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Medicinas Complementárias
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1.
Sci Rep ; 8(1): 12538, 2018 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-30135446

RESUMEN

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.


Asunto(s)
Péptidos de Penetración Celular/farmacología , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Animales , Bacteriófago T7 , Biotinilación , Células CHO , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/toxicidad , Dicroismo Circular , Cricetulus , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente , Distrofia Muscular de Duchenne/tratamiento farmacológico , Biblioteca de Péptidos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo
2.
Diabetologia ; 59(9): 2005-12, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27272237

RESUMEN

AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.


Asunto(s)
Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Resistencia a la Insulina/fisiología , Leptina/uso terapéutico , Animales , Ingestión de Energía/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Hígado Graso/sangre , Femenino , Insulina/metabolismo , Leptina/química , Ácidos Linoleicos Conjugados/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Lipodistrofia/inducido químicamente , Lipodistrofia/tratamiento farmacológico , Lipodistrofia/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL
3.
Endocrinology ; 157(1): 233-44, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26492472

RESUMEN

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.


Asunto(s)
Depresores del Apetito/uso terapéutico , Metabolismo Energético/efectos de los fármacos , Leptina/análogos & derivados , Obesidad/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Respuesta de Saciedad/efectos de los fármacos , Secuencias de Aminoácidos , Animales , Depresores del Apetito/administración & dosificación , Depresores del Apetito/efectos adversos , Depresores del Apetito/química , Relación Dosis-Respuesta a Droga , Ingestión de Energía/efectos de los fármacos , Femenino , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/patología , Inyecciones Subcutáneas , Leptina/administración & dosificación , Leptina/genética , Leptina/uso terapéutico , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Mutantes , Peso Molecular , Actividad Motora/efectos de los fármacos , Obesidad/metabolismo , Obesidad/patología , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Organismos Libres de Patógenos Específicos , Termogénesis/efectos de los fármacos , Pérdida de Peso/efectos de los fármacos
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