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1.
Bioanalysis ; 8(3): 193-204, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811930

RESUMO

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.


Assuntos
Cromatografia Líquida/métodos , Miostatina/análise , Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/economia , Análise Custo-Benefício , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miostatina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Espectrometria de Massas em Tandem/economia , Tripsina/metabolismo
2.
J Lipid Res ; 55(7): 1366-74, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24755647

RESUMO

A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.


Assuntos
Pelo Animal/metabolismo , Inibidores Enzimáticos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Doenças das Glândulas Sebáceas/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Mesocricetus , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ratos Sprague-Dawley , Doenças das Glândulas Sebáceas/induzido quimicamente , Estearoil-CoA Dessaturase/metabolismo
3.
Mol Cancer Res ; 9(11): 1551-61, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21954435

RESUMO

Emerging literature suggests that metabolic pathways play an important role in the maintenance and progression of human cancers. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. In the studies reported here, we aimed to understand whether tumor cells require the activity of either human isoform of stearoyl-CoA-desaturase (SCD1 or SCD5) for survival. Inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition, when tumor cells are cultured in reduced (2%) serum conditions, but has little impact on cells cultured in 10% serum. Depletion of SCD5 had minimal effects on cell growth or apoptosis. Consistent with the observed dependence on SCD1, but not SCD5, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids, the product of the enzymatic reaction catalyzed by SCD1. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that desaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Estearoil-CoA Dessaturase/biossíntese , Estearoil-CoA Dessaturase/deficiência , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Transfecção
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