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1.
Mol Cell Biochem ; 2023 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-38158493

RESUMO

Hypertrophic scar (HS) formation is a cutaneous fibroproliferative disease that occurs after skin injuries and results in severe functional and esthetic disability. To date, few drugs have shown satisfactory outcomes for the treatment of HS formation. Transforming growth factor-beta (TGF-ß)/Notch interaction via small mothers against decapentaplegic 3 (Smad3) could facilitate HS formation; therefore, targeting TGF-ß/ Notch interaction via Smad3 is a potential therapeutic strategy to attenuate HS formation. In addition, optic atrophy 1 (OPA1)-mediated mitochondrial fusion contributes to fibroblast proliferation, and TGF-ß/Smad3 axis and the Notch1 pathway facilitate OPA1-mediated mitochondrial fusion. Thus, the aim of this study was to investigate whether drugs targeting TGF-ß/Notch interaction via Smad3 suppressed fibroblast proliferation to attenuate HS formation through OPA1-mediated mitochondrial fusion. We found that the TGF-ß pathway, Notch pathway, and TGF-ß/Notch interaction via Smad3 were inhibited by pirfenidone, the gamma- secretase inhibitor DAPT, and SIS3 in human keloid fibroblasts (HKF) and an HS rat model, respectively. Protein interaction was detected by co-immunoprecipitation, and mitochondrial morphology was determined by electron microscopy. Our results indicated that pirfenidone, DAPT, and SIS3 suppressed the proliferation of HKFs and attenuated HS formation in the HS rat model by inhibiting TGF-ß/Notch interaction via Smad3. Moreover, pirfenidone, DAPT, and SIS3 hindered OPA1-mediated mitochondrial fusion through inhibiting TGF-ß/Notch interaction, thereby suppressing the proliferation of HS fibroblasts and HS formation. In summary, these findings investigating the effects of drugs targeting TGF-ß/Notch interaction on HS formation might lead to novel drugs for the treatment of HS formation.

2.
Pharm Biol ; 61(1): 356-361, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36728978

RESUMO

CONTEXT: Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown. OBJECTIVE: To study the interaction mechanism and pharmacokinetics of poziotinib on vonoprazan. MATERIALS AND METHODS: In vitro experiments were performed with rat liver microsomes (RLMs) and the contents of vonoprazan and its metabolite were then determined with UPLC-MS/MS after incubation of RLMs with vonoprazan and gradient concentrations of poziotinib. For the in vivo experiment, rats in the poziotinib treated group were given 5 mg/kg poziotinib by gavage once daily for 7 days, and the control group was only given 0.5% CMC-Na. On Day 8, tail venous blood was collected at different time points after the gavage administration of 10 mg/kg vonoprazan, and used for the quantification of vonoprazan and its metabolite. DAS and SPSS software were used for the pharmacokinetic and statistical analyses. RESULTS: In vitro experimental data indicated that poziotinib inhibited the metabolism of vonoprazan (IC50 = 10.6 µM) in a mixed model of noncompetitive and uncompetitive inhibition. The inhibitory constant Ki was 0.574 µM and the binding constant αKi was 2.77 µM. In vivo experiments revealed that the AUC(0-T) (15.05 vs. 90.95 µg/mL·h) and AUC(0-∞) (15.05 vs. 91.99 µg/mL·h) of vonoprazan increased significantly with poziotinib pretreatment. The MRT(0-∞) of vonoprazan increased from 2.29 to 5.51 h, while the CLz/F value decreased from 162.67 to 25.84 L/kg·h after pretreatment with poziotinib. CONCLUSIONS: Poziotinib could significantly inhibit the metabolism of vonoprazan and more care may be taken when co-administered in the clinic.


Assuntos
Microssomos Hepáticos , Espectrometria de Massas em Tandem , Ratos , Animais , Cromatografia Líquida , Interações Medicamentosas , Microssomos Hepáticos/metabolismo
3.
Proc Natl Acad Sci U S A ; 115(16): 4218-4222, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29610342

RESUMO

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid ß peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.


Assuntos
Guanina/análogos & derivados , Mutagênese/genética , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Peptídeos beta-Amiloides/genética , Anticódon/genética , Pareamento de Bases , Códon sem Sentido , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Técnicas de Silenciamento de Genes , Genes Reporter , Guanina/química , Células HeLa , Humanos , Luciferases/genética , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio
4.
Biochem Biophys Res Commun ; 527(4): 1057-1063, 2020 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-32439176

RESUMO

Salidroside possesses excellent anti-tumor activity in many types of malignant tumor. In present study, we focused on the effects of salidroside on hepatocellular carcinoma (HCC). The viability of human HCC cells was assayed using MTT. Apoptosis in the cells and tissues samples were detected by Annexin V/PI or TUNEL staining assays. The levels of apoptosis and endoplasmic reticulum (ER) stress related proteins were measured by western blotting analysis. We found salidroside significantly suppressed cell viability and promoted apoptosis in HCC cells. Salidroside could activate intrinsic and extrinsic apoptotic pathways, by increasing activities of caspase-3, caspase-8 and caspase-9, up-regulating levels of Bax, Cytochrome c and decreasing level of Bcl-2 in HepG2 cells. Moreover, it was found salidroside induced ER stress and increased expression of p-PERK, eIF2a, p-eIF2a, ATF-6 and CHOP in HepG2 cells. Interestingly, knockdown of CHOP impaired salidroside induced inhibitory effects on HepG2 cells, suggesting the important role of ER stress in cytotoxic effect of salidroside. Finally, we have confirmed salidroside induced ER stress and inhibited development of HepG2 in an xenograft mouse model. In conclusion, our data suggest salidroside inhibits viability and induces apoptosis of HCC both in vitro and vivo, and this effect is partially mediated by activation of ER stress.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucosídeos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Fenóis/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Glucosídeos/química , Glucosídeos/uso terapêutico , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenóis/química , Fenóis/uso terapêutico , Rhodiola/química
5.
FASEB J ; 31(6): 2533-2547, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28242773

RESUMO

Recent studies have shown that KIF5B (conventional kinesin heavy chain) mediates glucose transporter type 4 translocation and adiponectin secretion in 3T3-L1 adipocytes, suggesting an involvement of KIF5B in the homeostasis of metabolism. However, the in vivo physiologic function of KIF5B in adipose tissue remains to be determined. In this study, adipose-specific Kif5b knockout (F-K5bKO) mice were generated using the Cre-LoxP strategy. F-K5bKO mice had similar body weights to controls fed on a standard chow diet. However, F-K5bKO mice had hyperlipidemia and significant glucose intolerance and insulin resistance. Deletion of Kif5b aggravated the deleterious impact of a high-fat diet (HFD) on body weight gain, hepatosteatosis, glucose tolerance, and systematic insulin sensitivity. These changes were accompanied by impaired insulin signaling, decreased secretion of adiponectin, and increased serum levels of leptin and proinflammatory adipokines. F-K5bKO mice fed on an HFD exhibited lower energy expenditure and thermogenic dysfunction as a result of whitening of brown adipose due to decreased mitochondria biogenesis and down-regulation of key thermogenic gene expression. In conclusion, selective deletion of Kif5b in adipose tissue exacerbates HFD-induced obesity and its associated metabolic disorders, partly through a decrease in energy expenditure, dysregulation of adipokine secretion, and insulin signaling.-Cui, J., Pang, J., Lin, Y.-J., Gong, H., Wang, Z.-H., Li, Y.-X., Li, J., Wang, Z., Jiang, P., Dai, D.-P., Li, J., Cai, J.-P., Huang, J.-D., Zhang, T.-M. Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.


Assuntos
Tecido Adiposo/metabolismo , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/fisiologia , Cinesinas/metabolismo , Obesidade/induzido quimicamente , Animais , Intolerância à Glucose , Resistência à Insulina/genética , Cinesinas/genética , Masculino , Camundongos , Camundongos Knockout
6.
Pharmacology ; 100(1-2): 91-97, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28494448

RESUMO

AIMS: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro. METHODS: The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined. RESULTS: Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity. CONCLUSION: In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.


Assuntos
Citocromo P-450 CYP2C19/genética , Fluoxetina/farmacocinética , Inibidores Seletivos de Recaptação de Serotonina/farmacocinética , Alelos , Animais , Povo Asiático/genética , Cromatografia Líquida de Alta Pressão , Fluoxetina/análogos & derivados , Variação Genética , Humanos , Espectrometria de Massas , Células Sf9
7.
Can J Physiol Pharmacol ; 94(8): 895-9, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27203132

RESUMO

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.


Assuntos
Alelos , Antiarrítmicos/metabolismo , Povo Asiático/genética , Citocromo P-450 CYP2D6/genética , Variantes Farmacogenômicos/genética , Propafenona/metabolismo , Animais , Humanos , Insetos , Microssomos/metabolismo
8.
Xenobiotica ; 46(5): 439-44, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26391142

RESUMO

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.


Assuntos
Aripiprazol/farmacocinética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas , Microssomos Hepáticos/efeitos dos fármacos , Estilbenos/farmacocinética , Animais , Antipsicóticos/farmacocinética , Área Sob a Curva , Cromatografia Líquida , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacocinética , Humanos , Concentração Inibidora 50 , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Resveratrol , Espectrometria de Massas em Tandem
9.
Xenobiotica ; 46(5): 424-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26406933

RESUMO

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.


Assuntos
Citocromo P-450 CYP2D6/genética , Variação Genética , Cloridrato de Venlafaxina/metabolismo , Alelos , Animais , Catálise , Células Cultivadas , China , Cromatografia Líquida de Alta Pressão , Succinato de Desvenlafaxina/química , Relação Dose-Resposta a Droga , Humanos , Insetos/citologia , Microssomos/enzimologia , Farmacogenética , Polimorfismo Genético , Isoformas de Proteínas , Temperatura , Cloridrato de Venlafaxina/administração & dosagem
10.
Pharmacology ; 98(3-4): 124-33, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27251229

RESUMO

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.


Assuntos
Antipsicóticos/metabolismo , Povo Asiático/genética , Benzodiazepinas/metabolismo , Citocromo P-450 CYP2D6/genética , Variação Genética/genética , Vigilância da População , Relação Dose-Resposta a Droga , Humanos , Olanzapina , Polimorfismo Genético/genética , Vigilância da População/métodos
11.
Drug Metab Dispos ; 43(8): 1246-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25994031

RESUMO

CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-to-Phe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, Vmax, and/or increased Michaelis constant, Km, values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo.


Assuntos
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Alelos , Substituição de Aminoácidos , Animais , Anticoagulantes/efeitos adversos , Anticoagulantes/farmacocinética , Códon , Variação Genética , Vetores Genéticos , Genótipo , Humanos , Insetos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Pessoa de Meia-Idade , Modelos Moleculares , Polimorfismo Genético , Varfarina/efeitos adversos , Varfarina/farmacocinética
12.
Drug Metab Dispos ; 43(4): 561-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25614704

RESUMO

Our previous study detected totally 35 CYP2C9 allelic variants in 2127 Chinese subjects, of whom 21 novel alleles were reported for the first time in Chinese populations. The aim of the present study was to characterize the 13 CYP2C9 allelic variants both in vitro and in vivo. Different types of CYP2C9 variants were highly expressed in COS-7 cells, and 50 µM tolbutamide was added as the probing substrate to evaluate their metabolic abilities in vitro. Subsequently, the concentrations of tolbutamide and its metabolite in the plasma and urine within individuals with different types of genotypes were determined by HPLC to evaluate the catalytic activity of the 13 mutant CYP2C9 proteins in vivo. Our results showed that compared with *1/*1 wild-type subjects, subjects with *1/*40 genotype showed increased oral clearance (CL/F), whereas individuals with *1/*3, *1/*13, *3/*3, *3/*13, *1/*16, *1/*19, *1/*34, *1/*42, *1/*45, *1/*46, and *1/*48 genotype exhibited significantly decreased CL/F, and those with *1/*27, *1/*29, *1/*40, and *1/*41 genotype presented similar CL/F value. When expressed in COS-7 cells, the CYP2C9 variants showed similar pattern to the results in clinical study. The study suggests that, besides two typical defective alleles, *3 and *13, seven CYP2C9 allelic variants (*16, *19, *34, *42, *45, *46, and *48) cause defective effects on the enzymatic activities both in vitro and in vivo. In clinic, patients with these defective alleles should be paid close attention to.


Assuntos
Alelos , Povo Asiático/genética , Citocromo P-450 CYP2C9/genética , Frequência do Gene , Variação Genética , Animais , Área Sob a Curva , Células COS , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Frequência do Gene/genética , Genética Populacional , Genótipo , Humanos , Plasmídeos , Tolbutamida/sangue , Tolbutamida/urina , Transfecção
13.
Xenobiotica ; 45(11): 1030-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26153442

RESUMO

1. CYP2C19 is a highly polymorphic enzyme responsible for the metabolism of a wide range of clinical drugs. Alterations to the CYP2C19 gene contribute to the variability of CYP2C19 enzyme activity, which causes pharmacokinetics and drug efficacies to vary and adverse drug reactions to occur in different persons. Recently, we identified 24 novel CYP2C19 allelic variants in the Chinese Han population. The purpose of present study is to assess the impact of these newly found nucleotide mutations on the enzymatic activity of the CYP2C19 protein. 2. Dual-expression vectors were constructed and transiently transfected into 293FT cells. Forty-eight hours after transfection, cells were re-suspended and incubated with two typical probe substrates, omeprazole and S-mephenytoin, to determine the activities of each variant relative to the wild-type protein. 3. Immunoblotting results showed that the protein expression levels of the CYP2C19 variants were diverse. Enzymatic ability analysis showed that the variant 35FS exhibited no functional activity, and most of the other variants showed significantly decreased metabolic activities toward both omeprazole and S-mephenytoin compared with wild-type. 4. These findings greatly enrich the knowledge of biological effects of these newly found CYP2C19 mutations and aid the application of this knowledge to future individualized drug therapy in clinic.


Assuntos
Alelos , Povo Asiático/genética , Citocromo P-450 CYP2C19 , Mutação , Omeprazol/farmacocinética , Povo Asiático/etnologia , Linhagem Celular , China/etnologia , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Feminino , Humanos , Masculino , Omeprazol/farmacologia
14.
Pharmacology ; 95(5-6): 218-23, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25924705

RESUMO

BACKGROUND: Mestranol is a widely used estrogen, which is converted into its active metabolite ethinyl estradiol by cytochrome P450 (CYP) 2C9. To comprehensively examine the enzymatic activity of reported CYP2C9 variants in Chinese individuals in response to mestranol, wild-type CYP2C9*1 and 35 allelic variants were highly expressed in Sf21 insect cell microsomes and used for the detection of their enzymatic values in vitro. These results showed that the majority of tested variants exhibited decreased clearance values compared to wild type, except for CYP2C9*40 and *36. METHOD: Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.25-8 µmol/l mestranol for 30 min at 37°C. Then, the production of the metabolite of mestranol, ethinyl estradiol, was analyzed using high-performance liquid chromatography. RESULTS: Most CYP-catalyzed reactions were sufficiently described by classical Michaelis-Menten kinetic parameters (e.g., Km and Vmax), while 9 variants exhibited atypical or non-Michaelis-Menten kinetic values, which were largely due to the self-inhibitory effect in response to mestranol. CONCLUSION: This is the first report of these rare alleles for mestranol metabolism, which provides fundamental data for further clinical studies on CYP2C9 alleles for mestranol metabolism.


Assuntos
Povo Asiático/genética , Citocromo P-450 CYP2C9/genética , Estrogênios/metabolismo , Mestranol/metabolismo , Animais , Humanos , Insetos , Microssomos/metabolismo , Polimorfismo Genético
15.
Pharmacology ; 96(5-6): 290-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26544071

RESUMO

AIMS: Cytochrome P450 (CYP450) 2D6 is an important member of the P450 enzyme superfamily and responsible for clearing 25% of clinically important drugs. The aim of this study was to assess the catalytic characteristics of 24 CYP2D6 allelic isoforms found in the Chinese population and their effects on the metabolism of risperidone in vitro. METHODS: Insect microsomes expressing wild-type CYP2D6 and 24 CYP2D6 allelic variants were incubated with 20-1,000 µmol/l risperidone for 40 min at 37°C. After termination, risperidone and 9-OH risperidone, the metabolite of risperidone, were precipitated and used for signal collection by ultra-performance liquid-chromatography tandem mass spectrometry. RESULTS: Among 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be with no detectable activity. Two variants (E215K and R440C) exhibited higher intrinsic clearance values than the wild-type protein, while the remaining 20 CYP2D6 allelic variants exhibited significantly decreased clearance values (2.01-87.56%) compared to CYP2D6*1. CONCLUSION: These findings suggest that more attention should be directed to subjects carrying these infrequent CYP2D6 alleles when administering risperidone in the clinic. This is the first report of all these novel alleles for risperidone metabolism, providing fundamental data for further clinical studies on CYP2D6 alleles.


Assuntos
Antipsicóticos/metabolismo , Povo Asiático/genética , Citocromo P-450 CYP2D6/genética , Polimorfismo Genético , Risperidona/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas In Vitro , Insetos , Microssomos/enzimologia , Microssomos/metabolismo , Espectrometria de Massas em Tandem
16.
Drug Dev Ind Pharm ; 41(8): 1363-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25144335

RESUMO

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.


Assuntos
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Flurbiprofeno/metabolismo , Polimorfismo Genético/fisiologia , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Humanos , Insetos
17.
J Pharmacol Sci ; 125(2): 150-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25075423

RESUMO

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.


Assuntos
Anticoagulantes/administração & dosagem , Citocromo P-450 CYP2C9/genética , Estudos de Associação Genética , Erros Inatos do Metabolismo/genética , Mutação Puntual/genética , Varfarina/administração & dosagem , Idoso , Alelos , Substituição de Aminoácidos/genética , Anticoagulantes/metabolismo , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/metabolismo , Resistência a Medicamentos/genética , Feminino , Variação Genética , Humanos , Microssomos/enzimologia , Varfarina/metabolismo
18.
Neurol Sci ; 35(4): 531-6, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24057116

RESUMO

The objective of the study was to investigate the role of neuregulin-ErbB signaling in neuropathic pain in different types of injury. Neuregulin-1(NRG-1) was injected into animals with either formalin-induced pain model or spared nerve injury (SNI) model. Formalin tests or paw withdrawal tests were performed to study the role of NRG-1 in neuropathic pain. siRNA specific to different erbB receptors were then introduced to test which specific signaling pathway was required for NRG-1 signaling in the different pain models. NRG-1 inhibits neuropathic pain after SNI in a dose-dependent manner, while NRG-1 aggravates formalin-induced neuropathic pain. ErbB2 and erbB4 receptors were activated after neuregulin administration. Knockdown of ErbB2 relieves the aggravation of NRG-1 on formalin-induced neuropathic pain, and knockdown of ErbB4 could relieve the inhibition of NRG-1 on neuropathic pain in the SNI model. NRG-1 has two distinct functions depending on the different receptor activation in different models of neuropathic pain. These novel findings may provide new therapeutic approaches for the treatment of neuropathic pain in different injury types.


Assuntos
Neuralgia/metabolismo , Neuregulina-1/fisiologia , Receptor ErbB-2/metabolismo , Animais , Formaldeído , Hiperalgesia/complicações , Hiperalgesia/metabolismo , Masculino , Neuralgia/induzido quimicamente , Neuralgia/complicações , Neuregulina-1/farmacologia , Ratos
19.
Xenobiotica ; 44(3): 270-5, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23844998

RESUMO

Abstract 1. CYP2C9 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. Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.5-25 µM losartan for 30 min at 37 °C. Next, the products were extracted, and signal detection was performed using high-performance liquid chromatography. 3. Compared 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. These findings suggest that more attention should be paid to subjects carrying these infrequent CYP2C9 alleles when administering losartan in the clinic.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/farmacocinética , Povo Asiático/genética , Variação Genética/genética , Losartan/metabolismo , Análise de Variância , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2C9 , Humanos , Técnicas In Vitro , Cinética , Microssomos/metabolismo
20.
Int J Clin Pharmacol Ther ; 52(9): 732-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24986093

RESUMO

OBJECTIVE: Co-administration of anti-hypertension and anti-diabetic drugs is common in clinical settings. METHODS: In this study, we characterized the drug-drug interactions of losartan (LOS) and glimepiride (GLP) using recombinant cytochrome P450 (CYP) 2C9 enzymes (CYP2C9*1, CYP2C9*3, CYP2C9*13, and CYP2C9*16). RESULTS: Metabolism of losartan by recombinant CYP2C9* 1, CYP2C9*3, CYP2C9*13, and CYP2C9* 16 was inhibited by glimepiride competitively with IC50 values of 0.669 ± 0.055 µM, 0.424 ± 0.032 µM, 2.557 ± 0.058 µM, and 0.667 ± 0.039 µM, respectively. The inhibitory effect of glimepiride on losartan metabolism by CYP2C9*13 was marginal. The apparent Ki value of glimepiride with CYP2C9*3 (0.0416 ± 0.0059 µM) was significantly lower than with CYP2C9*1 (0.1476 ± 0.0219 µM) and CYP2C9*16 (0.2671 ± 0.0456 µM). On the other hand, losartan weakly inhibited the hydroxylation of glimepiride by P450 2C9 enzymes competitively. The potencies for inhibition of glimepiride hydroxylation were determined to be CYP2C9*1~CYP2C9*3~CYP2C9*16 > CYP2C9*13 by 4 µM losartan. No significant inhibition was observed when 0.5 µM losartan was used. CONCLUSIONS: Given these results, the potential inhibition of losartan metabolism by CYP2C9*3, CYP2C9*13, and CYP2C9*16 in vivo by glimepiride deserves further investigation. These results may provide valuable information for optimizing the anti-hypertension efficacy of losartan when glimepiride is co-administered to patients.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hipoglicemiantes/metabolismo , Losartan/metabolismo , Microssomos/enzimologia , Polimorfismo Genético , Compostos de Sulfonilureia/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/efeitos adversos , Hidrocarboneto de Aril Hidroxilases/genética , Biotransformação , Citocromo P-450 CYP2C9 , Interações Medicamentosas , Humanos , Hidroxilação , Hipoglicemiantes/efeitos adversos , Cinética , Losartan/efeitos adversos , Oxirredução , Farmacogenética , Fenótipo , Proteínas Recombinantes/metabolismo , Medição de Risco , Fatores de Risco , Compostos de Sulfonilureia/efeitos adversos
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