Functional evaluation of CYP2C19 and CYP3A4 gene polymorphism on ibuprofen metabolism.

AIM: Ibuprofen is the most commonly used analgesic. CYP polymorphisms are mainly responsible for the differences in drug metabolism among individuals. Variations in the ability of populations to metabolize ibuprofen can lead to drug exposure events. The aim of this study was to evaluate the effects of CYP2C19 and CYP3A4 polymorphisms on ibuprofen metabolism in a Chinese population. METHODS: First, 31 CYP2C19 and 12 CYP3A4 microsomal enzymes were identified using an insect expression system. Then, variants were evaluated using a mature incubation system. Moreover, ibuprofen metabolite content was determined via ultra-performance liquid chromatography-tandem mass spectrometry analysis. Finally, kinetic parameters of CYP2C19 and CYP3A4 genotypes were determined via Michaelis-Menten curve fitting. RESULTS: Most variants exhibited significantly altered intrinsic clearance compared to the wild type. In the CYP2C19 metabolic pathway, seven variants exhibited no significant alterations in intrinsic clearance (CLint), six variants exhibited significantly high CLint (121-291%), and the remaining 15 variants exhibited substantially reduced CLint (1-71%). In the CYP3A4 metabolic pathway, CYP3A4*30 was not detected in the metabolite content due to the absence of activity, and 10 variants exhibited significantly reduced CLint. CONCLUSION: To the best of our knowledge, this is the first study to assess the kinetic characteristics of 31 CYP2C19 and 12 CYP3A4 genotypes on ibuprofen metabolism. However, further studies are needed on poor metabolizers as they are more susceptible to drug exposure. Our findings suggest that the kinetic characteristics in combination with artificial intelligence to predict the toxicity of ibuprofen and reduce any adverse drug reactions.

Enzymatic activity of 38 CYP2C9 genotypes on ibuprofen.

BACKGROUND AND OBJECTIVE: Ibuprofen, a common non-steroidal anti-inflammatory drug, is used clinically for pain relief and antipyretic treatment worldwide. However, regular or long-term use of ibuprofen may lead to a series of adverse reactions, including gastrointestinal bleeding, hypertension and kidney injury. Previous studies have shown that CYP2C9 gene polymorphism plays an important role in the elimination of various drugs, which leads to the variation in drug efficacy. This study aimed to evaluate the effect of 38 CYP2C9 genotypes on ibuprofen metabolism. METHODS: Thirty-eight recombinant human CYP2C9 microsomal enzymes were obtained using a frugiperda 21 insect expression system according to a previously described method. Assessment of the catalytic function of these variants was completed via a mature incubation system: 5 pmol CYP2C9*1 and 38 CYP2C9 variants recombinant human microsomes, 5 µL cytochrome B5, ibuprofen (5-1000 µM), and Tris-HCl buffer (pH 7.4). The ibuprofen metabolite contents were determined using HPLC analysis. HPLC analysis included a UV detector, Plus-C18 column, and mobile phase [50% acetonitrile and 50% water (containing 0.05% trifluoroacetic acid)]. The kinetic parameters of the CYP2C9 genotypes were obtained by Michaelis-Menten curve fitting. RESULTS: The intrinsic clearance (CLint) of eight variants was not significantly different from CYP2C9*1; four CYP2C9 variants (CYP2C9*38, *44, *53 and *59) showed significantly higher CLint (increase by 35%-230%) than that of the wild-type; the remaining twenty-six variants exhibited significantly reduced CLint (reduced by 30%-99%) compared to that of the wild-type. CONCLUSION: This is the first systematic evaluation of the catalytic characteristics of 38 CYP2C9 genotypes involved ibuprofen metabolism. Our results provide a corresponding supplement to studies on CYP2C9 gene polymorphisms and kinetic characteristics of different variants. We need to focus on poor metabolizers (PMs) with severely abnormal metabolic functions, because they are more susceptible to drug exposure.

Enzymatic activity on valsartan of 38 CYP2C9 variants from the Chinese population.

BACKGROUND AND OBJECTIVE: Valsartan is widely used for the treatment of moderate hypertension. However, previous studies have found that efficacy of the valsartan depends on the dose and intake. Cytochrome P450 (CYP) 2C9 metabolizes ∼15% of the clinical drugs. Genetic polymorphisms of CYP2C9 markedly affect the safety and effectiveness of many drugs, which might lead to adverse reactions and therapeutic failure. Twenty-four novel CYP2C9 variants (*36-*60) had been previously discovered via gene sequencing in the Han population. Our study aims to evaluate the impact of 38 CYP2C9 variants from the Chinese population on valsartan metabolism compared with CYP2C9*1 in vitro. METHODS: Wild-type CYP2C9*1 and other CYP2C9 variants were expressed in Spodoptera frugiperda 21 insect cells. Incubations were performed at 37 °C with 20-2000 µM substrate for 30 min. The metabolite 4-OH valsartan was determined via UPLC-MS/MS. RESULTS: Among the 38 CYP2C9 variants, the enzymatic activities of most variants were significantly altered compared with the wild-type. Three variants (CYP2C9*27, *40 and *49) exhibited increased intrinsic clearance values (134-153% relative clearance). However, 12 variants (CYP *8, *13, *16, *19, *33, *36, *42, *43, *45, *52, *54, *58) caused >90% decreases in the relative clearance of valsartan compared to CYP2C9*1. CONCLUSIONS: Our research provides systematic data for evaluating the effects of CYP2C9 variants on valsartan metabolism in the Chinese population. These results will expand our understanding of the impact of CYP2C9 genetic polymorphisms on valsartan metabolism and will contribute to precision medicine.

Effects of 24 CYP2D6 variants found in Chinese population on the metabolism of clonidine in vitro.

BACKGROUND AND OBJECTIVES: Clonidine has been clinically used to treat Tourette's syndrome for decades. There was research finding that clonidine possessed the best risk-benefit ratio, especially for patients associated with attention deficit hyperactivity disorder. CYP2D6 is a significant member of Cytochrome P450 enzymes. The genetic polymorphisms of CYP2D6 greatly affect the clinical effects of drugs even lead to side effects and medical malpractice. Our goal is to research the effect of CYP2D6 genetic polymorphism on the metabolism of clonidine and evaluate the functions of 22 CYP2D6 allelic variants in vitro, which were discovered in Chinese Han population recently. METHODS: This study was carried out through a mature incubation system. The wild-type CYP2D6*1 and 24 variants (CYP2D6*2, CYP2D6*10 and 22 novel CYP2D6 variants) were expressed in insect cells, and the catalytic activity of all the variants were assessed by substrate clonidine. Metabolite 4-OH clonidine was accurately detected via ultra-performance liquid-chromatography tandem mass spectrometry to evaluate the effect of CYP2D6 genetic polymorphism on the clonidine. RESULT: Among the 22 novel CYP2D6 variants, the intrinsic clearance (Vmax/Km) of 21 variants were significantly decreased (from 1.53% to 83.25%) compared to the wild-type. In particular, the following seven variants (CYP2D6* 2, CYP2D6* 10, CYP2D6* 93, CYP2D6* 95, E215K, V327 M and R497C) attract more attention, of which the intrinsic clearance decreased more than 70% compared to the wild-type. Because the variants with significantly reduced intrinsic clearance are more likely to cause adverse reactions than the variants with increased or little changed intrinsic clearance. In addition, the related pharmacokinetic parameters of CYP2D6*92 and CYP2D6*96 could not be acquired for the defect of CYP2D6 nucleotide. CONCLUSION: We comprehensively evaluated the effect of 22 novel CYP2D6 variants on the metabolism of clonidine for the first time and hoped corresponding data provide a reference for metabolism of clonidine for further studies in vivo, and extend our understanding of the clinical drug toxicity or ineffectiveness by CYP2D6 genetic polymorphism.

An ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of obeticholic acid in rat plasma and its application in preclinical pharmacokinetic studies.

Currently, ursodeoxycholic acid (UDCA) is the only clear medical treatment for primary biliary cholangitis (PBC). However, approximately 40% of patients are not sensitive to UDCA. In recent years, obeticholic acid (OCA) combined with UDCA has been used in the PBC patients who were not sensitive to UDCA, or as monotherapy for PBC adult patients who are intolerant to UDCA. OBJECTIVE: To develop and validate a specific, sensitive and reliable tandem mass spectrometry (UPLC-MS/MS) method for the determination of obeticholic acid (OCA) in rat plasma. METHODS: Plasma samples were treated with liquid-liquid extraction. Diazepam was selected as the internal standard (IS). Chromatographic separation was achieved by an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 µm) and a mobile phase consisting of acetonitrile and ultrapure water (containing 0.1% formic acid). The analyte was detected in positive ion mode by electrospray ionization mass spectrometry (ESI-MS). Multiple reaction monitoring (MRM) methods were used to detect specific precursor and product ions. The target ion pair of OCA was 419.38 → 401.22, and the IS was 285.05 → 193.02. RESULTS: The linear range of OCA in rat plasma was 0.05-50 µg/mL (R2 = 0.992); the recovery rate was 91.34%-97.37%. This assay showed good intra- and inter-day precision and accuracy. No significant matrix effects in this study. CONCLUSION: A specific, sensitive and reliable quantitative analysis method was established to detect OCA after oral/intravenous administration in rat plasma via UPLC-MS/MS. It was appropriate for preclinical pharmacokinetic studies of OCA.

Determination of lesinurad in rat plasma by a UHPLC-MS/MS assay.

Lesinurad is an oral inhibitor of urate-anion exchanger transporter 1 and has been approved by the US Food and Drug Administration for combination therapy with a xanthine oxidase inhibitor for the treatment of hyperuricemia associated with refractory gout. In the present study, a sensitive and specific ultra high-performance liquid chromatography with tandem mass spectrometry assay was established and verified for the determination of lesinurad in rat plasma and was described in details for the first time. Chromatographic separation of lesinurad and diazepam (internal standard, IS) was performed on a Rapid Resolution HT C18 column (3.0 × 100 mm, 1.8 µm) using methanol-water (70:30, v/v) as the mobile phase at a flow rate of 0.3 mL/min. Lesinurad and IS were extracted from plasma by liquid-liquid extraction using ethyl acetate. The mass spectrometric detection was carried out using an electrospray ionization source in positive mode. Multiple reaction monitoring was used for quantification of the precursor to product ion at m/z 405.6 â†’ 220.9 for lesinurad and m/z 285.1 â†’ 192.8 for IS. The assay was well validated for selectivity, accuracy, precision, recovery, linearity, matrix effects, and stability. The verified method was applied to obtain the pharmacokinetic parameters and concentration-time profiles for lesinurad after oral/intravenous administration in rats. The study might provide an important reference and a necessary complement for the qualitative and quantitative evaluation of lesinurad.

Effects of CYP2C19 variants on methadone metabolism in vitro.

CYP2C19 is an important member of the cytochrome P450 (CYP450) enzyme super family and is responsible for clearing approximately 10% of commonly used clinical drugs that undergo phase I metabolism. Genetic polymorphisms of CYP2C19 significantly influence the efficacy and safety of some drugs, which might cause undesirable adverse effects or cure failure at standard dosages. The aim of this study was to clarify the catalytic activities of 31 CYP2C19 alleles on the oxidative in vitro metabolism of methadone. Insect microsomes expressing the CYP2C19 alleles were incubated with 50-2000 µM methadone for 30 min at 37 °C and terminated by cooling to -80 °C immediately. Methadone and its metabolite EDDP were analyzed by an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. Of the 31 tested CYP2C19 allelies variants, CYP2C19*1 is the wild-type. Compared with CYP2C19*1, two CYP2C19 variants (CYP2C19*3 and *35FS) had no detectable enzyme activity, one variant L16F exhibited slightly increased intrinsic clearance values, and one variant N277K showed no significant difference. In addition, 26 variants exhibited significantly decreased values (from 1.48% to 80.40%). These findings suggest that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2C19 alleles. Copyright © 2016 John Wiley & Sons, Ltd.

The Effect of Myricetin on Pharmacokinetics of Atomoxetine and its Metabolite 4-Hydroxyatomoxetine In Vivo and In Vitro.

BACKGROUND AND OBJECTIVES: Atomoxetine is the first non-stimulant drug to be approved for the treatment of ADHD, while the effect of myricetin on the pharmacokinetic of atomoxetine in rats or human is still unknown. The present work was to study the impact of myricetin on the metabolism of atomoxetine both in vivo and in vitro. METHODS: Twenty healthy male Sprague-Dawley rats were randomly divided into four groups: A (control group), B (100 mg/kg myricetin), C (50 mg/kg myricetin), and D (25 mg/kg myricetin). A single dose of atomoxetine (10 mg/kg) was administrated half an hour later. In addition, human and rat liver microsomes were performed to determine the effect of myricetin on the metabolism of atomoxetine in vitro. RESULTS: Group B, C, D all increased the C max and AUC of atomoxetine, but decreased the C max and AUC of 4-hydroxyatomoxetine. Moreover, myricetin showed inhibitory effect on human and rat microsomes, the IC50 of myricetin was 8.651 and 35.45 µmol/L, respectively. CONCLUSIONS: Our study showed that myricetin could significantly inhibit the formation of atomoxetine metabolite both in vivo and in vitro. It is recommended that the effect of myricetin on the metabolism of atomoxetine should be noted and atomoxetine plasma concentration should be monitored.

A rapid and sensitive UHPLC-MS/MS assay for the determination of trelagliptin in rat plasma and its application to a pharmacokinetic study.

This study aims to develop and validate a simple, rapid and sensitive ultra-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for exploring pharmacokinetic characteristics of trelagliptin. Protein precipitation by acetonitrile was used to prepare plasma sample. A RRHD Eclipse Plus C18 (2.1×50mm, 1.8µ) column with gradient mobile phase (containing acetonitrile and 0.1% formic acid) help to achieve the separation of trelagliptin and carbamazepine (IS) with high selectivity. Detection of target fragment ions m/z 358.2→133.9 for trelagliptin, and m/z 237.1→194.0 for IS was performed in positive-ion electrospray ionization mass spectrometry by multiple reaction monitoring. Linear calibration plots were achieved in the range of 5-4000ng/mL for trelagliptin (R(2)=0.999) in rat plasma. The recovery of trelagliptin ranged from 87.8% to 93.7%. The method was showed to be accurate, precise and stable. No obvious matrix effect was found. It has been fully validated and successfully applied to pharmacokinetic study of trelagliptin.

Effect of CYP2D6 genetic polymorphism on the metabolism of citalopram in vitro.

Genetic polymorphisms of CYP2D6 significantly influence the efficacy and safety of some drugs, which might cause adverse effects and therapeutic failure. We aimed at investigating the role of CYP2D6 in the metabolism of citalopram and identifying the effect of 24 CYP2D6 allelic variants we found in Chinese Han population on the metabolism of citalopram in vitro. These CYP2D6 variants expressed by insect cells system were incubated with 10-1000 µM citalopram for 30 min at 37 °C and the reaction was terminated by cooling to -80 °C immediately. Citalopram and its metabolites were analyzed by high-performance liquid chromatography (HPLC). The intrinsic clearance (Vmax/Km) values of the variants toward citalopram metabolites were significantly altered, 38-129% for demethylcitalopram and 13-138% for citalopram N-oxide when compared with CYP2D6*1. Most of the tested rare alleles exhibited significantly decreased values due to increased Km and/or decreased Vmax values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings suggest that more attention should be paid to subjects carrying these CYP2D6 alleles when administering citalopram in the clinic.