Guoxin Hu

In vitro assessment of 36 CYP2C9 allelic isoforms found in the Chinese population on the metabolism of glimepiride.

Of the 57 reported CYP2C9 alleles, to date, 36 of them have been identified in the Chinese population. The aim of this study was to assess the catalytic characteristics of these allelic isoforms and their effects on the metabolism of glimepiride in vitro. Baculovirus‐mediated expressing system was used to highly express wild‐type and the 35 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using glimepiride as the substrate. Reactions were performed at 37°C with the insect microsomes and 0.125–10 μM glimepiride for 40 min. After termination, the products were extracted and used for signal collection by LC‐MS/MS. Of the 36 tested CYP2C9 allelic isoforms, only four variants (CYP2C9.40, CYP2C9.47, CYP2C9.51 and CYP2C9.54) exhibited similar relative clearance values to that of wild‐type CYP2C9.1. In addition, one variant (CYP2C9.36) showed a higher intrinsic clearance value than the wild‐type protein, while the remaining 30 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.1% to 87.2%) compared to CYP2C9.1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used antidiabetic drug, glimepiride. Our results indicate that most of the tested rare alleles significantly decrease the catalytic activity of CYP2C9 variants towards glimepiride hydroxylation in vitro.

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

Abstract 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 apigenin on pharmacokinetics of imatinib and its metabolite N-desmethyl imatinib in rats.

The purpose of this study was to determine the effect of apigenin on the pharmacokinetics of imatinib and N-desmethyl imatinib in rats. Healthy male SD rats were randomly divided into four groups: A group (the control group), B group (the long-term administration of 165 mg/kg apigenin for 15 days), C group (a single dose of 165 mg/kg apigenin), and D group (a single dose of 252 mg/kg apigenin). The serum concentrations of imatinib and N-desmethyl imatinib were measured by HPLC, and pharmacokinetic parameters were calculated using DAS 3.0 software. The parameters of AUC(0−t), AUC(0−∞), T max, V z/F, and CLz/F for imatinib in group B were different from those in group A (P < 0.05). Besides, MRT(0−t) and MRT(0−∞) in groups C and D differed distinctly from those in group A as well. The parameters of AUC(0−t) and C max for N-desmethyl imatinib in group C were significantly lower than those in group A (P < 0.05); however, compared with groups B and D, the magnitude of effect was modest. Those results indicated that apigenin in the short-term study inhibited the metabolism of imatinib and its metabolite N-desmethyl imatinib, while in the long-term study the metabolism could be accelerated.

Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro

Abstract CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4′-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.

Effects of Cytochrome P450 2C9 Polymorphism on Bosentan Metabolism

Cytochrome P450 (P450) 2C9 is an important member of the P450 enzyme superfamily, with 58 CYP2C9 allelic variants previously reported. Genetic polymorphisms of CYP2C9 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. The aim of this study was to assess the catalytic activities of 38 human CYP2C9 alleles, including 24 novel alleles (*36–*60) found in the Han Chinese population, toward bosentan (BOS) in vitro. Insect microsomes expressing the 38 CYP2C9 alleles were incubated with 10–625 μM bosentan for 30 minutes at 37°C and terminated by cooling to −80°C immediately. BOS and hydroxyl bosentan, the major metabolite of BOS, were analyzed by ultra-performance liquid chromatography–tandem mass spectrometry system. Thirty-eight defective alleles can be classified into three categories according to the relative clearance value compared with wild type: nine alleles exhibited significantly increased intrinsic clearance values (Vmax/Km) compared with the wild type (1.5-fold–∼4.9-fold relative clearance); nine alleles exhibited significantly reduced intrinsic clearance values compared with the wild type (0.6–28.9% relative clearance). The remaining 20 alleles exhibited no significant difference (1-fold) in enzyme activity compared with the wild type. These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2C9 alleles when administering BOS in the clinic. This is the first report of all these rare alleles for BOS metabolism, providing fundamental data for further clinical studies on CYP2C9 alleles.

Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells

Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05–50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulated Cyp11a1 and Hsd3b1 expression levels at 50 nM. However, 50 μM DBP downregulated Star, Hsd3b1, and Hsd17b3 expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes.

Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC–MS/MS

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of pirfenidone was at 1.39 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 186.2→92.1 for pirfenidone and m/z 237.1→194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5-2000 ng/mL with a lower limit of quantitation (LLOQ) of 5 ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4-84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0mg/kg pirfenidone in rats.

Determination of Morphine and Codeine in Human Urine by Gas Chromatography-Mass Spectrometry

A sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the determination of morphine and codeine in human urine. The GC-MS conditions were developed. The analysis was carried out on a HP-1MS column (30 m × 0.25 mm, 0.25 μm) with temperature programming, and Helium was used as the carrier gas with a flow rate of 1.0 mL/min. Selected ion monitoring (SIM) mode was used to quantify morphine and codeine. The derivation solvent, temperature, and time were optimized. A mixed solvent of propionic anhydride and pyridine (5 : 2) was finally used for the derivation at 80°C for 3 min. Linear calibration curves were obtained in the concentration range of 25–2000.0 ng/mL, with a lower limit of quantification of 25 ng/mL. The intra- and interday precision (RSD) values were below 13%, and the accuracy was in the range 87.2–108.5%. This developed method was successfully used for the determination of morphine and codeine in human urine for forensic identification study.

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.

Inhibitory effect of ketoconazole and voriconazole on the pharmacokinetics of carvedilol in rats

Abstract The aim of this study was to investigate the effect of orally administered ketoconazole and voriconazole on the pharmacokinetics of carvedilol and its metabolites in rats. Fifteen healthy male Sprague–Dawley (SD) rats were randomly divided into three groups: A group (30 mg/kg ketoconazole), B group (30 mg/kg voriconazole) and C group (control group). A single dose of carvedilol was administered orally 30 min after administration of ketoconazole and voriconazole. Carvedilol and its metabolites plasma levels were measured by ultra-high performance liquid chromatography-mass spectrometry method (UPLC–MS/MS), and pharmacokinetic parameters were calculated by DAS 3.0 software. The co-administrated with ketoconazole could significantly increase the maximal plasma concentration (Cmax) and area under the curve (AUC) of carvedilol (p < 0.01). And the Cmax of its three metabolites 4′-hydroxyphenyl carvedilol (4′-HPC), 5′-hydroxyphenyl carvedilol (5′-HPC) and o-desmethyl carvedilol (o-DMC) decreased drastically by 39.4% (p < 0.01), 45.0% (p < 0.01) and 40.8% (p < 0.05), respectively. Following co-administered with voriconazole, Tmax of carvedilol and o-DMC increased, and the Cmax of 5′-HPC decreased by 27.7% (p < 0.05), while other drugs pharmacokinetic parameters performed no significant differences. Therefore, in clinical, when carvedilol was co-administrated with ketoconazole, dose adjustment of carvedilol should be taken into account.