determined by comparing the activity to the average activity absorbance (A386 nm–A418 nm) to a one-site ligand binding model as a function of of samples with CYP2A6 that did not contain NADPH or t-CA. (B) Determination 21 t-CA concentration. of kinact and KI by fitting kobs (min ) to eq. 1. 538 Chan et al. major drug-metabolizing P450s using recombinant enzymes. IC50 values for t-CA inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 are shown in Table 1. The IC50 value for CYP2A6 (6.1 mM) was 10.5-fold lower than the next-lowest IC50, which was for CYP2E1. IC50 values for all other P450s were 15.8-fold higher or more than the IC50 value for CYP2A6. Substantial inhibition of CYP2D6 was not observed except at the highest concentrations of t-CA used in the study (e.g., 95% activity was observed at 666 mM t-CA). Based on these results, t-CA was evaluated further for inhibition of coumarin hydroxylase activity in human liver microsomes, resulting in an IC50 of 14.2 6 1.1 mM (mean 6 standard deviation) and a 95% confidence interval of 7.6–26.6 mM (from two trials conducted in triplicate; N = 6). Spectral Analysis of t-Cinnamic Aldehyde Binding to Human CYP2A6. Spectral binding studies were conducted with recombinant CYP2A6 to evaluate the binding affinity between t-CA and CYP2A6. The spectra indicated that t-CA is a type I ligand of CYP2A6, consistent 2 with a compound deficient in sp hybridized nitrogen atoms. As shown Downloaded from in Fig. 2, fitting the spectral data from seven experiments to a one-site ligand binding model generated a KS = 14.9 6 6.6 mM(N = 7). Mechanism-Based Inhibition of CYP2A6-Mediated Coumarin Hydroxylation by t-CA. Mechanism-based inactivation was evaluated in two systems: recombinant CYP2A6 with cytochrome P450 reductase

and cytochrome b5 (Fig. 3) and human liver microsomes (Fig. 4). In dmd.aspetjournals.org both systems, inhibition was time- and NADPH-dependent (Table 2). The mechanism-based inhibition parameters generated from fitting kobs and t-CA concentration to eq. 1 using nonlinear regression were KI = 21 27.2 mM and kinact = 0.039 minute in recombinant CYP2A6 21 (rCYP2A6), and KI = 18.0 mM and kinact = 0.056 minute in human Fig. 4. (A) Inactivation of coumarin hydroxylase activity as a function of t-CA liver microsomes. The partition ratio for rCYP2A6 was 2600 (Supple- concentration, preincubation period, and NADPH in human liver microsomes (d, buffer; j, NADPH; m,10mM t-CA; .,20mM t-CA; ♦,40mM t-CA; s,80mM mental Fig. 2). u m t-CA; , 120 M t-CA). Each point is the mean of three individual experiments at ASPET Journals on October 2, 2021 Inhibition of CYP2A6-Mediated Letrozole Metabolism by t-CA. conducted on separate days (N = 3 for each point). The percentage of control activity Since substrate-dependent inhibition is often observed with the was determined by comparing the activity to the average activity of samples with xenobiotic-metabolizing P450s (VandenBrink et al., 2012), a second human liver microsomes that did not contain NADPH or t-CA. (B) Determination of k and K by fitting k (min21) to eq. 1. substrate was selected to evaluate NADPH-dependent inhibition of inact I obs CYP2A6 by t-CA. Similar to observations with coumarin hydroxylase respective trapping agent (data not shown). Unexpectedly, the addition activity, formation of 4,49-methanol-bisbenzonitrile from letrozole was of methoxylamine with t-CA enhanced inhibition compared with t-CA inhibited by t-CA in human liver microsomes, and this effect was alone, so a second substrate was evaluated. Methoxylamine had a similar concentration-dependent (Table 3). Inhibition was greatest in the effect on letrozole metabolism. Also, methoxylamine inhibited CYP2A6 presence of both NADPH and t-CA, supportive of mechanism-based in the absence of t-CA. Methoxylamine-containing samples exhibited inhibition, as well as a competitive component, based on the activity 28.2 6 7.1% coumarin hydroxylase activity (5.6 6 1.4 pmol/min/pmol) loss with t-CA in the absence of NADPH. compared with control activity (20.0 6 4.0 pmol/min/pmol; Impact of Nucleophilic Trapping Agents on CYP2A6 Inhibition by t-CA. The effect of glutathione and methoxylamine on CYP2A6 inhibition by t-CA was conducted to evaluate whether the reactive TABLE 2 metabolite formed from t-CA disassociated from the enzyme (Table 4). Coumarin hydroxylase activity following an 18-minute preincubation in rCYP2A6 Also, differences between the two traps can provide indicators of the and human liver microsomes chemical nature of the inhibitor, as glutathione and methoxylamine are Data points are the mean 6 standard deviation of individual experiments conducted on three separate days (N = 3). Control activity (i.e., samples that did not contain NADPH or t-CA) for “soft” and “hard” nucleophiles, respectively. For example, methoxylamine rCYP2A6 = 6.1 6 0.4 pmol/min/pmol; control activity in human liver microsomes = 879 6 reacts with aldehydes to generate stable adducts via Schiff base for- 55 pmol/mg protein/min. mation, and glutathione is more likely to react via conjugate addition Percentage of Control Activity at the b carbon of an a,b-unsaturated carbonyl (Coles, 1984–1985; Preincubation Components rCYP2A6 HLM Prakash et al., 2008; Lopachin et al., 2012). Glutathione (5 mM) moderately prevented CYP2A6 inactivation of coumarin hydroxylase rCYP2A6 or HLM 100 100 + NADPH 91.6 6 3.5 75.8 6 2.8*** activity; the effect was statistically significant. However, controls +80mM t-CA 79.8 6 3.4** 82.2 6 10.9** (without t-CA) were still substantially more active than the glutathione- + NADPH + 10 mM t-CA 84.1 6 4.2** 73.4 6 3.5*** containing samples (43 and 29% greater for the recombinant and + NADPH + 20 mM t-CA 69.3 6 10.8*** 60.8 6 5.8*** m 6 6 microsomal systems, respectively). Attempts were made to detect + NADPH + 40 M t-CA 56.6 3.9*** 48.2 6.8*** + NADPH + 80 mM t-CA 41.1 6 3.5*** 33.3 6 2.0*** glutathione and N-acetyl cysteine conjugates from incubations with + NADPH + 120 mM t-CA 34.9 6 3.4*** 29.1 6 5.3*** rCYP2A6 and t-CA using tandem mass spectrometry and scanning for HLM, human liver microsomes. neutral loss; no conjugates were identified following 60-minute **P , 0.01 in comparison with controls without NADPH and t-CA; ***P , 0.001 in incubations containing CYP2A6 (20 pmol), t-CA (20 mM), and the comparison with controls without NADPH and t-CA. CYP2A6 Inhibition by Cinnamic Aldehyde 539

TABLE 3 predicted for both nicotine and letrozole in the presence of unbound 4,49-Methanol-bisbenzonitrile formation from letrozole following a 9-minute 0.1 mM t-CA, and this increased to 3.3- and 3.7-fold, respectively, at preincubation in human liver microsomes (HLM) unbound 1 mM t-CA. Based on our literature search, the systemic t-CA Data points are the mean 6 standard deviation of individual experiments conducted on two concentration in human blood following oral administration has yet to separate days (N = 6; control activity = 0.74 6 31 pmol/mg protein/min). Percentage of control activity was determined by comparing the activity to samples with HLM that did not contain be reported. In rats, a dose of 250 mg/kg t-CA generated blood NADPH or t-CA. concentrations on the order of 7.6 mM(1mg/ml) (Yuan et al., 1992).

Preincubation Components Percentage of Control Activity Using these data and the body surface area normalization method (Food and Drug Administration, 2005; Reagan-Shaw et al., 2008), the human HLM 100 + NADPH 80.8 6 9.9** equivalent dose to generate a similar blood concentration was estimated + NADPH + 8 mM t-CA 79.2 6 5.3** to be 40.5 mg/kg or 2430 mg of t-CA per day for a 60-kg human. t-CA +80mM t-CA 70.9 6 4.8*** doses of 32 and 320 mg per day were estimated to achieve m 6 + NADPH + 80 M t-CA 51.9 6.9*** concentrations of 0.1 and 1 mM in humans. **P , 0.01 in comparison with controls without NADPH and t-CA; ***P , 0.001 in comparison with controls without NADPH and t-CA. Discussion Human exposure to t-CA is common due to its presence in the diet, three individual experiments conducted in triplicate; N =9;p , fragrances, electronic cigarettes, and use as a complementary treatment of 0.000001).

diabetes and other maladies (Friedman et al., 2000; Adams et al., 2004; Downloaded from Effect of t-CA and NADPH on CYP2A6 CO-Binding Spectra. Bickers et al., 2005; Crawford, 2009). From a toxicological perspective, it The CO-binding spectrum was measured using a reconstituted system contains a well known structural alert (i.e., a,b-unsaturated carbonyl), to investigate how the metabolism of t-CA impacts the level of reacts with skin proteins (Elahi et al.,2004),andisimplicatedincontact functional CYP2A6 (Fig. 5). The intensity of CO-binding spectra from dermatitis (Seite-Bellezza et al., 1994). However, considering the presence incubations containing both t-CA and NADPH (A –A = 0.004; 448 490 of two electrophilic sites, the risk of toxicity upon oral administration is A448–A490 = absorbance at 448 nm minus absorbance at 490 nm) was

relatively low. For example, the no-observed-adverse-effect dose in dmd.aspetjournals.org diminished to 8.5% of the intensity of spectra from incubations with chronic studies was 550 mg/kg per day, and oral LD values ranged t-CA and no NADPH (A –A = 0.047). NADPH in the absence of 50 448 490 from 1160 to 2200 mg/kg in studies of acute toxicity (Bickers et al., 2005). t-CA also diminished the peak intensity of the CO-binding spectrum, Differences in metabolic capacity and blood perfusion rates of skin versus but to a lesser degree (A –A = 0.018), analogous to observations 448 490 liver likely contribute to toxicity being associated more often upon dermal from the activity assays (Tables 2 and 3). The effect of NADPH alone exposure rather than oral administration. The characterization of t-CA was greater in the reconstituted system, likely due to decreased protein metabolism upon oral administration has been reported in several species stability, as compared with the more lipophilic environment present in (Bickers et al., 2005). The major metabolic route involves oxidation to microsomes. at ASPET Journals on October 2, 2021 cinnamic acid, b-oxidation, and conjugation of the acid metabolites. Prediction of t-CA–Mediated Changes in Nicotine and Letrozole Aldehyde dehydrogenase has been suggested as the enzyme responsible AUC. The potential for a drug–t-CA interaction with CYP2A6 for the conversion to cinnamic acid (Bickers et al., 2005), but there are also substrates was estimated with a mechanistic model (Mayhew et al., many examples of P450-mediated metabolism of aldehydes (Raner et al., 2000; Venkatakrishnan and Obach, 2007; Grimm et al., 2009; Mohutsky 1996; Amunom et al., 2007, 2011; Kaspera et al., 2012). and Hall, 2014) that accounts for MBI parameters (K and k ), the I inact Information about the impact of t-CA on human P450 enzymes and fraction of the dose metabolized (f ), and the in vivo degradation rate of m the risk for P450-mediated drug–t-CA diet interactions is limited. Our CYP2A6 (k ) (Yang et al., 2008). High and low values of both k and deg deg results indicate a metabolism-mediated interaction with cinnamon or f were used to estimate the range of possible AUC changes. m t-CA would be markedly more probable for CYP2A6 substrates than for Estimated changes in AUC for nicotine and letrozole in the presence drugs metabolized by the other P450 isoforms typically evaluated of unbound t-CA are shown in Tables 5 and 6. A 1.6-fold change was during drug discovery (Food and Drug Administration, 2012). Even though CYP2A6 and CYP2E1 exhibit overlap in substrate selectivity TABLE 4 (Yoo et al., 1990; Yamazaki et al., 1992; Chen et al., 1998; Harrelson Effect of nucleophilic trapping agents, glutathione or methoxylamine, on the et al., 2007), t-CA was 10.5-fold more selective for CYP2A6. The IC50 inhibition of CYP2A6 by t-CA in microsomal and recombinant systems values for other major drug-metabolizing P450s were at least 16-fold Data points are the mean 6 standard deviation of individual experiments conducted on three higher or more, and in some cases, substantially higher (i.e., 2D6 and separate days (N = 3 and 9) or two separate days (N = 6). The value of N is shown in parentheses. 2C19). Percentage of Remaining Activity The inhibitory mechanism is important in evaluating the likelihood of a metabolism-mediated interaction between a drug and a coadminis- Substrate/Trapping Agent rCYP2A6 HLM tered substance (Wienkers and Heath, 2005; Food and Drug Admin- Coumarin (80 mM t-CA) istration, 2012; Brantley et al., 2014a). Competitive/reversible 6 a 6 b None 41.1 3.5 (3) 31.7 1.9 (6) inhibition was a minor contributor to CYP2A6 inhibition by t-CA. Glutathione (5 mM) 57.4 6 9.5 (3)** 67.5 6 10.9 (6)*** Methoxylamine (1 mM) 0.8 6 0.2 (9)*** 9.5 6 1.5 (9)*** Time- and metabolism-dependent inhibition was a major contributor; Letrozole (80 mM t-CA) this is notable because, even though an inhibitor may not appear to be 6 c 6 d None 48.9 17.2 (9) 70.8 5.0 (9) potent as a reversible inhibitor, clinically relevant interactions can occur Methoxylamine (1 mM) 8.2 6 13.9 (9)*** 45.0 6 16.0 (9)*** due to prolonged inactivation (Mohutsky and Hall, 2014). Time- and HLM, human liver microsomes. metabolism-dependent inhibition often falls into the category of MBI. aControl activity = 5.7 pmol/min/pmol P450. bControl activity = 924.0 6 49.9 pmol/min/mg protein. MBI can involve metabolites coordinating to the heme iron and the cControl activity = 0.62 6 0.04 pmol/min/nmol P450. formation of a metabolite-intermediate complex; alkyl amines or dControl activity = 0.30 6 0.03 pmol/min/mg protein. **P , 0.01 in comparison with samples without trapping agent (i.e., with t-CA and NADPH); methylenedioxybenzene groups, which are not present in t-CA, are ***P , 0.001 in comparison with samples without trapping agent (i.e., with t-CA and NADPH). most often implicated in metabolite-intermediate complex formation. 540 Chan et al.

The mechanism for CYP2A6 degradation, as indicated by the loss in CO-binding spectra, could occur through covalent binding of the metabolite to the apoprotein, to the heme prosthetic group, or destruction of the heme (Hollenberg et al., 2008). Several aldehydes, including the structural analog of t-CA, 3-phenylpropionaldehyde, are susceptible to P450-catalyzed reactions that result in heme modification by radical mechanisms (Raner et al., 1997; Kuo et al., 1999). The decrease in CO-binding spectra without a simultaneous increase in P420 absorption supports a mechanism where it is degradation of the heme rather than covalent modification of the apoprotein (Foti et al., 2011). Recently, evidence for a t-CA epoxide that forms cysteine adducts with a model Fig. 5. Effect of t-CA and NADPH on CYP2A6 CO-binding spectra: 2A6 with hexapeptide was reported (Niklasson et al., 2014). The relevance of the t-CA (red line), 2A6 with NADPH (blue line), and 2A6 with t-CA and NADPH epoxide metabolite to CYP2A6 inhibition is uncertain since the conditions (green line). used to generate adducts in that study were different than the more physiologically relevant conditions used here. t-CA could also be bioactivated by CYP2A6 to a reactive metabolite Other important considerations for predicting potential drug-drug/ (Fig. 6) that covalently binds to the enzyme. The presence of two drug-herb interactions are the fraction of the “victim” drug metabolized electrophilic regions in t-CA suggests that direct covalent modification by the inhibited enzyme (fm) and the concentration of the “perpetrator” Downloaded from (i.e., metabolism independent) of CYP2A6 by t-CA is also a reasonable at the site of inhibition (Brantley et al., 2014a). Due to challenges possibility. associated with accurately measuring the concentration of a perpetrator t-CA contains an a,b-unsaturated aldehyde and is susceptible to at the site of inhibition, a conservative approach is to use systemic conjugate-addition reactions (also known as Michael addition) at the b plasma concentrations—that is, total concentration of free plus bound carbon, or reactions with the carbonyl carbon of the aldehyde, via Schiff drug (Food and Drug Administration, 2012)—whereas unbound

base formation (Prakash et al., 2008). Although CYP2A6 could be concentrations typically provide more accurate predictions (Grime dmd.aspetjournals.org inactivated by direct reaction with one of the enzyme’s nucleophilic and Riley, 2006). Presently, the availability of human pharmacokinetic side chains and t-CA, the results from this study indicate that direct data involving t-CA is unavailable. Thus, a range of concentrations was inactivation is not a major factor. Both NADPH and t-CA were required used for estimating AUC changes for CYP2A6 substrates. A 1.6-fold for potent inhibition of CYP2A6 in two different systems and change was predicted for both nicotine and letrozole in the presence of independent of substrate identity. CO-binding experiments also support 0.1 mM t-CA, and over 3.2-fold change at 1 mM t-CA. The guidance for a metabolism-dependent mechanism where a greater than 90% decrease drug interaction studies recommends an in vivo clinical study for AUC in CO-binding spectra, indicative of a loss of functional CYP2A6, was changes .1.25 (Food and Drug Administration, 2012). only observed in samples containing both t-CA and NADPH. A remaining question is whether humans are exposed to t-CA in at ASPET Journals on October 2, 2021 The results from experiments with glutathione support a metabolism- sufficient quantities to generate meaningful changes in AUC for dependent mechanism for inactivation of CYP2A6, rather than reaction CYP2A6 substrates. Human pharmacokinetic data are not available with a cysteine residue in CYP2A6 and the parent compound. A “soft” for t-CA. The oral doses estimated here (32–320 mg) to achieve blood nucleophile, glutathione could be expected to react with the b carbon of concentrations of 0.1–1 mM in humans are within the t-CA exposure t-CA in the absence of a bioactivation step, similar to a cysteine residue range when cinnamon powder is used for diabetes, based on a method in CYP2A6; however, even high concentrations (5 mM) only had (Reagan-Shaw et al., 2008) to extrapolate pharmacokinetic data from a modest effect. Notably, the concentration of t-CA used here was rats (Yuan et al., 1992) to humans. The estimated range of exposure 12.5-fold lower than the concentration that reacts spontaneously with when cinnamon powder (1–10 g/day) is used for diabetes (Kirkham glutathione and the cytotoxic threshold concentration of t-CA (Swales et al., 2009) is 8–275 mg of t-CA, based on the concentration of t-CA and Caldwell, 1996). in cinnamon powder (Friedman et al., 2000). Considering dietary

TABLE 5 Prediction of AUC changes for nicotine in the presence of increasing concentrations of t-CA Predictions are based on mechanism-based inhibition parameters determined in human liver microsomes.

AUC Fold Increase

21 21 fm kdeg = 0.00061 min (t1/2 = 19 hours) kdeg = 0.00031 min (t1/2 = 37 hours) Range 0.01 mM[t-CA] 0.77 1.04 1.08 1.03–1.08 0.60 1.03 1.06 0.1 mM[t-CA] 0.77 1.35 1.62 1.25–1.62 0.60 1.25 1.43 1 mM[t-CA] 0.77 2.77 3.29 1.99–3.29 0.60 1.99 2.19 10 mM[t-CA] 0.77 3.96 4.14 2.39–4.14 0.60 2.39 2.44 100 mM[t-CA] 0.77 4.17 4.25 2.45–4.25 0.60 2.45 2.48 CYP2A6 Inhibition by Cinnamic Aldehyde 541

TABLE 6 Prediction of AUC changes for letrozole in the presence of increasing concentrations of t-CA Predictions are based on mechanism-based inhibition parameters determined in human liver microsomes.

AUC Fold Increase

21 21 fm kdeg = 0.00061 min (t1/2 = 19 hours) kdeg = 0.00031 min (t1/2 = 37 hours) Range 0.01 mM[t-CA] 0.80 1.04 1.08 1.01–1.08 0.31 1.01 1.01 0.1 mM[t-CA] 0.80 1.37 1.66 1.06–1.66 0.31 1.06 1.11 1 mM[t-CA] 0.80 2.97 3.61 1.27–3.61 0.31 1.27 1.34 10 mM[t-CA] 0.80 4.47 4.71 1.41–4.71 0.31 1.41 1.43 100 mM[t-CA] 0.80 4.76 4.87 1.44–4.87

0.31 1.44 1.45 Downloaded from

exposure, t-CA concentrations in cinnamon-containing foods range (described earlier). Similarly, the results suggest patients with diabetes from 0.05 to 31.1 mg per 100 g. Based on these estimates, a diet high in who use cinnamon and are being treated with letrozole for breast cancer t-CA could also represent a reasonable risk for CYP2A6 modulation. may be at risk for changes in letrozole metabolism that could lead to

For example, 30 g of food (a mass corresponding to approximately a elevated letrozole plasma concentrations. Letrozole is typically dosed dmd.aspetjournals.org slice of bread) with high t-CA content is estimated to generate an daily for several years, and although it is effective, many patients exposure between 7 and 9 mg of t-CA (Friedman et al., 2000). Four to experience musculoskeletal effects that adversely influence medication five servings of food containing high amounts of t-CA could achieve adherence (Henry et al., 2012). The contribution of variable pharma- the 32-mg dose threshold necessary to generate a blood concentration of cokinetics to these processes is an active area of research (Desta et al., 0.1 mM. 2011). Although the mechanisms are poorly understood, individuals In summary, t-CA selectively inhibits CYP2A6 by a metabolism- with type 2 diabetes mellitus have a greater risk of being diagnosed with

dependent mechanism that involves degradation of the functional breast cancer (Hardefeldt et al., 2012). Important next steps for at ASPET Journals on October 2, 2021 enzyme. Substantial changes in AUC were predicted for nicotine and determining the relevance and mechanism of the interaction between letrozole at low t-CA concentrations. A population more likely to t-CA and CYP2A6 include determining the concentration of t-CA (total experience a drug–t-CA interaction are individuals with diabetes who and unbound) in humans upon oral administration of t-CA and/or use cinnamon powder as an approach to lower blood sugar and who also cinnamon powder, the influence of cytosolic enzymes on t-CA stability/ smoke cigarettes or use nicotine replacement therapy, since CYP2A6 potency, and further characterization of CYP2A6 degradation and t-CA activity is known to modulate smoking behavior and cessation metabolism by CYP2A6.

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. 542 Chan et al.

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