Proton-pump inhibitors in patients requiring antiplatelet therapy: new FDA labeling.

Proton-pump inhibitors (PPIs) are recommended for patients who require antiplatelet therapy and have a history of upper gastrointestinal bleeding. Proton-pump inhibitors should also be considered for patients receiving antiplatelet therapy who have other risk factors for gastrointestinal bleeding, including use of aspirin. Thus, evidence of pharmacokinetic and pharmacodynamic interactions between PPIs and consequent impaired effectiveness of the antiplatelet agent clopidogrel has caused concern. Here, we discuss comparative studies suggesting that the extent to which a PPI reduces exposure to the active metabolite of clopidogrel and attenuates its antithrombotic effect differs among PPIs. Although a clinically meaningful effect of the interaction between PPIs and clopidogrel on cardiovascular outcomes has not been established, these studies provided the basis for recent changes in US Food and Drug Administration (FDA) labeling for several PPIs and clopidogrel. New labeling suggests that PPI use among patients taking clopidogrel be limited to pantoprazole, rabeprazole, lansoprazole, or dexlansoprazole. Because comparative studies indicate that omeprazole and esomeprazole have a greater effect on the CYP2C19-mediated conversion of clopidogrel to its active metabolite and, consequently, clopidogrel's effect on platelet reactivity, FDA labeling recommends avoiding omeprazole and esomeprazole in patients taking clopidogrel. Even a 12-hour separation of dosing does not appear to prevent drug interactions between omeprazole and clopidogrel.

Isolation of volatiles from Nigella sativa seeds using microwave-assisted extraction: effect of whole extracts on canine and murine CYP1A.

The volatile components of Nigella sativa seeds were isolated using microwave-assisted extraction (MAE) and identified using gas chromatography. Further investigations were carried out to demonstrate the effects of whole extracts on canine (dog) and murine (rat) cytochrome P450 1A (CYP1A). The optimal extraction conditions of MAE were as follows: 25 mL of water, medium level of microwave oven power and 10 min of extraction time. A total of 32 compounds were identified under the conditions using GC-FID and GC-MS. Thymoquinone (38.23%), p-cymene (28.61%), 4-isopropyl-9-methoxy-1-methyl-1-cyclohexene (5.74%), longifolene (5.33%), α-thujene (3.88) and carvacol (2.31%) were the main compounds emitted from N. sativa seeds. Various extracts including pure compounds, essential oil, nonpolar partition, relatively high-polar/nonpolar partition, and polar partition extracts effectively inhibited the reaction of ethoxyresorufin O-de-ethylation, which is specified for CYP1A activity both in dog and rat. This in vitro data should be heeded as a signal of possible in vivo interactions. The use of human liver preparations would considerably strengthen the practical impact of the data generated from this study.

Pyranoflavones: a group of small-molecule probes for exploring the active site cavities of cytochrome P450 enzymes 1A1, 1A2, and 1B1.

Selective inhibition of P450 enzymes is the key to block the conversion of environmental procarcinogens to their carcinogenic metabolites in both animals and humans. To discover highly potent and selective inhibitors of P450s 1A1, 1A2, and 1B1, as well as to investigate active site cavities of these enzymes, 14 novel flavone derivatives were prepared as chemical probes. Fluorimetric enzyme inhibition assays were used to determine the inhibitory activities of these probes toward P450s 1A1, 1A2, 1B1, 2A6, and 2B1. A highly selective P450 1B1 inhibitor 5-hydroxy-4'-propargyloxyflavone (5H4'FPE) was discovered. Some tested compounds also showed selectivity between P450s 1A1 and 1A2. α-Naphthoflavone-like and 5-hydroxyflavone derivatives preferentially inhibited P450 1A2, while ß-naphthoflavone-like flavone derivatives showed selective inhibition of P450 1A1. On the basis of structural analysis, the active site cavity models of P450 enzymes 1A1 and 1A2 were generated, demonstrating a planar long strip cavity and a planar triangular cavity, respectively.

Pharmacogenomic biomarkers in dermatologic drugs.

Biomarkers are becoming increasingly important when considering the efficacy, toxicology, mechanism of action, and risk of adverse events in certain drugs. As availability of bio-genomic information increases, more treatments can be tailored to specific individuals, with a net effect of improved health outcomes. Many dermatology drugs have pharmacogenomic information on their labels. Knowing the risks and benefits associated with genomic biomarkers can aid physicians to make more knowledgeable decisions when identifying treatments for their patients.

Co-administration of vismodegib with rosiglitazone or combined oral contraceptive in patients with locally advanced or metastatic solid tumors: a pharmacokinetic assessment of drug-drug interaction potential.

PURPOSE: Vismodegib, a first-in-class oral hedgehog pathway inhibitor, is an effective treatment for advanced basal cell carcinoma. Based on in vitro data, a clinical drug-drug interaction (DDI) assessment of cytochrome P450 (CYP) 2C8 was necessary; vismodegib's teratogenic potential warranted a DDI study with oral contraceptives (OCs). METHODS: This single-arm, open-label study included two cohorts of patients with locally advanced or metastatic solid malignancies [Cohort 1: rosiglitazone 4 mg (selective CYP2C8 probe); Cohort 2: OC (norethindrone 1 mg/ethinyl estradiol 35 µg; CYP3A4 substrate)]. On Day 1, patients received rosiglitazone or OC. On Days 2-7, patients received vismodegib 150 mg/day. On Day 8, patients received vismodegib plus rosiglitazone or OC. The effect of vismodegib on rosiglitazone and OC pharmacokinetic parameters (primary objective) was evaluated through pharmacokinetic sampling over a 24-h period (Days 1 and 8). RESULTS: The mean ± SD vismodegib steady-state plasma concentration (Day 8, N = 51) was 20.6 ± 9.72 µM (range 7.93-62.4 µM). Rosiglitazone AUC(0-inf) and C(max) were similar with concomitant vismodegib [≤8% change in geometric mean ratios (GMRs); N = 24]. Concomitant vismodegib with OC did not affect ethinyl estradiol AUC(0-inf) and C(max) (≤5% change in GMRs; N = 27); norethindrone C(max) and AUC(0-inf) GMRs were higher (12 and 23%, respectively) with concomitant vismodegib. CONCLUSIONS: This DDI study in patients with cancer demonstrated that systemic exposure of rosiglitazone (a CYP2C8 substrate) or OC (ethinyl estradiol/norethindrone) is not altered with concomitant vismodegib. Overall, there appears to be a low potential for DDIs when vismodegib is co-administered with other medications.

[Clopidogrel resistance--risk factor in patients with acute coronary syndromes]. / Rezistenta la clopidogrel--factor de risc pentru pacientii cu sindroame coronariene acute.

Clopidogrel is an ADP receptor antagonist, an inhibitor of platelet activation and aggregation. The effect is due to its selective and irreversible blockade of the P2Y12 receptor. The concept of clopidogrel resistance emerged since several years ago. This biological finding has a very important impact especially in patients with acute coronary syndromes, explaining by this their worse evolution. Mechanisms of this resistance are very complex including: differences in intestinal absorption, hepatic conversion to the active metabolite through cytochrome 3A4 (CYP3A4) activity, drug interactions, individual variations in the activity of hepatic cytochrome P450 izoenzymes, platelet receptor polymorphisms and also clinical factors. According to this resistance patients are divided in two groups : "low or non-responders" (with a high risk of thrombosis) and "high--responders" (with a high risk of bleeding). Carriers of CYP2C19*2 or *3 alleles have been shown to respond poorly to standard doses of clopidogrel due to reduced metabolic activation of the drug. So, they are likely to develop subsequent thrombotic events. Starting from these, a lot of studies have been performed in order to elucidate the mechanisms of this resistance offering new perspectives regarding a proper administration of antiplatelet therapy in patients with acute coronary syndromes.

Underlying genetic structure impacts the association between CYP2B6 polymorphisms and response to efavirenz and nevirapine.

OBJECTIVE: CYP2B6 variation predicts pharmacokinetic characteristics of its substrates. Consideration for underlying genetic structure is critical to protect against spurious associations with the highly polymorphic CYP2B6 gene. DESIGN: The effect of CYP2B6 variation on response to its substrates, nonnucleoside reverse transcriptase inhibitors (NNRTIs), was explored in the Women's Interagency HIV Study. METHODS: Five putative functional polymorphisms were tested for associations with virologic suppression within 1 year after NNRTI initiation in women naive to antiretroviral agents (n = 91). Principal components were generated to control for population substructure. Logistic regression was used to test the joint effect of rs3745274 and rs28399499, which together indicate slow, intermediate, and extensive metabolizers. RESULTS: Rs3745274 was significantly associated with virologic suppression [odds ratio = 3.61, 95% confidence interval (CI) 1.16-11.22, P trend = 0.03]; the remaining polymorphisms tested were not significantly associated with response. Women classified as intermediate and slow metabolizers were 2.90 (95% CI 0.79-12.28) and 13.44 (95% CI 1.66 to infinity) times as likely to achieve virologic suppression compared to extensive metabolizers after adjustment for principal components (P trend = 0.005). Failure to control for genetic ancestry resulted in substantial confounding of the relationship between the metabolizer phenotype and treatment response. CONCLUSION: The CYP2B6 metabolizer phenotype was significantly associated with virologic response to NNRTIs; this relationship would have been masked by simple adjustment for self-reported ethnicity. Given the appreciable genetic heterogeneity that exists within self-reported ethnicity, these results exemplify the importance of characterizing underlying genetic structure in pharmacogenetic studies. Further follow-up of the CYP2B6 metabolizer phenotype is warranted, given the potential clinical importance of this finding.

Exposure to potentially dangerous drug-drug interactions involving antipsychotics.

OBJECTIVE: Antipsychotic drug therapy is the cornerstone of treatment of persons with schizophrenia. Because most antipsychotics are metabolized by the hepatic cytochrome P450 system, concomitant use of an antipsychotic and medications that are competitively metabolized by the same system may cause a potentially harmful drug-drug interaction. This study used a large state's Medicaid claims database to examine the proportion of patients exposed to such interactions and the risk factors associated with exposure. METHODS: Claims from January 2000 through December 2003 for adult patients with a diagnosis of schizophrenia and at least one prescription for an antipsychotic (N=27,909) were examined for pairs of medications identified as potentially causing moderate or severe adverse drug effects. Logistic regression models were estimated to determine potential risk factors associated with exposure to the interaction pairs. RESULTS: A total of 6,417 (23%) patients were exposed to 14,213 potentially harmful interactions; 4,725 patients had at least one exposure from the same pharmacy, and 4,032 patients were exposed by the same physician. The greatest number of exposures (N=1,353) to potentially harmful combinations involved olanzapine and haloperidol. Patients prescribed risperidone were most likely to be exposed to an interaction (13.1%), followed by patients prescribed olanzapine (10.3%), quetiapine (3.3%), and clozapine (3.2%). A higher risk of exposure was associated with being female (odds ratio [OR]=.94), being white (OR=1.43), having depression (OR=1.21), or having impulse-control disorder (OR=1.98). CONCLUSIONS: Interventions by physicians and pharmacies to reduce the prescribing and dispensing of potentially harmful pairs of medications to patients with schizophrenia are recommended.

Genetic considerations.

Dual antiplatelet therapy with aspirin and a P2Y(12) receptor antagonist improves outcomes in patients with acute coronary syndrome and in those treated with percutaneous coronary intervention (PCI) and a coronary stent. Candidate gene and genome-wide association studies have found that common genetic polymorphisms of the cytochrome P450 (CYP) 2C19 isoenzyme that result in a loss of functional activity are associated with less exposure of clopidogrel active metabolite and a diminished antiplatelet effect. Meta-analyses of registries and genetic substudies of randomized clinical trials demonstrate that carriers of these polymorphisms who are treated with clopidogrel are at an increased risk of cardiovascular events, particularly stent thrombosis, compared with noncarriers. This deleterious effect appears to be attenuated in patients not treated with PCI. The influence of polymorphisms of other genes, such as ABCB1, is inconsistent across clinical studies. The clinical efficacy of the newer P2Y(12) antagonists prasugrel and ticagrelor do not appear to be affected by the CYP2C19 genotype, but these agents increase major bleeding not related to coronary artery bypass surgery. Although data from randomized clinical trials are currently lacking, these observations suggest that a pharmacogenomic-guided approach to antiplatelet therapy in acute coronary syndrome could potentially maximize ischemic benefit while minimizing bleeding risk.

Polyunsaturated omega-3 fatty acids improve responsiveness to clopidogrel after percutaneous coronary intervention in patients with cytochrome P450 2C19 loss-of-function polymorphism.

BACKGROUND: Antiplatelet properties of omega-3 polyunsaturated fatty acids (PUFA) have been demonstrated in patients with coronary artery disease (CAD). It is unknown whether omega-3 PUFA can enhance platelet inhibition on standard aspirin and clopidogrel treatment in the setting of CYP2C19 loss-of-function polymorphism. AIM: To investigate whether omega-3 PUFA are able to modify platelet responsiveness to clopidogrel therapy in patients with CYP2C19 loss-of-function polymorphism undergoing percutaneous coronary intervention (PCI). METHODS: 63 patients with stable CAD undergoing PCI (48 males, mean age 63.2 ± 9.6 years) were enrolled into an investigator- initiated, prospective, single-centre, double-blind, placebo-controlled, randomised study. Patients on standard dual antiplatelet therapy (aspirin 75 mg daily and clopidogrel 600 mg loading dose followed by 75 mg daily) were assigned to receive the addition of 1 g of omega-3 ethyl esters (n = 33) or placebo (n = 30) for 1 month. Platelet function was measured serially by light transmittance aggregometry in response to 5 and 20 µmol/L ADP at baseline, 12 h, 3-5 days and 30 days after randomisation. CYP2C19*2 was genotyped at baseline. RESULTS: No significant differences were found in baseline variables, including the frequency of CYP2C19 genetic variants. At least one loss-of-function variant of CYP2C19*2 was found in 19 (30.2%) patients. In patients with CYP2C19*1/*2 and *2/*2 variants, maximal platelet aggregation induced by 5 and 20 µmol/L ADP was reduced by 21.4% (p = 0.006) and 14.3% (p = 0.041), respectively, after 1 month of treatment with omega-3 PUFA as compared to placebo. The beneficial effect of omega-3 PUFA was demonstrated in carriers of CYP2C19 loss-of-function polymorphism, whereas no differences in platelet aggregation between the omega-3 PUFA and placebo groups were found in patients with the 1*/1* variant. CONCLUSIONS: The addition of omega-3 ethyl esters significantly potentiates platelet response to clopidogrel after PCI mostly in patients with CYP2C19 loss-of-function polymorphism.

2,3,7,8-Tetrachlorodibenzo-p-dioxin activates the aryl hydrocarbon receptor and alters sex steroid hormone secretion without affecting growth of mouse antral follicles in vitro.

The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis and growth of intact mouse antral follicles in vitro. Specifically, these studies tested the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Since TCDD acts through binding to the aryl hydrocarbon receptor (AHR), and the AHR has been identified as an important factor in ovarian function, we also conducted experiments to confirm the presence and activation of the AHR in our tissue culture system. To do so, we exposed mouse antral follicles for 96 h to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture, which also encompass environmentally relevant and pharmacological exposures (0.1-100 nM), to determine a dose response for TCDD in our culture system for growth, hormone production, and expression of the Ahr and Cyp1b1. The results indicate that TCDD decreases progesterone, androstenedione, testosterone, and estradiol levels in a non-monotonic dose response manner without altering growth of antral follicles. The addition of pregnenolone substrate (10 µM) restores hormone levels to control levels. Additionally, Cyp1b1 levels were increased by 3-4 fold regardless of the dose of TCDD exposure, evidence of AHR activation. Overall, these data indicate that TCDD may act prior to pregnenolone formation and through AHR transcriptional control of Cyp1b1, leading to decreased hormone levels without affecting growth of antral follicles.

Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC50) values ranging from 4.4 to 61.8 µM), with the exception of CYP2E1 (IC50 > 200 µM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 µM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids.

Biomarkers and bioaccumulation of clam Ruditapes philippinarum in response to combined cadmium and benzo[α]pyrene exposure.

Biochemical and molecular biomarkers (the contents of metallothionein (MT), glutathione (GSH), the activities of aryl hydrocarbon hydroxylase (AHH), glutathione S-transferase (GST) and superoxide dismutase (SOD) and the mRNA expressions of GST-pi and Cu, Zn-SOD) were evaluated in clams Ruditapes philippinarum exposed to cadmium (Cd, 15 µg/L) and benzo[α]pyrene (BaP, 0.01 µg/L) individually and in combination (15 µg/L Cd+0.01 µg/L BaP) for 21 days. The accumulation of Cd, BaP and the biomarkers measured in the gills and digestive glands of the clam showed significant increase in combination treatment and it was significantly higher than the Cd or BaP treatment (P>0.05). The contents of MT increased in Cd and Cd+BaP treatment, while AHH activities were increased in Bap and Cd+BaP treatment (P>0.05). GSH levels enhanced in Cd group and declined significantly in Cd+BaP treatment (P>0.05). The activities of GST, SOD, and mRNA expressions of GST-pi, Cu, Zn-SOD increased remarkably in the clams exposed to combined pollutants. In this study, a significant interaction was observed for Cd and BaP accumulation in the clam and the current findings demonstrate the differences in antioxidant response of the biomarkers in clam to single contaminant and the mixtures.

Assessment of a novel ß2-adrenoceptor agonist, trantinterol, for interference with human liver cytochrome P450 enzymes activities.

The effect of a novel ß(2)-adrenoceptor agonist, trantinterol on the activities of cytochrome P450 (CYP450) was investigated with human liver microsomes and human cryohepatocytes in order to assess the potential for drug-drug interactions. The ability of trantinterol to inhibit CYP450 activities was evaluated in vitro in human liver microsomes. Trantinterol did not inhibit CYP2C19, CYP2D6, and CYP3A4/5 (IC(50)>100 µM). It acted as a weak inhibitor of CYP1A2 and CYP2C9 with IC(50) of 70.8 and 81.9 µM, respectively. No time-dependent inhibitions were observed in the present research. To evaluate CYP450 induction, human cryohepatocytes (n=3) were used and treated once daily for 3 days with trantinterol (0.01, 0.1, and 1 ng/ml), after which CYP450 activities were measured. At concentration of 0.01 ng/ml, which is close to the C(max) at maximal recommended doses (50 µg), trantinterol was about 8% as effective as omeprazole (CYP1A2 inducer) only with donor 2. At concentration of 1 ng/ml, trantinterol was about 3.6 ± 3.1% as effective as rifampin (CYP3A4/5 inducer). These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol will not produce clinically significant CYP450 inhibition or induction.

Effects of CYP2C9*1/*13 on the pharmacokinetics and pharmacodynamics of meloxicam.

AIMS: To determine the effects of the CYP2C9*1/*13 genotype on the pharmacokinetics and pharmacodynamics of meloxicam in Korean subjects. METHODS: Meloxicam (15 mg) was orally administered to 21 healthy Korean volunteers with either the CYP2C9*1/*1 or the CYP2C9*1/*13 genotype. Plasma meloxicam concentrations were analysed by HPLC-UV for 72 h after drug administration. The pharmacodynamic effects of meloxicam were determined by measuring TXB(2) generated in blood. RESULTS: The AUC(0,∞) and C(max) of meloxicam were 2.43- and 1.46-fold higher in the CYP2C9*1/*13 group than in the CYP2C9*1/*1 group, respectively. The oral clearance of meloxicam was significantly lower in the CYP2C9*1/*13 group (37.9% of wild type) than in the CYP2C9*1/*1 group. The t(1/2) of meloxicam was 1.84-fold longer in the CYP2C9*1/*13 group than in the CYP2C9*1/*1 group. The rate of TXB(2) production was significantly lower in the CYP2C9*1/*13 group than in the CYP2C9*1/*1 group. CONCLUSIONS: The CYP2C9*1/*13 genotype is associated with decreased metabolism and increased pharmacodynamic effects of meloxicam.

Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats.

Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

In vivo effect of dried chicory root (Cichorium intybus L.) on xenobiotica metabolising cytochrome P450 enzymes in porcine liver.

Cytochrome P450 (CYP) enzymes are widely studied for their involvement in metabolism of drugs and endogenous compounds. In porcine liver, CYP1A2, 2A and 2E1 are important for the metabolism of skatole. Feeding chicory roots to pigs is known to decrease the skatole concentration in plasma and fat. In the present study we investigated the effect of chicory on CYP mRNA and protein expression, as well as their activity. Male pigs were feed dried chicory root for 16 days before liver samples were collected. By the use of RT-PCR and Western blotting we showed that the mRNA and protein expression of CYP1A2 and 2A were increased in chicory fed pigs. The mRNA expression of CYP2E1 was increased, while there was no effect on protein expression. Activity of CYP1A2 and 2A were increased in chicory feed pigs; this was not the case for CYP2E1 activity. In conclusion; oral administration of chicory root for 16 days to pigs increased the mRNA expression of CYP1A2, 2A and 2E1; and the protein expression of CYP1A2 and 2A. The activities of CYP1A2 and 2A were increased.

Flavonoids diosmetin and hesperetin are potent inhibitors of cytochrome P450 2C9-mediated drug metabolism in vitro.

The aim of this study was to examine in vitro, by means of kinetic analysis and molecular docking simulations, the effects of the flavone diosmetin and its flavanone analog hesperetin on CYP (cytochrome P450) 2C9-mediated drug metabolism. To this purpose, the conversion of diclofenac to 4'-hydroxydiclofenac by human liver microsomes was used as a model assay for assessing the CYP2C9 inhibitory activity of these two flavonoids. Kinetic analyses showed that diosmetin and hesperetin were reversible, dead-end inhibitors of 4'-hydroxydiclofenac formation; their mean K(i) (inhibitor dissociation constant) values were 1.71 ± 0.58 and 21.50 ± 3.62 µM, respectively. Diosmetin behaved as a competitive inhibitor, since it increased markedly the K(m) (substrate concentration yielding 50% of V(max)) of the reaction without affecting the V(max) (maximum velocity of reaction). Hesperetin modified markedly K(m) and to a lesser extent also modified V(max), thus acting as a mixed competitive-noncompetitive inhibitor. The results of molecular docking simulations were consistent with those of kinetic analysis, since they showed that the putative binding sites of both diosmetin and hesperetin coincided with the CYP2C9 substrate binding site. The demonstration that diosmetin and hesperetin inhibit CYP2C9-mediated diclofenac metabolism at low micromolar concentrations is of potential clinical relevance because CYP2C9 is responsible for the biotransformation of various therapeutically important drugs that have narrow therapeutic indexes.