OBJECTIVE Lithocholic acid,which is a secondary bile acid,has been reported to be hepatotoxic and carcinogenic.It is metabolized by human cytochrome P450 3A(CYP3A)to form 3-ketocholanoic acid.A previous study suggests...OBJECTIVE Lithocholic acid,which is a secondary bile acid,has been reported to be hepatotoxic and carcinogenic.It is metabolized by human cytochrome P450 3A(CYP3A)to form 3-ketocholanoic acid.A previous study suggests that vitamin E isomers(tocotrienols and tocopherols)are metabolized by CYP3 A.Given that substrates of an enzyme may competitively inhibit the enzyme,we determined whether alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,tocotrienol-rich mixture(a mixture consisting of 25.7% d-α-tocotrienol,2.6% d-β-tocotrienol,28.6% d-γ-tocotrienol,8.4% d-δ-tocotrienol,25.6% d-α-tocopherol,and 4.3% d-α-tocomonoenol),and alpha-tocopherol inhibit human liver microsomal CYP3Aactivity,as assessed by the enzymatic conversion of lithocholic acid to 3-ketocholanoic acid and of testosterone to6β-hydroxytestosterone.METHODS Enzymatic formation of 3-ketocholanoic acid via lithocholic acid 3-oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5.Enzyme inhibition assay was conducted in a mixture containing potassium phosphate buffer(pH 7.4),human liver microsomes,NADPH,lithocholic acid,and various concentrations of a test chemical.The amount of 3-ketocholanoic acid formed was quantified by a novel,validated ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS-MS)method.RESULTS Lithocholic acid was metabolized to 3-ketocholanoic acid by human recombinant CYP3A4 and CYP3A5enzymes and human liver microsomes.Alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,and tocotriernol-rich mixture,but not alpha-tocopherol,inhibited 3-ketocholanoic acid formation in human liver microsomes.Concentration-response experiments indicated that tocotrienol-rich mixture and delta-tocotrienol inhibited 3-ketocholanoic acid formation with IC50 values of 6.6±2.1μg·mL-1 and 19.0±1.0μmol·L-1,respectively.CONCLUSION Tocotrienols inhibited CYP3A-catalyzed lithocholic acid 3-oxidation but not CYP3A-catalyzed testosterone 6-beta-hydroxylation.This suggests that lithocholic acid and testosterone bind to different CYP3 Abinding sites and that tocotrienols preferentially inhibit the lithocholic acid binding site on CYP3 Aenzymes.展开更多
OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After ad...OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.展开更多
The cytochrome P450 superfamily,one of the most important drug-metabolizing enzyme systems in humans,is responsible for the metabolism of numerous xenobiotic substances as well as endogenous substrates.Induction or in...The cytochrome P450 superfamily,one of the most important drug-metabolizing enzyme systems in humans,is responsible for the metabolism of numerous xenobiotic substances as well as endogenous substrates.Induction or inhibition of the cytochrome P450 enzymes,after exposure to different drugs and chemicals,is directly linked to a number of drug-induced toxicity and drug interactions leading to treatment failure.As we know,the traditional Chinese medicine(TCM)has complicated components and is always coadministered with other drugs.Through searching and summing up the literatures about the effects of TCM on cytochrome P450 at home andabroad,the results show that the activity of cytochrome P450 was inhibited or induced by some single herbs,traditional Chinese prescriptions andthe active components of the TCM,such as the flavonoids,flavonoid derivatives,alkaloids,saponins,terpenoids,anthraquinones and lignans,and so on,which therefore might slow or speed metabolism of other drugs.The paper tips that much attention should be paid to the research fields of TCM on cytochrome P450,which is meaningful to clinical reasonable medicine treatment and new drug development.展开更多
Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration ...Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.展开更多
Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were inv...Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.展开更多
The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomy...The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomyocyte death.Inhibition of endogenous Puma by siRNA attenuated H/R-induced cell death.Puma stimulated caspase-8 activation.展开更多
The difference in protein content, MDH,POD and COD isozymes have been seenbetween lowland rice roots and upland rice roots during root growth and development. High pro-tein content has been examined in upland rice roo...The difference in protein content, MDH,POD and COD isozymes have been seenbetween lowland rice roots and upland rice roots during root growth and development. High pro-tein content has been examined in upland rice roots,but isozyme bands in lowland rice roots aremore than that in upland rice roots.Total area of POD isozyme bands at seedling and tilleringstages of upland rice is larger than that of lowland rice.The POD isozymes zymogram in the rootof upland rice is more stable compared with that of lowland rice.More COD isozyme numbershave been showed in roots of upland rice,but the relative activity of COD isozyme in roots of up-land rice at the seedling and tillering stages is lower than that of lowland rice.COD<sub>7</sub> band can beshown in upland rice root at flowering stage,but it is absent in lowland rice.Therefore,COD<sub>7</sub>,band can be taken as an index for resistance of the upland rice to drought environment.展开更多
基金The project supported by National University of Singapore Grant(R-148-000-185-133)
文摘OBJECTIVE Lithocholic acid,which is a secondary bile acid,has been reported to be hepatotoxic and carcinogenic.It is metabolized by human cytochrome P450 3A(CYP3A)to form 3-ketocholanoic acid.A previous study suggests that vitamin E isomers(tocotrienols and tocopherols)are metabolized by CYP3 A.Given that substrates of an enzyme may competitively inhibit the enzyme,we determined whether alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,tocotrienol-rich mixture(a mixture consisting of 25.7% d-α-tocotrienol,2.6% d-β-tocotrienol,28.6% d-γ-tocotrienol,8.4% d-δ-tocotrienol,25.6% d-α-tocopherol,and 4.3% d-α-tocomonoenol),and alpha-tocopherol inhibit human liver microsomal CYP3Aactivity,as assessed by the enzymatic conversion of lithocholic acid to 3-ketocholanoic acid and of testosterone to6β-hydroxytestosterone.METHODS Enzymatic formation of 3-ketocholanoic acid via lithocholic acid 3-oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5.Enzyme inhibition assay was conducted in a mixture containing potassium phosphate buffer(pH 7.4),human liver microsomes,NADPH,lithocholic acid,and various concentrations of a test chemical.The amount of 3-ketocholanoic acid formed was quantified by a novel,validated ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS-MS)method.RESULTS Lithocholic acid was metabolized to 3-ketocholanoic acid by human recombinant CYP3A4 and CYP3A5enzymes and human liver microsomes.Alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,and tocotriernol-rich mixture,but not alpha-tocopherol,inhibited 3-ketocholanoic acid formation in human liver microsomes.Concentration-response experiments indicated that tocotrienol-rich mixture and delta-tocotrienol inhibited 3-ketocholanoic acid formation with IC50 values of 6.6±2.1μg·mL-1 and 19.0±1.0μmol·L-1,respectively.CONCLUSION Tocotrienols inhibited CYP3A-catalyzed lithocholic acid 3-oxidation but not CYP3A-catalyzed testosterone 6-beta-hydroxylation.This suggests that lithocholic acid and testosterone bind to different CYP3 Abinding sites and that tocotrienols preferentially inhibit the lithocholic acid binding site on CYP3 Aenzymes.
基金The project supported by National Natural Science Foundation of China(81473579,81273654,81102879)the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2013ZX09103002-022)
文摘OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.
文摘The cytochrome P450 superfamily,one of the most important drug-metabolizing enzyme systems in humans,is responsible for the metabolism of numerous xenobiotic substances as well as endogenous substrates.Induction or inhibition of the cytochrome P450 enzymes,after exposure to different drugs and chemicals,is directly linked to a number of drug-induced toxicity and drug interactions leading to treatment failure.As we know,the traditional Chinese medicine(TCM)has complicated components and is always coadministered with other drugs.Through searching and summing up the literatures about the effects of TCM on cytochrome P450 at home andabroad,the results show that the activity of cytochrome P450 was inhibited or induced by some single herbs,traditional Chinese prescriptions andthe active components of the TCM,such as the flavonoids,flavonoid derivatives,alkaloids,saponins,terpenoids,anthraquinones and lignans,and so on,which therefore might slow or speed metabolism of other drugs.The paper tips that much attention should be paid to the research fields of TCM on cytochrome P450,which is meaningful to clinical reasonable medicine treatment and new drug development.
文摘Isoflavones which mainly distributed in leguminous plants have plenty of health benefits.Isoflavone synthase(IFS)is a membrane-associated cytochrome P450 enzyme(CYP450)which carries out the unique aryl-ring migration and hydroxylation.So far,few crystal structures of plant P450s have been obtained.We determined the crystal structure of IFS from Medicago truncatula at 1.9 by MAD method using a selenomethionine substituted crystal and conducted molecular docking and mutagenesis study.The structure of IFS complexed with imidazole exhibits the helix Iα-loop-helix Iβmotif which corresponds to helix I of other P 450s.Compared with structures of common P450s,IFS/imidazole structure contains an extra domain,i.e.,theγ-domain.The structure reveals a homodimer in which theγ-domain of one molecule interacts with theβ-domain of another.The plane of heme group makes an angle of approximately 40°with the helix Iα-loop-helix Iβmotif.Molecular docking combined with mutagenesis study suggested that Trp-128 and Asp-300 might play important roles in substrate binding and recognition.Phe-301,Ser-303 and Gly-305 from the helix Iα-loop-helix Iβmotif may play important roles in the aryl-ring migration.These novel structural features reveal insights into the unique reaction mechanism of IFS and provide a basis for engineering IFS in leguminous crops for health purpose.
文摘Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.
文摘The present study is to investigate the role of Puma in cardiomyocyte death induced by hypoxia/reoxygenation(H/R).We found that H/R increased the level of Puma mRNA and protein accompanied by the elevation of cardiomyocyte death.Inhibition of endogenous Puma by siRNA attenuated H/R-induced cell death.Puma stimulated caspase-8 activation.
文摘The difference in protein content, MDH,POD and COD isozymes have been seenbetween lowland rice roots and upland rice roots during root growth and development. High pro-tein content has been examined in upland rice roots,but isozyme bands in lowland rice roots aremore than that in upland rice roots.Total area of POD isozyme bands at seedling and tilleringstages of upland rice is larger than that of lowland rice.The POD isozymes zymogram in the rootof upland rice is more stable compared with that of lowland rice.More COD isozyme numbershave been showed in roots of upland rice,but the relative activity of COD isozyme in roots of up-land rice at the seedling and tillering stages is lower than that of lowland rice.COD<sub>7</sub> band can beshown in upland rice root at flowering stage,but it is absent in lowland rice.Therefore,COD<sub>7</sub>,band can be taken as an index for resistance of the upland rice to drought environment.
