RESUMO
Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.
Assuntos
Insulina Glargina/química , Células 3T3 , Adipogenia/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacologia , Células CHO , Dicroísmo Circular , Cricetulus , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Humanos , Insulina Glargina/farmacologia , Lipólise/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS).
Assuntos
Proteínas/química , Selênio/análise , Cromatografia/métodos , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas/análise , Selenometionina/farmacologia , Análise de Sequência de ProteínaRESUMO
Stroke is the leading cause of disability in the USA. Rapid, reliable diagnosis via a point-of-care blood test may facilitate earlier treatment to reduce disability. We have recently reported detailed methods of chromatographic separations of plasma samples coupled with nanoESI-ion trap a list of proteins which are viable candidates for further investigation as stroke biomarkers.
Assuntos
Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Acidente Vascular Cerebral/sangue , HumanosRESUMO
Stroke is the leading cause of adult disability, worldwide. Metalloproteins and metals play key roles in epigenetic events in living organisms, including hypertension, the most important modifiable risk factor for stroke. Thus, metalloproteins may be important target biomarkers for disease diagnosis. The primary goal of this study was to assess metal containing proteins in blood plasma, detected by ICP-MS, followed by ESIMS for peptide/protein identification. We then compared the relative concentration differences between samples from patients with ischemic stroke, hemorrhagic stroke and stroke mimics. In 29 plasma samples (10 stroke mimics, 10 ischemic stroke and 9 hemorrhagic stroke patients) previously collected from patients who presented to the University of Cincinnati Emergency Department within 12 hours of symptom onset for a plasma banking project. For the metal associated protein study, Mg, Mn, Cu, Se concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and ischemic stroke patients vs. hemorrhagic stroke patients. Pb concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and Mo levels were statistically the same among the three groups. In addition, we also report concentration levels and preliminary correlation studies for total elemental analysis among the three sets of patients. This pilot study demonstrates that mass spectrometry methods may be highly valuable in detecting novel stroke biomarkers in blood plasma. Expanded studies are warranted to confirm these findings.
Assuntos
Espectrometria de Massas/métodos , Metaloproteínas/sangue , Metais/sangue , Acidente Vascular Cerebral/sangue , Idoso , Biomarcadores/sangue , Cromatografia Líquida , Feminino , Humanos , Masculino , Metaloproteínas/química , Metais/química , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Projetos PilotoRESUMO
Stroke is the most common cause of morbidity and death in the Western world, following ischemic heart disease and cancer. Stroke can be of two types, ischemic or hemorrhagic, with ischemic stroke accounting for approximately 85% of the total number of strokes. Well-recognized environmental risk factors for stroke include hypertension, smoking, diabetes mellitus, atrial fibrillation, and atherosclerosis. Computed tomography (CT) scanning is used to diagnose hemorrhagic stroke but is relatively ineffective and may remain normal in patients with mild ischemic strokes. Magnetic Resonance Imaging (MRI) is more sensitive in detecting ischemia than CT, especially in the diagnosis of mild stroke but it is still not 100% sensitive or precise. A simple and low-cost, rapid blood test to confirm a clinical and imaging diagnosis of ischemic stroke would be extremely useful. Based on this, the central idea of this paper is to develop a method that would be applicable to a statistically viable sample set to provide candidate biomarkers for distinguishing stroke types. In search of these candidate biomarkers, different analytical separation techniques have been used to screen for major differences in the proteomes of patients plasma samples with proteomics for identification.
Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/análise , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Acidente Vascular Cerebral/sangue , Biomarcadores/análise , Proteínas Sanguíneas/isolamento & purificação , Feminino , Humanos , Masculino , Projetos Piloto , Acidente Vascular Cerebral/diagnósticoRESUMO
Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.