Effect of quercetin on the pharmacokinetics of selexipag and its active metabolite in beagles.

CONTEXT: As an inhibitor cytochrome P450 family 2 subfamily C polypeptide 8 (CYP2C8), quercetin is a naturally occurring flavonoid with its glycosides consumed at least 100 mg per day in food. However, it is still unknown whether quercetin and selexipag interact. OBJECTIVE: The study investigated the effect of quercetin on the pharmacokinetics of selexipag and ACT-333679 in beagles. MATERIALS AND METHODS: The ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to investigate the pharmacokinetics of orally administered selexipag (2 mg/kg) with and without quercetin (2 mg/kg/day for 7 days) pre-treatment in beagles. The effect of quercetin on the pharmacokinetics of selexipag and its potential mechanism was studied through the pharmacokinetic parameters. RESULTS: The assay method was validated for selexipag and ACT-333679, and the lower limit of quantification for both was 1 ng/mL. The recovery and the matrix effect of selexipag were 84.5-91.58% and 94.98-99.67%, while for ACT-333679 were 81.21-93.90% and 93.17-99.23%. The UPLC-MS/MS method was sensitive, accurate and precise, and had been applied to the herb-drug interaction study of quercetin with selexipag and ACT-333679. Treatment with quercetin led to an increased in Cmax and AUC0-t of selexipag by about 43.08% and 26.92%, respectively. While the ACT-333679 was about 11.11% and 18.87%, respectively. DISCUSSION AND CONCLUSION: The study indicated that quercetin could inhibit the metabolism of selexipag and ACT-333679 when co-administration. Therefore, the clinical dose of selexipag should be used with caution when co-administered with foods high in quercetin.

Assessment of 25 CYP2D6 alleles found in the Chinese population on propafenone metabolism in vitro.

Cytochrome P450 enzyme 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily, with more than 100 CYP2D6 allelic variants being previously reported. The aim of this study was to assess the catalytic characteristics of 25 alleles (CYP2D6.1 and 24 CYP2D6 variants) and their effects on the metabolism of propafenone in vitro. Twenty-five CYP2D6 alleles were expressing in 21 Spodoptera frugiperda (Sf) insect cells, and each variant was evaluated using propafenone as the substrate. Reactions were performed at 37 °C with 1-100 µmol/L propafenone for 30 min. After termination, the product 5-OH-propafenone was extracted and used for signal collection by ultra-performance liquid chromatography (UPLC). Compared with wild type CYP2D6.1, the intrinsic clearance (Vmax and Km) values of all variants were significantly altered. Three variants (CYP2D6.87, CYP2D6.90, CYP2D6.F219S) exhibited markedly increased intrinsic clearance values (129% to 165%), whereas 21 variants exhibited significantly decreased values (16% to 85%) due to increased Km and (or) decreased Vmax values. These results indicated that the majority of tested alleles had significantly altered catalytic activity towards propafenone hydroxylation in this expression system. Attention should be paid to subjects carrying these rare alleles when treated with propafenone.

The effect of resveratrol on pharmacokinetics of aripiprazole in vivo and in vitro.

1. The objective of this study were to investigate the effect of orally administered resveratrol on the pharmacokinetics of aripiprazole (APZ) in rat, and the inhibitory effects of resveratrol on APZ dehydrogenation activity in liver microsomes and human cytochrome P450 3A4 and 2D6. 2. Twenty-five healthy male Sprague-Dawley rats were randomly divided into five groups: A (control group), B (multiple dose of 200 mg/kg resveratrol), C (multiple dose of 100 mg/kg resveratrol), D (a single dose of 200 mg/kg resveratrol) and E (a single dose of 100 mg/kg resveratrol). A single dose of 3 mg/kg APZ administered orally 30 min after administration of resveratrol. In addition, CYP2D6*1, CYP3A4*1, human and rat liver microsomes were performed to determine the effect of resveratrol on the metabolism of APZ in vitro. 3. The multiple dose of 200 or 100 mg/kg resveratrol significantly increased the AUC and Cmax of APZ. The resveratrol also obviously decreased the CL, but without any significant difference on t1/2 in vivo. On the other hand, resveratrol showed inhibitory effect on CYP3A4*1, CYP2D6*1, human and rat microsomes, the IC50 of resveratrol was 6.771, 87.87, 45.11 and 35.59 µmol l(-1), respectively. 4. Those results indicated more attention should be paid when APZ was administrated combined with resveratrol.

Effect of 24 Cytochrome P450 2D6 Variants Found in the Chinese Population on Atomoxetine Metabolism in vitro.

OBJECTIVE: The aim of this article was to assess the catalytic activities of 24 cytochrome P450 2D6 (CYP2D6) variants found in the Chinese population toward atomoxetine in vitro as well as CYP2D6.1. METHODS: In this study, the co-expression enzyme of human recombinant CYPOR, CYPb5, and CYP2D6.1 or other CYP2D6 variants with the baculovirus-mediated insect cells (Sf21) was used to study the catalytic activities of 24 CYP2D6 variants toward atomoxetine metabolism. The metabolite of atomoxetine (4-hydroxyatomoxetine) was detected by ultra-high performance liquid chromatography-mass spectrometry method. RESULTS: The intrinsic clearance (Vmax/Km) values of most variants were significantly altered when compared with CYP2D6.1. CYP2D6.94, CYP2D6.D336N, CYP2D6.R440C exhibited marked increased values 172, 126, 121% respectively. CYP2D6.89 and CYP2D6.98 exhibited similar catalytic activity as the wild type, whereas 17 variants exhibited significantly decreased values (from 5 to 87%) due to increase Km and/or decrease Vmax values. However, CYP2D6.92 and CYP2D6.96 showed no or few activity because of producing nothing. CONCLUSIONS: Our results suggest that most of these newly found variants exhibit significantly changed catalytic activities compared with the wild type. And these findings provide valuable information for the growth and development of personalized medicine in China.

Effects of 22 CYP2D6 Genetic Variations Newly Identified in Chinese Population on Olanzapine Metabolism in vitro.

The objective of this study was to assess the catalytic activity of 22 novel CYP2D6 allelic variants (2D6*87-*98, R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C and R497C) to olanzapine in vitro. Their protein products expressed in Spodoptera frugiperda 21 (Sf21) insect cells were incubated with olanzapine 100-2,000 µmol/l for 30 min. The kinetic parameters of Km, Vmax and intrinsic clearance were determined by 2-hydroxymethylolanzapine, the metabolite of olanzapine mediated by CYP2D6, using ultra-performance liquid chromatography tandem mass spectrometry. Results showed that the kinetic parameters of 2 alleles, CYP2D6*92 and 2D6*96, could not be detected; 17 allelic variants, CYP2D6*87-*88, 2D6*90-*91, 2D6*93-*95, 2D6*97, R25Q, F164L, E215K, F219S, V327M, V342M, R344Q, R440C and R497C, significantly reduced the intrinsic clearance of olanzapine; 2 variants, CYP2D6*89 and 2D6*98, increased the intrinsic clearance of olanzapine; no difference was found in intrinsic clearance of D336N. Furthermore, 6 alleles, CYP2D6*87, 2D6*88, 2D6*91, 2D6*93, 2D6*97 and R497C, exhibited higher Km values in a range of 120.80-217.56% relative to wild-type CYP2D6*1. The research demonstrated the metabolic phenotype of the 22 novel CYP2D6 variants for olanzapine that were different from probe drugs we used previously and might provide beneficial information to the personalized medicine of olanzapine.

Inhibitory Effect of Apigenin on Pharmacokinetics of Venlafaxine in vivo and in vitro.

OBJECTIVE: This study was conducted to investigate the effects of orally administered apigenin on the pharmacokinetics of venlafaxine (VEN) in rats and on the metabolism of VEN in human and rat liver microsomes in vitro. METHODS: Ten healthy male SD rats were randomly divided into 2 groups: A group (control group), B group (a single dose of 250 mg/kg apigenin). A single dose of 20 mg/kg VEN was administered orally 30 min after administration of apigenin (250 mg/kg). VEN plasma levels were measured by HPLC with fluorescence detection, and pharmacokinetic parameters were calculated by DAS 3.0 software. RESULTS: The single dose of 250 mg/kg apigenin significantly increased the AUC0-t of VEN by 40.9% (p < 0.05) and obviously increased the peak plasma concentration (Cmax) of VEN (p < 0.05). Furthermore, apigenin showed inhibitory effect on human and rat microsomes and the IC50 of apigenin was 58.37 and 25.73 µmol/l, respectively. CONCLUSIONS: Our results indicated that an intake of apigenin could increase VEN plasma levels and some of its pharmacokinetic parameters (AUC, Tmax). Thus, more attention should be paid when VEN was administrated combined with apigenin.

Pharmacokinetics of Anti-rheumatic Drugs Methotrexate and Tofacitinib with its Metabolite M9 in Rats by UPLC-MS/MS.

BACKGROUND: Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. METHODS: Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. RESULTS: The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. CONCLUSION: The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

A high throughput method for Monitoring of Sorafenib, regorafenib, cabozantinib and their metabolites with UPLC-MS/MS in rat plasma.

As multi-targeted tyrosine kinase inhibitors, sorafenib, regorafenib and cabozantinib are widely used in hepatocellular carcinoma (HCC) for systemic therapies with anti-proliferative and anti-angiogenic effects. Nevertheless, adverse effects or insufficient efficacy appear frequently due to the plasma concentration with individual variability of these drugs. To ensure the curative effect and safety by therapeutic drug monitoring (TDM), this study developed a high throughput method to quantify sorafenib, regorafenib, cabozantinib and their active metabolites in plasma simultaneously. The chromatographic separation analysis achievement was performed on a Waters-ACQUITY UPLC BEH C18 column by UPLC-MS/MS system using a gradient elution of solvent A (acetonitrile) and solvent B (water with 0.1% formic acid) in 3.0 min. This method presented satisfactory results of specificity, precision (the intra-day coefficient of variation was between 2.5% and 6.6%, and the inter-day coefficient of variation was between 4.0% and 11.1%) and accuracy (within ±15% for intra-day and inter-day), as well as the stability under certain conditions, the matrix effect in plasma, and extraction recovery (75.6%-94.4%). The linearity of each analyte in the proper concentration scope indicated excellent. This study strictly complied with the performance rules of assay validation in biological medium proposed by FDA and was successfully applied to the pharmacokinetic study in rats. Thus, it would be an advantageous option to research the relationship between concentration-efficacy and concentration-toxic in HCC patients who were supposed to take these medications.

A Novel UPLC-MS/MS Assay for the Measurement of Linezolid and its Metabolite PNU-142300 in Human Serum and its Application to Patients With Renal Insufficiency.

The contribution of the metabolites of linezolid to the associated myelosuppression is unknown in patients who are renal impairment. In this research, the purpose of our experiment was to explore and develop a quick and robust ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the determination of linezolid and its metabolite PNU-142300 in human serum simultaneously. The analytes were prepared using a simple and convenient approach with acetonitrile for protein crash, and then separated from the matrix on a Waters Acquity Ultra performance liquid chromatography (UPLC) BEH C18 (2.1 mm × 50 mm, 1.7 µm) column in a program of gradient elution, where the mobile phase was consisted of water with 0.1% formic acid and acetonitrile, and was placed at 0.40 ml/min flow rate. Multiple reaction monitoring (MRM) was employed and conducted for UPLC-MS/MS detection with ion transitions at m/z 338.01 → 296.03 for linezolid, m/z 369.96 → 327.98 for PNU-142300 and m/z 370.98 → 342.99 for tedizolid (Internal standard, IS), respectively. This method had good linearity respectively in the calibration range of 0.01-20 µg/ml for linezolid, and 0.05-100 µg/ml for PNU-142300. In the intra- and inter-day, the precision of linezolid and PNU-142300 was below 14.2%, and the accuracy in this method was determined to be from -9.7 to 12.8%. In addition, recovery and matrix effect of the analytes were all found to be acceptable, and the analytes during the assay and storage in serum samples were observed to be stable. The novel optimized UPLC-MS/MS assay was also successfully employed to determine the concentration levels of linezolid and PNU-142300 in human serum. The results showed that linezolid-associated myelosuppression occurs more frequently in patients with renal insufficiency, and the metabolite-to-parent concentration ratio of PNU-142300 is predicted to reduce this toxicity of myelosuppression.

The effects of cytochrome P450 2C19 polymorphism on the metabolism of voriconazole in vitro.

BACKGROUND: CYP/CYP450 2C19 (CYP2C19) is a highly polymorphic enzyme and exhibits individual differences in metabolic activity. The purpose of this research was mainly to explore the catalytic activities of 30 CYP2C19 variants on the substrate voriconazole in vitro, including 24 novel CYP2C19 variants (2C19.2E-.2H, .2J, .3C, .29-.33, L16F, 35FS, R124Q, R125G, T130M, N231T, M255T, R261W, N277K, S303N, I327T, N403I, and A430V) found in Chinese Han population for the first time. METHODS: These CYP2C19 variants were expressed in Spodoptera frugiperda (Sf) 21 insect cells using the baculovirus-mediated expression system. The substrate voriconazole was incubated with the abovementioned proteins at 37°C for 30 minutes in an appropriate designed system. Then through detecting its major metabolite voriconazole N-oxide by ultra-performance liquid chromatography tandem mass spectrometry, available data were obtained to explain the influence of CYP2C19 polymorphisms on voriconazole. RESULTS: From the results, when compared to CYP2C19.1, most variants exhibited either reduced Vmax and/or increased Km value, indicating that the intrinsic clearance (Vmax/Km ) values of most variants were significantly altered. The catalytic activities of 20 novel variants exhibited decreases in different degrees compared to CYP2C19.1, with relative clearance values ranging from 1.11% to 83.78%. However, L16F exhibited the increased catalytic activity for 135.68%. In addition, the kinetic parameters of four variants (2C19.2H, .3, 35FS, and R124Q) could not be detected, due to the defective gene. CONCLUSION: This is the first study to report the effects of CYP2C19 polymorphisms on vori-conazole metabolism in vitro, and we hope these data could lay the foundation for the early clinical research and individualized treatment.

Effects of 22 novel CYP2D6 variants found in Chinese population on the metabolism of dapoxetine.

BACKGROUND: CYP2D6 is one of the most important members of the cytochrome P450 superfamily. Its genetic polymorphism significantly influences the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. METHODS AND RESULTS: The aim of this research was mainly to explore the catalytic activities of 22 newly reported CYP2D6 isoforms (2D6*87, *88, *89, *90, *91, *92, *93, *94, *95, *96,*97, *98, *R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C, R497C) on dapoxetine in vitro. The research was designed with an appropriate incubation system in test tubes and carried out in the constant temperature water. Through detecting its two metabolites desmethyldapoxetine and dapoxetine-N-oxide, the available data were obtained to explain the influence of CYP2D6 polymorphism on the substrate drug dapoxetine. As a result, the intrinsic clearance (Vmax/Km) values of most variants were significantly altered when compared with the counterpart of CYP2D6*1, with most of these variants exhibiting either reduced Vmax and/or increased Km values. For dapoxetine demethylation pathway (which produces desmethyldapoxetine), 2D6*89 and E215K exhibited no markedly decreased relative clearance of 92.81% and 97.70%, respectively. The relative clearance of rest 20 variants exhibited decrease in different levels, ranging from 20.44% to 90.90%. For the dapoxetine oxidation pathway (which produces dapoxetine-N-oxide), the relative clearance values of three variants, 2D6*90, *94, and V342M, exhibited no markedly increased relative clearance of 106.17%, 107.78%, and 109.98%, respectively; the rest 19 variants exhibited significantly decreased levels ranging from 27.56% to 84.64%. In addition, the kinetic parameters of two CYP2D6 variants (2D6*92 and 2D6*96) could not be detected, due to the defect of the CYP2D6 gene. CONCLUSION: As the first report of all aforementioned alleles for dapoxetine metabolism, these data may help in the clinical assessment of the metabolic elimination of dapoxetine and may provide fundamental information for further clinical studies.

Effect of 22 Novel Cytochrome P450 2D6 (CYP2D6) Variants Found in the Chinese Population on Hemangeol Metabolism In Vitro.

BACKGROUND AND OBJECTIVES: Hemangeol, approved for the treatment of proliferative infantile hemangiomas requiring systemic therapy, is metabolized by cytochrome P450 2D6 (CYP2D6), which is a highly polymorphic enzyme that metabolizes a large number of drugs. More than 100 CYP2D6 allelic variants have been reported so far, including 22 novel variants that discovered in our lab in the Chinese population. Our study aimed to probe the enzymatic activity of these variants toward hemangeol in vitro with recombinant microsomes that expressed in sf21 insect cells using a baculovirus-mediated expression system. METHODS: The wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) were incubated with 1-200 µM hemangeol for 50 min at 37 °C. Then the products were extracted, and signal detection was performed by high-performance liquid chromatography with fluorescence detector. RESULTS: All of the variants exhibited changed apparent Michaelis-Menten constant (Km) or maximum velocity of the reaction (V max) values compared with that of wild-type protein. The intrinsic clearances (V max /Km) were significantly decreased by 0.37 to 42.74 %. However, CYP2D6.92 and CYP2D6.96 showed no or minimal enzymatic activity as no concentration of 4'-hydroxypropranolol was detected. CONCLUSIONS: The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards hemangeol in vivo.

Evaluation of the Effects of Ketoconazole and Voriconazole on the Pharmacokinetics of Oxcarbazepine and Its Main Metabolite MHD in Rats by UPLC-MS-MS.

Oxcarbazepine (OXC), a second-generation antiepileptic drug, undergoes rapid reduction with formation of the active metabolite 10,11-dihydro-10-hydroxy-carbazepine (MHD) in vivo. In this study, a method for simultaneous determination of OXC and MHD in rat plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS-MS) was developed and validated. Under given chromatographic conditions, OXC, MHD and internal standard diazepam were separated well and quantified by electrospray positive ionization mass spectrometry in the multiple reaction monitoring transitions mode. The method validation demonstrated good linearity over the range of 10-2,000 ng/mL for OXC and 5-1,000 ng/mL for MHD. The lower limit of quantification was 5 ng/mL for OXC and 2.5 ng/mL for MHD, respectively. The method was successfully applied to the evaluation of the pharmacokinetics of OXC and MHD in rats, with or without pretreatment by ketoconazole (KET) and voriconazole (VOR). Statistics indicated that KET and VOR significantly affected the disposition of OXC and MHD in vivo, whereas VOR predominantly interfered with the disposition of MHD. This method is suitable for pharmacokinetic study in small animals.

An UPLC-MS/MS method for the quantitation of vortioxetine in rat plasma: Application to a pharmacokinetic study.

In this work, a simple, sensitive and fast ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitative determination of vortioxetine in rat plasma. Plasma samples were processed with a protein precipitation. The separation was achieved by an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7µm) column with a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The validated method had an excellent linearity in the range of 0.05-20ng/mL (R(2)>0.997) with a lower limit of quantification (0.05ng/mL). The extraction recovery was in the range of 78.3-88.4% for vortioxetine and 80.3% for carbamazepine (internal standard, IS). The intra- and inter-day precision was below 8.5% and accuracy was from -11.2% to 9.5%. No notable matrix effect and astaticism was observed for vortioxetine. The method has been successfully applied to a pharmacokinetic study of vortioxetine in rats for the first time, which provides the basis for the further development and application of vortioxetine.

Simultaneous quantification of carvedilol and its metabolites in rat plasma by ultra performance liquid chromatography tandem mass spectrometry and pharmacokinetic application.

A rapid-resolution ultra high-performance liquid chromatography-tandem mass spectrometry separation method (UPLC-MS/MS) was developed for the simultaneous determination of carvedilol, and its three metabolites: 4'-hydroxyphenyl-carvedilol, 5'-hydroxyphenyl-carvedilol, o-desmethyl-carvedilol. The effective UPLC-MS/MS separation of the examined compounds was applied on an Acquity BEH C18 column (2.1 mm × 5 0 mm, 1.7 µm particle size) column with a gradient mobile phase system. The analysis was performed in less than 6 min with a flow rate of 0.4 mL/min. The assay was validated over concentration ranges of 0.500-100 ng/mL for carvedilol and 0.0500-10.0 ng/mL for its three metabolites. Intra- and inter-assay precision values for replicate quality control samples were within 11.4% for all analytes during the assay validation. Mean quality control accuracy values were within ±11.5% of nominal values for all analytes. Assay recoveries were high (>91%) and internal standard normalized matrix effects were minimal. The four analytes were stable in rat plasma for at least 24h at room temperature, 89 days at -20 °C and -80 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of carvedilol and its pharmacologically active metabolites in rat pharmacokinetic study. The accurate and simple method we developed could be applied to human pharmacokinetic study in the near future.