Objective: An in vivo cocktail probe drug method was conducted to evaluate the effect of luteolin on CYP450 enzyme activity. Methods: The rats were randomly divided into two groups, the control and the luteolin groups were respectively given gavage administration of CMC-Na (2.0 mL·kg-1) and luteolin (30.0 mg·kg-1) for 7 successive days. On the 7th day, all rats were injected intraperitoneally with the mixed solution of 3 probe drugs (10.0 mg·kg-1 of phenacetin and testosterone and 2.5 mg·kg-1 of tolbutamide), the plasma samples were collected at different time points and precipitated with isopropanol, then determined by LC-MS/MS. The chromatographic separation was achieved on a ZORBAX SB-C18(2.1 mm×30 mm, 3.5 μm) column by gradient elution of acetonitrile-0.1% formic acid in water, and the MS instrument was operated in multiple reaction monitoring (MRM) with positive and negative electrospray ionization (ESI) modes. The UHPLC-MS/MS method was established for the determination of the concentrations of three probe drugs in plasma, the acquired data were calculated for pharmacokinetic parameters by using DAS software subsequently. Further, the protein expression levels of the three CYP450 enzymes were tested by western blot. Results: Methodological results suggested that the linear range of phenacetin, testosterone, and tolbutamide were in the concentration of 2.0-2 000.0 ng·mL-1, 0.3-300.0 ng·mL-1, 1.7-1 700.0 ng·mL-1, and the lower limit of quantification (LLOQ) were 2.0 ng·mL-1, 0.3 ng·mL-1, and 1.7 ng·mL-1, respectively. For all analytes, the accuracy was in the range of 96.9%-108.0%, and the RSD of intra-day and inter-day precision were within 8.9%. The extraction recovery, matrix effect and stability all met the criteria. The pharmacokinetic results showed that luteolin could significantly reduce the AUC value of phenacetin, and increase the AUC value of tolbutamide. But there was few significant variation in pharmacokinetic parameters of testosterone. The western blot results showed that the expression of CYP1A2 was significantly induced by luteolin, while the levels of CYP2C9 and CYP3A4 were inhibited, which verified the LC-MS/MS results. Conclusion: The luteolin is a moderate inducer on CYP1A2 and a weak inhibitor on CYP2C9, but has no significant effect on CYP3A4 according to the FDA guidance document.
SONG Ke-xin, YI Jie, GAO Lin, REN Ji-ping, LI Qian
. Investigation on effect of luteolin on CYP450 enzymes in rats using cocktail method*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(2)
: 279
-286
.
DOI: 10.16155/j.0254-1793.2022.02.12
[1] JUSZCZAK AM, ZOVKO-KONČIĆ M, TOMCZYK M. Recent trends in the application of chromatographic techniques in the analysis of luteolin and its derivatives[J].Biomolecules, 2019, 9(11): 731
[2] NABAVI SF, BRAIDY N, GORTZI O, et al. Luteolin as an anti-inflammatory and neuroprotective agent: a brief review[J].Brain Res Bull, 2015, 119(Pt A): 1
[3] FAN XY, DU KX, LI N, et al. Evaluation of anti-nociceptive and anti-inflammatory effect of luteolin in mice[J].J Environ Pathol Toxicol Oncol, 2018, 37(4): 351
[4] RAKARIYATHAM K, WU X, TANG Z, et al. Synergism between luteolin and sulforaphane in anti-inflammation[J].Food Funct, 2018, 9(10): 5115
[5] IMRAN M, RAUF A, ABU-IZNEID T, et al. Luteolin, a flavonoid, as an anticancer agent: a review[J].Biomed Pharmacother, 2019, 112: 108612
[6] LIN Y, SHI R, WANG X, et al. Luteolin, a flavonoid with potential for cancer prevention and therapy[J].Curr Cancer Drug Targets, 2008, 8(7): 634
[7] YANG SC, CHEN PJ, CHANG SH, et al. Luteolin attenuates neutrophilic oxidative stress and inflammatory arthritis byinhibiting raf1 activity[J].Biochem Pharmacol, 2018, 154: 384
[8] WEI B, LIN Q, JI YG, et al. Luteolin ameliorates rat myocardial ischaemia-reperfusion injury through activation of peroxiredoxin Ⅱ[J].Br J Pharmacol, 2018, 175(16): 3315
[9] YANG JT, WANG J, ZHOU XR, et al. Luteolin aleviates cardiac ischemia/reperfusion injury in the hypercholesterolemic rat via activating Akt/Nrf2 signaling[J].Naunyn Schmiedebergs Arch Pharmacol, 2018, 391(7): 719
[10] FENG S, HE X. Mechanism-based inhibition of CYP450: an indicator of drug-induced hepatotoxicity[J].Curr Drug Metab, 2013, 14(9): 921
[11] ALMAZROO OA, MIAH MK, VENKATARAMANAN R. Drug metabolism in the liver[J].Clin Liver Dis, 2017, 21(1):1
[12] FUHR U, JETTER A, KIRCHHEINER J. Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the “cocktail” approach[J].Clin Pharmacol Ther, 2007, 81(2): 270
[13] DANIELSON PB. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans[J].Curr Drug Metab, 2002, 3(6): 561
[14] DMITRIEV AV, LAGUNIN AA, KARASEV DA, et al. Prediction of drug-drug interactions related to inhibition or induction of drug-metabolizing enzymes[J].Curr Top Med Chem, 2019, 19(5): 319
[15] MARTINY VY, MITEVA MA. Advances in molecular modeling of human cytochrome p450 polymorphism[J].J Mol Biol, 2013, 425(21): 3978
[16] FDA-2017-D-5961 In Vitro Drug Interaction Studies —Cytochrome p450 Enzyme- and Transporter-Mediated Drug Interactions[S].2020: 20
[17] SPAGGIARI D, DAALI Y, RUDAZ S. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure[J].Toxicol Appl Pharmacol, 2016, 302: 41
[18] OTTEN JN, HINGORANI GP, HARTLEY DP, et al. An in vitro, high throughput, seven cyp cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS[J].Drug Metab Lett, 2011: 5(1): 17
[19] CHEN L, VAN BREEMEN RB. Validation of a sensitive UHPLC-MS/MS method for cytochrome P450 probe substrates caffeine, tolbutamide, dextromethorphan, and alprazolam in human serum reveals drug contamination of serum used for research[J].J Pharm Biomed Anal, 2020, 179: 112983
[20] SAMY RP, GOPALAKRISHNAKONE P, IGNACIMUTHU S. Anti-tumor promoting potential of luteolin against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats[J].Chem Biol Interact, 2006, 164(1-2):1
[21] YANG MD, LIN SY, CHIU TH, et al. Effects of luteolin on distribution and metabolism of 2-aminofluorene in male Sprague-Dawley rats[J].In Vivo,2008, 22(6):729
[22] FDA-2017-D-5961 Clinical Drug Interaction Studies-Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry[S].2020: 18
