Time‐dependent Inhibition of Cytochrome P450 2A13 (CYP2A13): by Cinnamaldehyde as a Lung Cancer Preventive Agent
Authors: San Brandon S. , Wong Isabelle , Li Jeffery , Nguyen Aiden , Harrelson John 
Published: April 2020

Nicotine is the addictive substance found in various tobacco products CYP2A13, an enzyme localized in the lungs, metabolizes tobacco‐specific nitrosamine carcinogens that contribute to lung cancer. Therefore, pinpointing CYP2A13 inhibitors is an approach to lower tobacco‐based lung cancer risk. Cinnamaldehyde is a common flavoring agent in the fluids of electronic nicotine vaping devices. Cinnamaldehyde was found to be a potent inhibitor of CYP2A6, another enzyme that metabolizes nicotine. Because CYP2A13 and CYP2A6 exhibit overlap in substrate/inhibitor selectivity, the goal here was to evaluate the inhibition of CYP2A13 by cinnamaldehyde. A time‐dependent inhibition coumarin assay was performed to determine the kinetic parameters for cinnamaldehyde in recombinant CYP2A13. Primary incubations contained cinnamaldehyde (5 to 200 micromolar) of (or DMSO for control), CYP2A13 Supersomes, and potassium phosphate buffer. Incubations were initiated with NADPH. Secondary incubations contained coumarin, NADPH, and potassium phosphate buffer. At selected time points (0 to 18 minutes), an aliquot of the primary incubation mixture was transferred to the secondary incubation tubes, which were terminated with trichloroacetic acid after heating at 37 degree C for 5.5 minutes. A linearity study o was conducted to determine the appropriate termination time. CYP2A13 activity was measured by detection of hydroxycoumarin using high performance liquid chromatography (HPLC) and a fluorescence detector. Standard curves of hydroxycoumarin were used to quantify product formation from experimental samples.

Hydroxycoumarin formation decreased with time and inhibitor concentrations. Maximal inhibition following a 18 minute incubation was 38.3 ± 1.6 and 4.0 ± 0.6 % at 20 and 200 micromolar cinnamaldehyde, respectively. The maximal rate of inhibition was 0.109 per minute. The results provide evidence that cinnamaldehyde is a time‐dependent inhibitor of CYP2A13. Furthermore, cinnamaldehyde appears to be a more potent inhibitor of CYP2A13 than CYP2A6, based on the maximal rate of inhibition (0.109 and 0.056 per minute, respectively). The results imply that cinnamaldehyde could interfere with the bioactivation of nitrosamine lung carcinogens.. Additional kinetic studies are needed to confirm the results of this study, and to evaluate the safety and toxicity profiles of cinnamaldehyde in more complex physiological models.

Figure 1

Standard Curve (7‐OH‐Coumarin)

Percent

uM

Area

uM

Stds 1

Stds 2

Average

Change

Stds 1

4

1.1E+07

4

1.1E+07

11028898

11028898

0.0

2

5684098

2

5684098

5684098

5684098

0.0

Slope

2,764,695.99

1

2773892

1

2773892

2784915

2779404

0.4

Y‐intercept

29,108.47

0.5

1410325

0.5

1410325

1425656

1417991

1.1

R2

0.9998

0.25

713352

0.25

713352

724841

719097

1.6

0.125

355993

0.125

355993

364369

360181

2.4

0.0625

181197

0.0625

181197

182438

181818

0.7

0.03125

92767

0.03125

92767

92341

92554

−0.5

Stds 2

4

1.1E+07

AUC

E

20

200

E

20

200

2

5684098

0

966263

897472

321950

873055

768870

272624

1

2784915

3

810370

715528

193710

871857

651272

169610

0.5

1425656

6

862911

554828

144832

3746286

804730

133450

0.25

724841

9

884632

520572

119480

841174

461582

109707

0.125

364369

12

853112

496090

87451

834152

423436

81136

0.0625

182438

15

871382

390425

76171

799519

326200

85743

0.03125

92341

18

855074

279226

66930

761978

340103

60431

OH‐Cou Concentration

E

20

200

E

20

200

0

0.339

0.314

0.106

0.305

0.268

0.088

3

0.283

0.248

0.060

0.305

0.225

0.051

6

0.302

0.190

0.042

1.345

0.281

0.038

9

0.309

0.178

0.033

0.294

0.156

0.029

12

0.298

0.169

0.021

0.291

0.143

0.019

15

0.305

0.131

0.017

0.279

0.107

0.020

18

0.299

0.090

0.014

0.265

0.112

0.011