Peiwu Geng

In vitro functional characterization of 37 CYP2C9 allelic isoforms found in Chinese Han population

Aim:Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for the metabolism of approximately 15% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 37 CYP2C9 allelic isoforms found in Chinese Han population on the metabolism of tolbutamide in vitro.Methods:The wild-type and 36 CYP2C9 variants were expressed in sf21 insect cells using a baculovirus-mediated expression system. Then the insect microsomes were prepared for assessing the metabolic characteristics of each variant toward the CYP2C9-specific drug substrate tolbutamide.Results:Of 36 allelic variants tested, the intrinsic clearance values of 2 allelic isoforms (CYP2C9.36 and CYP2C9.51) were much higher than the wild-type CYP2C9.1 protein, 3 allelic isoforms (CYP2C9.11, CYP2C9.56 and N418T) exhibited similar intrinsic clearance values as the wild-type enzyme, whereas the other 31 variants showed significantly reduced intrinsic clearance values, ranging from 0.08% to 66.88%, for tolbutamide.Conclusion:Our study provides the most comprehensive data concerning the enzymatic activity of the CYP2C9 variants that are present in the Chinese Han population, and our data suggest that most of the carriers of these alleles might be paid more attention when using CYP2C9 mediated drugs clinically.

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.

Effect of 36 CYP2C9 variants found in the Chinese population on losartan metabolism in vitro

Abstract 1.u2002CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, with 57 CYP2C9 allelic variants being previously reported. Among these variants, we recently identified 21 novel alleles (*36–*56) in the Han Chinese population. The aim of this study was to assess the catalytic activities of 36 CYP2C9 variants found in the Chinese population toward losartan in vitro. 2.u2002Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.5–25u2009μM losartan for 30u2009min at 37u2009°C. Next, the products were extracted, and signal detection was performed using high-performance liquid chromatography. 3.u2002Compared with wild-type CYP2C9.1, the intrinsic clearance (Vmax/Km) values of all variants except for CYP2C9.56 were significantly altered. One variant exhibited markedly increased values (>250%), whereas 33 variants exhibited significantly decreased values (from 20 to 96%) due to increased Km and/or decreased Vmax values. 4.u2002These findings suggest that more attention should be paid to subjects carrying these infrequent CYP2C9 alleles when administering losartan in the clinic.

Determination of metoprolol and its two metabolites in human plasma and urine by high performance liquid chromatography with fluorescence detection and its application in pharmacokinetics.

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol and its two metabolites in human plasma and urine. Separation of metoprolol, α-hydroxymetoprolol, O-desmethylmetoprolol and esmolol (internal standard) was achieved on an Agilent XDB-C18 column (150mm×4.6mm, 5μm) using fluorescence detection with Ex 216nm and Em 312nm. The mobile phase consisted of ACN-H2O-0.1%TFA. The analysis was performed in less than 16min with a flow rate of 0.8mL/min. The assay was linear over the concentration range of 5-600ng/mL and 2.5-300ng/mL for metoprolol and its metabolites, respectively. The LOQ were 5.0 and 2.5ng/mL for plasma and urine, respectively. Good precision and accuracy for metoprolol and its two metabolites were obtained. The extraction recoveries were found to be more than 86.91% both in plasma and urine. At the same time, the method was successfully applied to nine healthy volunteers who had been given an oral tablet of 100mg metoprolol.

In vitro functional assessment of 22 newly identified CYP2D6 allelic variants in the Chinese population.

Cytochrome P450 2D6 (CYP2D6) is one of the most widely investigated CYPs related to genetic polymorphisms and is responsible for one‐quarter of the currently used clinical drugs. We previously detected 22 novel, non‐synonymous, mutated sites in the Chinese population, but nothing is known about the functional effects of these mutations in terms of specific CYP2D6 substrates. In this study, wild‐type CYP2D6, two common allelic variants and 22 newly reported CYP2D6 isoforms were transiently expressed in 293FT cells, and the enzymatic activities of these variants were systematically assessed using dextromethorphan and bufuralol as the probing substrates. Consequently, 19 and 21 allelic variants were found to exhibit significantly decreased enzymatic activities for dextromethorphan and bufuralol, respectively. Of 22 novel CYP2D6 variants, six allelic isoforms (CYP2D6.89, CYP2D6.92, CYP2D6.93, CYP2D6.96, E215K and R440C) exhibited absent or extremely reduced metabolic activities compared with those observed for the wild‐type enzyme. Our in vitro functional data can be useful for CYP2D6 phenotype prediction and provide valuable information for the study of clinical impact of these newly found CYP2D6 variants in China.

Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus‐mediated expression system was used to express wild‐type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high‐performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1′‐hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O‐demethylation than those of the wild‐type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild‐type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations.

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 165u2009mg/kg apigenin for 15 days), C group (a single dose of 165u2009mg/kg apigenin), and D group (a single dose of 252u2009mg/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.

Metabolic changes in rat urine after acute paraquat poisoning and discriminated by support vector machine

Paraquat is quick-acting and non-selective, killing green plant tissue on contact; it is also toxic to human beings and animals. In this study, we developed a urine metabonomic method by gas chromatography-mass spectrometry to evaluate the effect of acute paraquat poisoning on rats. Pattern recognition analysis, including both partial least squares discriminate analysis and principal component analysis revealed that acute paraquat poisoning induced metabolic perturbations. Compared with the control group, the levels of benzeneacetic acid and hexadecanoic acid of the acute paraquat poisoning group (intragastric administration 36 mg/kg) increased, while the levels of butanedioic acid, pentanedioic acid, altronic acid decreased. Based on these urinary metabolomics data, support vector machine was applied to discriminate the metabolomic change of paraquat groups from the control group, which achieved 100% classification accuracy. In conclusion, metabonomic method combined with support vector machine can be used as a useful diagnostic tool in paraquat-poisoned rats.

Validated UPLC-MS/MS method for determination of hordenine in rat plasma and its application to pharmacokinetic study.

Hordenine is an active compound found in several foods, herbs and beer. In this work, a sensitive and selective UPLC-MS/MS method for determination of hordenine in rat plasma was developed. After addition of caulophylline as internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 μm) with acetonitrile (containing 10mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 166.1 → 121.0 for hordenine and m/z 205.1 → 58.0 for IS. Calibration plots were linear over the range of 2-2000 ng/mL for hordenine in rat plasma. Mean recoveries of hordenine in rat plasma were in the range of 80.4-87.3%. RSD of intra-day and inter-day precision were both <8%. The accuracy of the method ranged from 97.0% to 107.7%. The method was successfully applied to pharmacokinetic study of hordenine after oral and intravenous administration.

Pharmacokinetic study of dendrobine in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry.

Dendrobine, considered as the major active alkaloid compound, has been used for the quality control and discrimination of Dendrobium which is documented in the Chinese Pharmacopoeia. In this work, a sensitive and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for determination of dendrobine in rat plasma is developed. After addition of caulophyline as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 (2.1 ×100u2009mm, 1.7u2009µm) column with acetonitrile and 0.1% formic acid as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 264.2 → 70.0 for dendrobine and m/z 205.1 → 58.0 for IS. Calibration plots were linear throughout the range 2-1000u2009ng/mL for dendrobine in rat plasma. The RSDs of intra-day and inter-day precision were both <13%. The accuracy of the method was between 95.4 and 103.9%. The method was successfully applied to pharmacokinetic study of dendrobine after intravenous administration. Copyright