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1.
Biomed Chromatogr ; 24(7): 752-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19908207

RESUMO

Biomarkers are an increasingly important constituent of the drug development process, offering the potential of increased efficiency through reduced compound attrition and earlier proof of mechanism and/or efficacy. Assays developed for compound screening that can be directly translated for clinical trials are especially valuable, but their successful adoption requires a careful balance between assay performance and implementation costs. One such 'fit-for-purpose' biomarker assay, the indirect measurement of pharmacological modulation of sphingolipid biosynthesis and disposition, is presented here. Among sphingolipids, numerous ceramide species are readily detectable in different lipoprotein fractions of mammalian plasma, but their parallel quantification can be prohibitively expensive and time consuming. Ceramides differ in their fatty acid moiety, which is readily removed by hydrolysis, yielding a common sphingosine derivative, the measurement of which serves as an indicator of total ceramide. When followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) for detection, robust analyte quantification becomes relatively straightforward. The practical utility of a method developed to be fit for the purpose of rapidly and quantitatively measuring treatment-induced variations in total ceramide from hamster plasma and individual lipoprotein fractions is described. With a linear calibration range from 0.003 to 33.4 microm sphingosine, precision and accuracy error in plasma-based quality controls spiked with ceramides was less than 15%. The specificity of the assay for ceramides was also assessed. The simplicity of the method would allow for its potential translation to other preclinical species, as well as for clinical applications in later-stage drug development.


Assuntos
Ceramidas/análise , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Esfingosina/análise , Pesquisa Translacional Biomédica , Animais , Ceramidas/sangue , Cricetinae , Masculino , Mesocricetus , Esfingosina/sangue
2.
Bioorg Med Chem Lett ; 18(2): 546-53, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18063367

RESUMO

Cholesterol absorption inhibition (CAI) represents an important treatment option for hypercholesterolemia. Herein, we report the design and evaluation of a series of substituted oxazolidinones as ligands for the Niemann Pick C1 Like 1 (NPC1L1) protein, a key mediator of cholesterol transport. Novel analogs were initially evaluated in a brush border membrane NPC1L1 binding assay; subsequently, promising compounds were evaluated in vivo for acute inhibition of cholesterol absorption. These studies identified analogs with low micromolar NPC1L1 binding affinity and acute in vivo efficacy of >50% absorption inhibition at 3mg/kg.


Assuntos
Colesterol/metabolismo , Absorção Intestinal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Oxazolidinonas/farmacologia , Animais , Ligantes , Microvilosidades/metabolismo , Oxazolidinonas/química , Oxazolidinonas/metabolismo , Oxazolidinonas/farmacocinética , Ratos , Difração de Raios X
3.
Atherosclerosis ; 189(2): 264-72, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16458317

RESUMO

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.


Assuntos
Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Ácidos Graxos Monoinsaturados/uso terapêutico , Esfingomielinas/antagonistas & inibidores , Esfingomielinas/biossíntese , Animais , Apolipoproteínas E/deficiência , Western Blotting , HDL-Colesterol/sangue , LDL-Colesterol/sangue , VLDL-Colesterol/sangue , Modelos Animais de Doenças , Regulação da Expressão Gênica , Imunossupressores/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , RNA/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
4.
Circulation ; 110(22): 3465-71, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15545514

RESUMO

BACKGROUND: In clinical studies, sphingomyelin (SM) plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels. We hypothesized that inhibition of SM synthesis would have antiatherogenic effects. To test this hypothesis, apolipoprotein E (apoE)-knockout (KO) mice were treated with myriocin, a potent inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in SM biosynthesis. METHODS AND RESULTS: Diet-admix treatment of apoE-KO mice with myriocin in Western diet for 12 weeks lowered SM and sphinganine plasma levels. Decreases in sphinganine and SM concentrations were also observed in the liver and aorta of myriocin-treated animals compared with controls. Inhibition of de novo sphingolipid biosynthesis reduced total cholesterol and triglyceride plasma levels. Cholesterol distribution in lipoproteins demonstrated a decrease in beta-VLDL and LDL cholesterol and an increase in HDL cholesterol. Oil red O staining of total aortas demonstrated reduction of atherosclerotic lesion coverage in the myriocin-treated group. Atherosclerotic plaque area was also reduced in the aortic root and brachiocephalic artery. CONCLUSIONS: Inhibition of de novo SM biosynthesis in apoE-KO mice lowers plasma cholesterol and triglyceride levels, raises HDL cholesterol, and prevents development of atherosclerotic lesions.


Assuntos
Aciltransferases/antagonistas & inibidores , Apolipoproteínas E/deficiência , Arteriosclerose/prevenção & controle , Ácidos Graxos Monoinsaturados/uso terapêutico , Esfingomielinas/biossíntese , Esfingosina/análogos & derivados , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/enzimologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/genética , Arteriosclerose/sangue , Arteriosclerose/enzimologia , Arteriosclerose/etiologia , Arteriosclerose/patologia , Colesterol/sangue , Cromatografia Líquida de Alta Pressão , Dieta Aterogênica , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/enzimologia , Hiperlipoproteinemia Tipo II/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/sangue , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Serina C-Palmitoiltransferase , Esfingomielinas/sangue , Esfingosina/sangue , Linfócitos T/patologia , Triglicerídeos/sangue
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