Inhibitory Effect of Apigenin on Losartan Metabolism and CYP2C9 Activity in vitro.

BACKGROUND: CYP2C9 is one of the most important phase I drug-metabolizing enzymes in liver. The objective of this work was to investigate the effects of apigenin on the metabolism of losartan and human CYP2C9 and rat CYP2C11 activity in vitro. METHODS: Different concentrations of apigenin were added to a 100 mmol/l Tris-HCl reaction mixture containing 2 pmol/ml recombinant human CYP2C9.1, 0.25 mg/ml human liver microsomes or 0.5 mg/ml rat liver microsomes to determine the half maximal inhibition or a half-maximal inhibitory concentration (IC50) on the metabolism of losartan. In addition, diclofenac used as CYP2C9 substrate was performed to determine the effects of apigenin on CYP2C9. RESULTS: The results showed that apigenin has the inhibitory effect on the metabolism of losartan in vitro, the IC50 was 7.61, 4.10 and 11.07 µmol/l on recombinant CYP2C9 microsomes, human liver microsomes and rat liver microsomes, respectively. Meanwhile, apigenin's mode of action on human CYP2C9 activity was competitive for the substrate diclofenac. In contrast to its potent inhibition of CYP2C9 in humans (9.51 µmol/l), apigenin had lesser effects on CYP2C11 in rat (IC50 = 15.51 µmol/l). CONCLUSION: The observations imply that apigenin has the inhibitory effect on the metabolism of losartan and CYP2C9 activity in vitro. More attention should be paid as to when losartan should be administrated combined with apigenin.

UPLC-MS/MS determination of voriconazole in human plasma and its application to a pharmacokinetic study.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine voriconazole in human plasma. Sample preparation was accomplished through a simple one-step protein precipitation with methanol. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column using an isocratic mobile phase system composed of acetonitrile and water containing 1% formic acid (45:55, v/v) at a flow rate of 0.50 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electrospray ionization source in the positive ion mode. The multiple reaction monitoring transitions of m/z 351.0 → 281.5 and m/z 237.1 → 194.2 were used to quantify voriconazole and carbamazepine (internal standard), respectively. The linearity of this method was found to be within the concentration range of 2.0-1000 ng/mL with a lower limit of quantification of 2.0 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 200 mg voriconazole to 20 Chinese healthy male volunteers.

Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.

The effect of clopidogrel on pharmacokinetics of ivabradine and its metabolite in rats.

The present study aimed to investigate the effect of clopidogrel (CLO) on pharmacokinetics of ivabradine (IVA) and its metabolite in rats and develop a reliable method to determine IVA and its metabolite N-demethyl ivabradine in serum. Healthy male SD rats were randomized to be given 0.8 mg/kg IVA or IVA combined with 8 mg/kg CLO. Blood samples were collected at 0.083, 0.16, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 h after administration. The serum concentrations of IVA and N-demethyl ivabradine were determined by ultra-performance liquid chromatography-mass spectrometry and pharmacokinetic parameters were calculated using DASver3.0 software. The parameters of AUC(0 - t), AUC(0 - ∞), and Cmax for IVA in the group of IVA + CLO were significantly higher than those in the group of IVA (p < 0.01); the half-time (t1/2) in the IVA + CLO group was extended compared to IVA (p < 0.01) and CL/F was dropped obviously (p < 0.01). The decreases in AUC(0 - t), AUC(0 - ∞), and Cmax for N-demethyl ivabradine in the group of IVA + CLO was significantly compared to the group of IVA (p < 0.01). CL/F was higher than IVA (p < 0.01) and the t1/2 was slightly increased. In this study, we find that CLO restrains the metabolism of IVA into N-demethyl ivabradine, which may be related to its competitive inhibition effect on cytochrome P450 isoform 3A4(CYP3A4).

The CYP3A inducer dexamethasone affects the pharmacokinetics of sunitinib by accelerating its metabolism in rats.

Sunitinib, a novel anti-tumor small molecule targeting VEGFR, is prescribed for advanced RCC and GISTs. Sunitinib is primarily metabolized by the CYP3A enzyme. It is well-known that dexamethasone serves as a potent inducer of this enzyme system. Nonetheless, the effect of dexamethasone on sunitinib metabolism remains unclear. This study examined the effect of dexamethasone on the pharmacokinetics of sunitinib and its metabolite N-desethyl sunitinib in rats. The plasma levels of both compounds were measured using UHPLC-MS/MS. Pharmacokinetic parameters and metabolite ratio values were calculated. Compare to control group, the low-dose dexamethasone group and high-dose dexamethasone group decreased the AUC(0-t) values of sunitinib by 47 % and 45 %, respectively. Meanwhile, the AUC(0-t) values of N-desethyl sunitinib were increased by 2.2-fold and 2.4-fold in low-dose dexamethasone group and high-dose dexamethasone group, respectively. The CL values for sunitinib were both approximately 45 % higher in the two dexamethasone groups. Remarkably, metabolite ratio values increased over 5-fold in both low-dose dexamethasone group and high-dose dexamethasone group, indicating a significant enhancement of sunitinib metabolism by dexamethasone. Moreover, the total levels of sunitinib and its metabolite are also significantly increased. The impact of interactions on sunitinib metabolism, as observed with CYP3A inducers such as dexamethasone, is a crucial consideration for clinical practice. To optimize the dosage and prevent adverse drug events, therapeutic drug monitoring can be employed to avoid the toxicity from such interactions.

Determination of CYP450 activities in diabetes mellitus rats by a UHPLC-MS/MS method.

In this study, we investigated the effect of type 1 diabetes mellitus on the modulation of the activities of CYP450s in dynamics by a UHPLC-MS/MS method. The diabetic rat model was constructed by an intraperitoneal single injection of streptozotocin. Fasting blood glucose levels > 16.7 mmol/L were considered as diabetic. The rats were given a cocktail of four probe drugs (10 mg/kg phenacetin, 1 mg/kg tolbutamide, 10 mg/kg metoprolol, and 10 mg/kg midazolam) by oral administration for the pharmacokinetic study. Thereafter, the metabolic ratio (MR) of the metabolites to probe substrates were determined. The results indicated that two weeks after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin (CYP1A2) and 4-hydroxyl tolbutamide/tolbutamide (CYP2C9); however, it decreased the MRs of α-hydroxy metoprolol/metoprolol (CYP2D6) and 1-hydroxy midazolam/midazolam (CYP3A4). Two months after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin and 4-hydroxyl tolbutamide/tolbutamide. The MR of α-hydroxy metoprolol/metoprolol was decreased and the MR of 1-hydroxy midazolam/midazolam was increased but the difference was not significant. According to the results, CYP1A2 and CYP2C9 activities were enhanced in the diabetic rats. and CYP2D6 activity was inhibited in a short period of diabetes; however, the decrease in CYP2D6 activity was not significant in the long period. CYP3A4 activity was decreased in a short period of diabetes and increased in a long period of diabetes but was not significant in the two periods. This study suggests the activity change rule of the CYP450 enzyme system in diabetes mellitus, which can provide a reference for precise clinical medication.

Effects of CYP3A inhibitors ketoconazole, voriconazole, and itraconazole on the pharmacokinetics of sunitinib and its main metabolite in rats.

Sunitinib is a small molecule inhibitor of multiple receptor tyrosine kinases such as platelet derived growth factor receptor, vascular endothelial growth factor receptor, kit receptor and other receptors. The US Food and Drug Administration (FDA) has approved sunitinib for the treatment of advanced renal cell carcinoma and gastrointestinal stromal tumors. It has been reported that sunitinib was mainly metabolized by CYP3A but its pharmacokinetic interactions have not been revealed. In this study, we investigated whether CYP3A inhibitors (ketoconazole, voriconazole, and itraconazole) could influence the pharmacokinetics of sunitinib and its equipotent metabolite N-desethyl sunitinib in a drug-drug interaction study in Sprague Dawley (SD) rats. The results showed that ketoconazole and voriconazole significantly increased the exposure of sunitinib, decreased the exposure of N-desethyl sunitinib, and inhibited the metabolism of sunitinib in rats. However, itraconazole showed only a weak effect on pharmacokinetics and metabolism. Coadministration of sunitinib with ketoconazole and voriconazole should be avoided if possible or if not, there should be therapeutic drug monitoring of the levels of sunitinib and N-desethyl sunitinib. Therefore, drug-drug interaction should be considered when sunitinib is administered in conjunction with CYP3A inhibitors, which might lead to toxicity.

A UHPLC-MS/MS method coupled with liquid-liquid extraction for the quantitation of phenacetin, omeprazole, metoprolol, midazolam and their metabolites in rat plasma and its application to the study of four CYP450 activities.

Drug-drug interactions (DDIs) are thought to be associated with the inhibition of cytochrome P450 activities. The cocktail method with analysis of the metabolism of two or more probe drugs is used to determine CYP450 activities. In this study, we established a UHPLC-MS/MS method for simultaneous quantitation of four CYP450 probe drugs (phenacetin, omeprazole, metoprolol and midazolam) and their metabolites (acetaminophen, 5'-hydroxy omeprazole, α-hydroxy metoprolol and 1'-hydroxy midazolam) in rat plasma. Sample preparation by plasma protein precipitation was combined with a liquid-liquid extraction method. The separation was carried out on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column with a gradient elution, using water containing 0.1% formic acid (A) and acetonitrile (B) in a run time of only 3.0 min. Detection was conducted with a 6420 series triple-quadrupole tandem mass spectrometer, using ESI in positive ion mode with multiple reaction monitoring (MRM). The calibration curves were linear over the concentration range 10-5000 ng/mL for phenacetin, omeprazole, metoprolol and midazolam, and 1-500 ng/mL for their metabolites. Intra- and inter-day precisions were within 15%, and the accuracies were in the range of 87-112%. The method was successfully applied to the pharmacokinetic study of probe drugs/metabolites and DDIs with 3-n-butylphthalide (NBP) after administration of a single oral dose of phenacetin, omeprazole, metoprolol and midazolam in rats.

Determination of Anlotinib, a Tyrosine Kinase Inhibitor, in Rat Plasma by UHPLC-MS/MS and Its Application to a Pharmacokinetic Study.

Anlotinib is a novel inhibitor of receptor kinase tyrosine with multitargets and has a broad spectrum of inhibitory action on tumor angiogenesis and growth. A simple and rapid UHPLC-MS/MS bioanalytical method was validated for the determination of anlotinib in rat plasma, using imatinib as an internal standard. An Acquity BEH C18 column was used to separate analytes. The eluents consisted of formic acid/water (0.1 : 100, v/v) and acetonitrile with a mobile phase. A triple quadrupole mass spectrometer was operated for the quantification with multiple reaction monitoring (MRM) to determine transitions: 408.2 ⟶ 339.1 for anlotinib, and 494.3 ⟶ 394.1 for imatinib. The validated range was 0.1-50 ng/mL for anlotinib. Mean recovery rate of anlotinib in plasma was ≥99.32% and reproducible. Also, the intra- and interday precisions were both below 15%. This robust method was successfully applied to support the pharmacokinetic study of anlotinib in rats.

Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro.

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 µM to 50 µM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95%) compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to personalized medicine.

Pharmacokinetics interaction between imatinib and genistein in rats.

The objective of this work was to investigate the effect of orally administered genistein on the pharmacokinetics of imatinib and N-desmethyl imatinib in rats. Twenty-five healthy male SD (Sprague-Dawley) rats were randomly divided into five groups: A group (control group), B group (multiple dose of 100 mg/kg genistein for consecutive 15 days), C group (multiple dose of 50 mg/kg genistein for consecutive 15 days), D group (a single dose of 100 mg/kg genistein), and E group (a single dose of 50 mg/kg genistein). A single dose of imatinib is administered orally 30 min after administration of genistein (100 mg/kg or 50 mg/kg). The pharmacokinetic parameters of imatinib and N-desmethyl imatinib were calculated by DAS 3.0 software. The multiple dose of 100 mg/kg or 50 mg/kg genistein significantly (P < 0.05) decreased the AUC0-t and C max of imatinib. AUC0-t and the C max of N-desmethyl imatinib were also increased, but without any significant difference. However, the single dose of 100 mg/kg or 50 mg/kg genistein has no effect on the pharmacokinetics of imatinib and N-desmethyl imatinib. Those results indicated that multiple dose of genistein (100 mg/kg or 50 mg/kg) induces the metabolism of imatinib, while single dose of genistein has no effect.

UPLC-MS/MS determination of thiamphenicol in human plasma and its application to a pharmacokinetic study.

A sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to determine thiamphenicol (TAP) in human plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate to precipitation of plasma protein, and to a 0.1 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 354.3→185.1 for TAP and m/z 168.1→132.1 for IS. The linearity of this method was found to be within the concentration range of 10-8000 ng/mL with a lower limit of quantification of 10 ng/mL. Only 1.5 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of TAP in healthy Chinese volunteers after oral administration.