RESUMEN

Protection of endothelial integrity has been recognized as a frontline approach to alleviating sepsis progression, yet no effective agent for preserving endothelial integrity is available. Using an unusual anti-angiopoietin 2 (ANG2) antibody, ABTAA (ANG2-binding and TIE2-activating antibody), we show that activation of the endothelial receptor TIE2 protects the vasculature from septic damage and provides survival benefit in three sepsis mouse models. Upon binding to ANG2, ABTAA triggers clustering of ANG2, assembling an ABTAA/ANG2 complex that can subsequently bind and activate TIE2. Compared with a conventional ANG2-blocking antibody, ABTAA was highly effective in augmenting survival from sepsis by strengthening the endothelial glycocalyx, reducing cytokine storms, vascular leakage, and rarefaction, and mitigating organ damage. Together, our data advance the role of TIE2 activation in ameliorating sepsis progression and open a potential therapeutic avenue for sepsis to address the lack of sepsis-specific treatment.

Asunto(s)

Anticuerpos/uso terapéutico , Receptor TIE-2/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Angiopoyetina 2/metabolismo , Animales , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Infiltración Neutrófila/efectos de los fármacos , Ribonucleasa Pancreática/metabolismo , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Hydrophobically modified glycol chitosan (HGC) nanoparticles loaded with mono-lithocholic acid-conjugated exendin-4 at the Lys(27) residue (LAM1-Ex4) were prepared and characterized by particle size measurement, proteolytic stability, in vitro drug-release profile, and in vivo antidiabetic effects in a db/db diabetic mouse model. Compared with Ex-4-loaded HGC nanoparticles (Ex4/HGC NPs) prepared as a control, LAM1-Ex4-loaded HGC nanoparticles (LAM1-Ex4/HGC NPs) showed improved drug-loading efficiency, small particle size, enhanced resistance against proteolytic digestion, and an extended in vitro drug release profile. These findings may be attributable to the strong hydrophobic interaction between LAM1-Ex4 and the inner core of HGC. Furthermore, LAM1-Ex4/HGC NPs showed prolonged hypoglycemic efficacy in db/db mice, lasting 1 week after a single subcutaneous administration. The present study demonstrated that LAM1-Ex4/HGC NPs have considerable potential as a long-acting sustained-release antidiabetic system for type 2 diabetes.

Asunto(s)

Quitosano/administración & dosificación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Ácido Litocólico/química , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Péptidos/uso terapéutico , Ponzoñas/administración & dosificación , Ponzoñas/uso terapéutico , Animales , Quitosano/química , Liberación de Fármacos , Exenatida , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Lisina/química , Ratones , Nanopartículas/química , Tamaño de la Partícula , Péptidos/química , Succinimidas/química , Ponzoñas/química

RESUMEN

OBJECTIVE: To test the hypothesis that Notch signalling plays a role in the pathogenesis of rheumatoid arthritis (RA) and to determine whether pharmacological inhibition of Notch signalling with γ-secretase inhibitors can ameliorate the RA disease process in an animal model. METHODS: Collagen-induced arthritis was induced in C57BL/6 or Notch antisense transgenic mice by immunisation with chicken type II collagen (CII). C57BL/6 mice were administered with different doses of inhibitors of γ-secretase, an enzyme required for Notch activation, at disease onset or after onset of symptoms. Severity of arthritis was monitored by clinical and histological scores, and in vivo non-invasive near-infrared fluorescence (NIRF) images. Micro-CT was used to confirm joint destruction. The levels of CII antibodies and cytokines in serum were determined by ELISA and bead-based cytokine assay. The expression levels of cytokines were studied by quantitative PCR in rheumatoid synovial fibroblasts. RESULTS: The data show that Notch signalling stimulates synoviocytes and accelerates their production of proinflammatory cytokines and immune responses involving the upregulation of IgG1 and IgG2a. Pharmacological inhibition of γ-secretase and antisense-mediated knockdown of Notch attenuates the severity of inflammatory arthritis, including arthritis indices, paw thickness, tissue damage and neutrophil infiltration, and reduces the levels of active NF-κB, ICAM-1, proinflammatory cytokines and matrix metalloproteinase-3 activity in the mouse model of RA. CONCLUSIONS: These results suggest that Notch is involved in the pathogenesis of RA and that inhibition of Notch signalling is a novel approach for treating RA.

Asunto(s)

Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Citocinas/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Membrana Sinovial/inmunología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Citocinas/efectos de los fármacos , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Notch/antagonistas & inhibidores , Receptores Notch/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos

RESUMEN

Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.

Asunto(s)

Leucina Zippers/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación de Dinámica Molecular , Complejos Multiproteicos/ultraestructura , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/ultraestructura , Alineación de Secuencia , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/ultraestructura

RESUMEN

The successful clinical translation of siRNA-based therapeutics requires efficient carrier systems that can specifically deliver siRNA within the cytosol of the target cells. Although numerous polymeric nanocarriers forming ionic complexes with siRNA have been investigated for cancer therapy, their poor stability and lack of tumor targetability have impeded their in vivo applications. To surmount these limitations, we synthesized a novel type of biodegradable hyaluronic acid-graft-poly(dimethylaminoethyl methacrylate) (HPD) conjugate that can form complexes with siRNA and be chemically crosslinked via the formation of the disulfide bonds under facile conditions. The crosslinked siRNA-HPD (C-siRNA-HPD) complexes exhibited high stability in a 50% serum solution, as compared to the uncrosslinked siRNA-HPD (U-siRNA-HPD) complexes and free siRNA. Both the C-siRNA-HPD and U-siRNA-HPD complexes were efficiently taken up by the CD44-overexpressing melanoma cells (B16F10), but not by the normal fibroblast cells (NIH3T3). When the RFP-expressing B16F10 cells were treated with the complexes or free siRNA, the C-siRNA-HPD complexes showed the highest decrease in RFP expression. In vivo studies demonstrated the selective accumulation of C-siRNA-HPD complexes at the tumor site after their systemic administration into tumor-bearing mice, resulting in an efficient gene silencing effect. Overall, these results suggest that the HPD conjugate could be used as an efficient carrier for the tumor-targeted delivery of siRNA.

Asunto(s)

Portadores de Fármacos/química , Ácido Hialurónico/análogos & derivados , Metacrilatos/química , Neoplasias/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Silenciador del Gen , Haplorrinos , Melanoma/genética , Melanoma/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células 3T3 NIH , Neoplasias/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , ARN Interferente Pequeño/uso terapéutico

RESUMEN

Heparin decomplexation experiments, as well as all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations, were performed to determine the effect of the size of arginine(Arg)-rich peptides on the structure and binding strength of the siRNA-peptide complex. At a fixed peptide/siRNA mole ratio of 5:1 or 10:1, the siRNA complexes with peptides longer than nine Arg residues are more easily decomplexed by heparin than are those with nine Arg residues. At these mole ratios, peptides longer than nine Arg residues have cationic/anionic charge ratios in excess of unity, and produce more weakly bound complexes than nine Arg residue ones do. AA simulations of mixtures of peptides with a single siRNA show formation of an electrostatically induced complex, and the longer peptides produce a larger complex, but with no significant increase in the number of Arg residues bound to the siRNA. Larger-scale CG-MD simulations show that multiple siRNAs can be linked together by peptides into a large complex, as observed in the experiments. The peptides longer than nine residues, which at mole ratio 5:1 yield a peptide/siRNA charge ratio in excess of unity, include many noninteracting Arg residues, which repel each other electrostatically. This leads to a less dense complex than for 9-residue peptides, which can explain why these longer complexes are more easily decomplexed by heparin molecules, as observed in the experiments. The key role of the charge ratio is supported by simulations that show that, at a mole ratio of 2.5 peptides per siRNA, the longer 18-residue peptide has a charge ratio of roughly unity and also shows a tight complex, just as the 9-residue peptide does at a 5:1 mole ratio, where its charge ratio is also unity.

Asunto(s)

Péptidos/química , ARN Interferente Pequeño/química , Arginina/química , Heparina/química , Heparina/metabolismo , Simulación de Dinámica Molecular , Péptidos/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Electricidad Estática

RESUMEN

The in vivo stability and tumor targetability of self-assembled polymeric nanoparticles are crucial for effective drug delivery. In this study, to develop biostable nanoparticles with high tumor targetability, poly(ethylene glycol)-conjugated hyaluronic acid nanoparticles (PEG-HANPs) were mineralized through controlled deposition of inorganic calcium and phosphate ions on the nanoparticular shell via a sequential addition method. The resulting nanoparticles (M-PEG-HANPs) had a smaller size (153.7±4.5nm) than bare PEG-HANPs (265.1±9.5nm), implying that mineralization allows the formation of compact nanoparticles. Interestingly, when the mineralized nanoparticles were exposed to acidic buffer conditions (

Asunto(s)

Antibióticos Antineoplásicos/química , Doxorrubicina/química , Portadores de Fármacos/química , Ácido Hialurónico/química , Nanopartículas/química , Polietilenglicoles/química , Animales , Antibióticos Antineoplásicos/administración & dosificación , Fosfatos de Calcio/química , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Ácido Hialurónico/farmacocinética , Ratones , Ratones Desnudos , Nanopartículas/administración & dosificación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Distribución Tisular , Carga Tumoral/efectos de los fármacos

RESUMEN

Cancer chemotherapy is often limited, since more than one molecule is usually involved with the cancer pathogenesis. A combination of therapeutic drugs would be a promising approach to overcome the complexity of tumors. In this study, a conjugate (DA3) of deoxycholic acid and low molecular weight polyethylenimine (PEI 1.8 kDa), which has a property that mimics that of cell penetrating peptides (CPPs), was used for simultaneous delivery of an anticancer drug and siRNA. When complexed with siRNA, DA3 showed significantly higher target gene silencing efficiency than PEI 25 kDa. The gene silencing efficiency of DA3 was maintained even in the presence of endocytosis inhibitors, suggesting that the polymeric carrier can mediate an endocytosis-independent macromolecular transduction similar to CPPs. The capability of forming a micelle-like core-shell structure enables the conjugates to encapsulate and dissolve paclitaxel (PTX), a water-insoluble drug. The drug-loaded cationic micelles can then interact with siRNA to form stable complexes (PTX/DA3/siRNA). The PTX/DA3/siRNA showed significantly enhanced inhibition of cancer cell growth. When administered into tumor-bearing animals, the PTX/DA3/siRNA demonstrated significant suppression of tumor growth, providing potential usefulness in clinical settings.

Asunto(s)

Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Paclitaxel/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Péptidos de Penetración Celular/química , Neoplasias Colorrectales/patología , Ácido Desoxicólico/química , Sistemas de Liberación de Medicamentos , Endocitosis , Silenciador del Gen , Células HCT116 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Micelas , Peso Molecular , Paclitaxel/química , Paclitaxel/farmacología , Polietileneimina/química , Solubilidad

RESUMEN

Clinical applications of RNA interference-based therapeutics such as small interfering RNAs (siRNAs) have been limited mainly due to low intracellular delivery efficiency in vitro and in vivo. In this study, facially amphipathic deoxycholic acid (DA)-modified polyethyleneimine (PEI(1.8)) (DA-PEI(1.8)) was synthesized and used as a potent carrier system for siRNA targeted against matrix metalloproteinase-2 (MMP-2) to inhibit the migration of vascular smooth muscle cells (SMCs), which is the major pathomechanism in the development of atherosclerosis and restenosis after arterial injury. A representative facial amphipathic bile acid DA having a high membrane permeability was conjugated to the terminal amine groups of the low molecular weight PEI(1.8) via amide bonds. The DA-PEI(1.8) conjugates formed self-assembled nanoparticles with siRNA molecules in an aqueous phase and the DA-PEI(1.8)/siRNA polyplexes became stabilized and condensed as particle incubation time increased from 0 to 4h. Both cellular internalization and target gene silencing were enhanced as the DA-PEI(1.8)/siRNA polyplexes stabilized. When vascular SMCs were transfected with MMP-2 siRNA, the DA-PEI(1.8)/siRNA polyplex formulation led to a significant decrease in MMP-2 gene expression, resulting in the suppression of cell migration. These results suggest that the DA-PEI(1.8)/MMP-2 siRNA delivery system may be useful in anti-restenotic treatment for various vasculoproliferative disorders such as atherosclerosis, in-stent restenosis, and vein graft failure.

Asunto(s)

Ácido Desoxicólico/química , Metaloproteinasa 2 de la Matriz/genética , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Movimiento Celular , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Nanopartículas , Interferencia de ARN , Factores de Tiempo , Transfección

RESUMEN

The extensive use of human growth hormone (hGH), emerging as protein therapeutics, has been limited by its instability in biological fluids and short biological half-life. In this study, thiolated glycol chitosan bearing α-cyclodextrin (TGC-CD), in situ cross-linked by poly(ethylene glycol)-diacrylate (PEG-DA), was synthesized to develop a sustained release system for PEGylated hGH (PEG-hGH). TGC-CD could form a stable inclusion complex with PEG-hGH by the physical interaction between the inner cavity of CD and PEG. Such a complex was readily cross-linked in the presence of PEG-DA via a Michael-type addition reaction. From the in vitro release experiments of PEG-hGH, it was confirmed that PEG-hGH was completely released from the complex for 12 h in PBS (pH 7.4), whereas the release rate of PEG-hGH was significantly reduced by the chemical cross-linking of the complex. PEG-hGH, released from the cross-linked complexes, maintained its structural integrity, which was demonstrated using circular dichroism spectroscopy. Overall, TGC-CD might be useful for sustained delivery of PEG-hGH.

Asunto(s)

Quitosano , Hormonas/administración & dosificación , Hormona de Crecimiento Humana/administración & dosificación , Polietilenglicoles , alfa-Ciclodextrinas , Quitosano/síntesis química , Quitosano/química , Dicroismo Circular , Preparaciones de Acción Retardada/síntesis química , Preparaciones de Acción Retardada/química , Liberación de Fármacos , Hormonas/farmacocinética , Hormona de Crecimiento Humana/farmacocinética , Humanos , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Espectroscopía de Protones por Resonancia Magnética , Difracción de Rayos X , alfa-Ciclodextrinas/síntesis química , alfa-Ciclodextrinas/química

RESUMEN

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics.

Asunto(s)

Biomimética , Péptidos de Penetración Celular/metabolismo , Técnicas de Transferencia de Gen , Polímeros/metabolismo , Endocitosis/fisiología , Genes Reporteros , Polímeros/química , Transfección

RESUMEN

A biodegradable amphiphilic pentablock copolymer PAE-PCL-PEG-PCL-PAE with a pH-sensitive unit was synthesized for use as a nontoxic, biodegradable carrier for gene delivery by forming nanocapsules entrapping nucleic acid drugs. The PAE block can interact with plasmid DNA to form polyelectrolyte complexes in an acidic environment. At physiological pH, the PAE blocks are deprotonated and form an insoluble skin, resulting in the formation of nanocapsules that encapsulate plasmid DNA. The surface charges of the nanocapsules became almost neutral at pH = 7.4, and their size ranged from 210 to 280 nm. The nanocapsule maintained most of its transfection efficiency even in the presence of serum. These nanocapsules are therefore potential carriers for systemic gene therapy.

Asunto(s)

Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , ADN/farmacología , Técnicas de Transferencia de Gen , Nanocápsulas/química , Plásmidos/farmacología , Poliésteres/química , Polietilenglicoles/química , Polímeros/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , ADN/genética , ADN/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Microscopía Electrónica de Transmisión , Neoplasias/tratamiento farmacológico , Tamaño de la Partícula , Plásmidos/genética , Plásmidos/metabolismo , Poliésteres/metabolismo , Polietilenglicoles/metabolismo , Polímeros/metabolismo , Protones , Electricidad Estática , Transfección

RESUMEN

The clinical applications of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), an emerging therapeutic protein for cancer and rheumatoid arthritis (RA), are limited by its instability and short biological half-life. In this study, efficient therapeutic modalities for RA treatment were developed in the form of nano-sized complexes (nanocomplexes) based on hyaluronic acid (HA) and polyethylene glycol (PEG)-derivatized TRAIL (PEG-TRAIL) formed by N-terminal specific PEGylation. The nanocomplexes were prepared by simply mixing the positively charged PEG-TRAIL and negatively charged HA, and showed negligible loss of bioactivity compared with the PEG-TRAIL. The in vivo biodistribution and diffusion kinetics of Cy5.5-labeled PEG-TRAIL in mice were observed using a near-infrared optical imaging system after subcutaneous injection of three different formulations: PEG-TRAIL in phosphate-buffered saline (PBS, pH 7.4), nanocomplex in PBS, or nanocomplex in 1% HA solution. The results suggested that PEG-TRAIL is released slowly in vivo from the nanocomplex in 1% HA. Experiments in a collagen-induced arthritis mouse model demonstrated that the magnitudes of therapeutic effects, as judged by clinical scores and histology, were significantly enhanced by the sustained delivery of PEG-TRAIL, with the order of nanocomplex in 1% HA>nanocomplex in PBS>PEG-TRAIL in PBS. In addition, sustained delivery of PEG-TRAIL from the nanocomplex in 1% HA resulted in significant reduction of serum inflammatory cytokines and collagen-specific antibodies that are responsible for the pathogenesis of RA. These results imply that HA/PEG-TRAIL nanocomplex formulations are promising therapeutic modalities for the treatment of RA.

Asunto(s)

Artritis Reumatoide/tratamiento farmacológico , Ácido Hialurónico/uso terapéutico , Polietilenglicoles/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Animales , Artritis Reumatoide/sangre , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Difusión/efectos de los fármacos , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Iones , Cinética , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/patología , Ratones , Ratones Desnudos , Microscopía Fluorescente , Nanoestructuras/uso terapéutico , Tamaño de la Partícula , Bazo/citología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacocinética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Distribución Tisular/efectos de los fármacos

RESUMEN

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, its low stability, solubility, unexpected side effects, and weak pharmacokinetic profiles restrict its successful clinical application. To develop efficient TRAIL-based anticancer biotherapeutics, a new version of trimeric TRAIL was constructed by incorporating trimer-forming zipper sequences (HZ-TRAIL), and then NH(2)-terminal-specific PEGylation was done to produce PEGylated TRAIL (PEG-HZ-TRAIL). The biological, physicochemical, and pharmaceutical characteristics of PEG-HZ-TRAIL were then investigated using various in vitro and in vivo experiments, including a cell-based cytotoxicity test, a solubility test, pharmacokinetic analysis, and antitumor efficacy evaluations. Although slight activity loss occurred after PEGylation, PEG-HZ-TRAIL showed excellent tumor cell-specific cytotoxic effects via apoptotic pathways with negligible normal cell toxicity. The stability and pharmacokinetic problems of HZ-TRAIL were successfully overcome by PEGylation. Furthermore, in vivo antitumor tests revealed that PEG-HZ-TRAIL treatment enhanced therapeutic potentials compared with HZ-TRAIL in tumor xenograft animal models, and these enhancements were attributed to its better pharmacokinetic properties and tumor-targeting performance. These findings show that PEG-HZ-TRAIL administration provides an effective antitumor treatment, which exhibits superior tumor targeting and better inhibits tumor growth, and suggest that PEG-HZ-TRAIL should be considered a potential candidate for antitumor biotherapy.

Asunto(s)

Neoplasias/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Receptores Tipo I de Factores de Necrosis Tumoral/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Imagen Molecular , Neoplasias/patología , Polietilenglicoles/química , Ratas , Ratas Sprague-Dawley , Receptores Tipo I de Factores de Necrosis Tumoral/química , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacocinética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Distribución Tisular/efectos de los fármacos

RESUMEN

Despite clinical exploitation of exendin 4 for the treatment of type 2 diabetes, the significantly short half-life requiring twice a day injection has limited the wide applications. In this work, a protocol for the synthesis of long acting hyaluronate (HA) - exendin 4 conjugate was successfully developed using Michael addition chemistry between vinyl sulfone modified HA (HA-VS) and thiolated exendin 4. The exendin 4 content could be controlled in the range of 5-30 molecules per single HA chain with a bioconjugation efficiency higher than 90%. The conjugation of exendin 4 with HA resulted in about 20 times improved in vitro serum stability maintaining the hypoglycemic and gluco-regulatory bioactivities of exendin 4. HA - exendin 4 conjugates showed excellent glucose-lowering capabilities in type 2 db/db mice demonstrating protracted hypoglycemic effect up to 3 days after a single subcutaneous injection. Furthermore, insulin immunohistochemical analysis of islets in db/db mice confirmed the improved insulinotropic activity of HA - exendin 4 conjugates. The HA - exendin 4 conjugates will be investigated further as a twice a week injection dosage form for clinical applications.

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ácido Hialurónico/uso terapéutico , Péptidos/uso terapéutico , Ponzoñas/uso terapéutico , Animales , Cromatografía en Gel , Diabetes Mellitus Tipo 2/sangre , Exenatida , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Ácido Hialurónico/sangre , Ácido Hialurónico/síntesis química , Ácido Hialurónico/química , Inmunohistoquímica , Inyecciones Intraperitoneales , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Péptidos/sangre , Péptidos/química , Sulfonas/síntesis química , Sulfonas/química , Ponzoñas/sangre , Ponzoñas/química

RESUMEN

Improved glucagon-like peptide-1 (GLP-1) receptor activation is considered one of the most effective targets for antidiabetic therapy. For this purpose, we modified the GLP-1 analog of exendin-4 using two fatty acids (FA) either lauric acid (LUA, C12) or palmitic acid (PAA, C16) at its two lysine residues, to produce; Lys(12)-FA-Exendin-4 (FA-M2), Lys(27)-FA-Exendin-4 (FA-M1), or Lys(12,27)-diBA-Exendin-4 (FA-Di). The structural, biological, and pharmaceutical characteristics of these exendin-4 analogs were then investigated. Biological activity tests demonstrated that LUA-M1 had well-preserved in vivo antidiabetic activity and in vitro insulinotropic activity with minimum GLP-1 receptor binding affinity loss as compared with exendin-4. Furthermore, pharmacokinetic studies in rats revealed that s.c. administration of LUA-M1 significantly enhanced pharmacokinetic parameters, such as, elimination half-life, mean residence time, and AUC values as compared with exendin-4. The protracted antidiabetic effects of LUA-M1 were also confirmed by prolonged normoglycemia observed in type 2 diabetic mice (20nmol/mouse single injection of exendin-4 or LUA-M1 induced normoglycemia for 6 or 24h, respectively). These findings suggest that FA conjugated exendin-4s should be considered potential candidates for the treatment of diabetes.

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Animales , Antígenos/metabolismo , Antígenos/uso terapéutico , Diabetes Mellitus , Exenatida , Ácidos Grasos/uso terapéutico , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/uso terapéutico , Receptor del Péptido 1 Similar al Glucagón , Semivida , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Lisina/metabolismo , Lisina/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ácido Palmítico/uso terapéutico , Péptidos , Ratas , Ratas Sprague-Dawley , Receptores de Glucagón , Ponzoñas

RESUMEN

This study focused on the potential therapeutic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on collagen-induced arthritis (CIA) and on the elucidation of the mechanisms involved. DBA/1J mice with established CIA were treated with various amount of recombinant soluble human TRAIL. The effects of TRAIL on the development and severity of CIA in this DBA/1J mouse model were assessed clinically and histologically, and a detailed investigation was conducted on proinflammatory cytokine and anticollagen-specific antibody levels. Cellular immunity was evaluated by investigating the proliferative responses and cytokine release profiles of splenocytes after TRAIL treatment. TRAIL treatment significantly reduced the severity and incidence of CIA, joint swelling, erythema, and edema. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in joints of TRAIL-treated mice with dose-dependent manner. TRAIL treatment also strongly decreased and/or normalized the productions of proinflammatory cytokines and of anti-collagen-specific antibodies in the sera of CIA mice. Furthermore, in vitro studies with primary splenocytes showed the cytotoxic effect of TRAIL on activated lymphocytes, with reduction of inflammatory cytokine release. These findings show that TRAIL administration is an effective anti-inflammatory treatment that prevents the development and progression of CIA in DBA/1J mice, and they suggest that TRAIL might be considered a potential treatment for human RA.

Asunto(s)

Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Articulaciones/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Apoptosis , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Autoanticuerpos/sangre , Citocinas/sangre , Humanos , Inmunidad Humoral/efectos de los fármacos , Articulaciones/inmunología , Articulaciones/patología , Células Jurkat , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico

RESUMEN

Alterations in the physicochemical characteristics of peptide drugs can transform their biological and pharmaceutical features. In the present study, we explored the potentials of lithocholic acid (LCA)-modified exendin-4 derivatives as novel long-acting GLP-1 receptor agonists. Exendin-4 was modified with lithocholic acid at two lysine residues to produce three derivatives that were obtained by reverse-phase HPLC separation, namely, Lys(12)-LCA-exendin-4 (LCA-M2), Lys(27)-LCA-exendin-4 (LCA-M1), and Lys(12,27)-LCA-exendin-4 (LCA-Di)). The biological, pharmacological, and physicochemical characteristics of these three exendin-4 analogues were then investigated. Although slight reductions in the GLP-1 receptor binding capacity and insulinotropic activity of exendin-4 were observed after derivatization, the mono-LCA substitutions, especially LCA-M1, well-preserved antidiabetic activity in type 2 diabetic mice when administered subcutaneously or intraperitoneally. Furthermore, the pharmacokinetic characteristics were dramatically enhanced, that is, absorption was delayed and elimination half-life was increased (1.6+/-0.4 and 9.7+/-1.4h by exendin-4 and LCA-M1, respectively). The enhanced long-acting characteristics of the derivative was found to be due to albumin binding and nanoparticle formation, and these were verified by the restoration of normoglycemia in type 2 diabetic mice after single injection (>24h, >10 nmol/kg, s.c.) and daily injections (15 nmol/kg/day) maintained normoglycemia for the 4-week administration period. Furthermore, antidiabetic potentials, such as, glucose clearance kinetics and percentage areas occupied by pancreatic beta-cells were also enhanced by long-term LCA-M1 administration. The present study demonstrates that the derivatization of exendin-4 with LCA offers a possible means of producing a long-acting GLP-1 receptor agonist.

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Ácido Litocólico/química , Péptidos/farmacocinética , Péptidos/uso terapéutico , Ponzoñas/farmacocinética , Ponzoñas/uso terapéutico , Animales , Exenatida , Hipoglucemiantes/química , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Ratas , Ratas Sprague-Dawley , Ponzoñas/química

RESUMEN

To develop an effective long-acting antidiabetic, the GLP-1 analogue of exendin-4 was modified with three different bile acids (BAs; cholic, deoxycholic, or lithocholic acid), at its two lysine residues. The biological, pharmaceutical, and physicochemical characteristics of these exendin-4 analogues were carefully investigated. Biological activity tests demonstrated that the monobile acid substitutions of exendin-4 showed well preserved receptor binding efficacy without noticeable insulinotropic or antidiabetic activity loss. However, physicochemical and pharmacokinetic studies revealed that the albumin-binding properties and in vivo elimination half-lives of BAM1-Ex4s (Lys(27)-BA-Ex4s) were significantly enhanced by increasing the hydrophobicities of the conjugated BAs. Furthermore, the protracted antidiabetic effects of the BAM1-Ex4s were also verified by the prolonged restoration of normoglycemia in type 2 diabetic mice. Accordingly, the present study suggests that the derivatization of exendin-4 with BAs offers a means of producing long-acting GLP-1 receptor agonists for type 2 diabetic therapy.

Asunto(s)

Ácidos Cólicos/síntesis química , Hipoglucemiantes/síntesis química , Péptidos/síntesis química , Receptores de Glucagón/agonistas , Ponzoñas/síntesis química , Animales , Línea Celular Tumoral , Ácidos Cólicos/farmacocinética , Ácidos Cólicos/farmacología , Ácido Desoxicólico/análogos & derivados , Ácido Desoxicólico/síntesis química , Ácido Desoxicólico/farmacocinética , Ácido Desoxicólico/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Exenatida , Receptor del Péptido 1 Similar al Glucagón , Prueba de Tolerancia a la Glucosa , Interacciones Hidrofóbicas e Hidrofílicas , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Técnicas In Vitro , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ácido Litocólico/análogos & derivados , Ácido Litocólico/síntesis química , Ácido Litocólico/farmacocinética , Ácido Litocólico/farmacología , Masculino , Ratones , Péptidos/farmacocinética , Péptidos/farmacología , Unión Proteica , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Albúmina Sérica/metabolismo , Relación Estructura-Actividad , Ponzoñas/farmacocinética , Ponzoñas/farmacología

RESUMEN

A pH- and temperature-sensitive hydrogel of poly(beta-amino ester)-poly(epsilon-caprolactone)-poly (ethylene glycol)-poly(epsilon-caprolactone)-poly(beta-amino ester) (PAE-PCL-PEG-PCL-PAE) pentablock copolymer was evaluated as a sustained injectable insulin delivery system. Insulin was readily loaded into the matrix, forming an ionically linked insulin-PAE complex. Complex mixtures containing various concentrations of insulin and copolymer were subcutaneously injected into male Sprague-Dawley rats to study the profile of insulin release in vivo. The insulin-release profile showed that insulin was maintained at a constant steady-state level for 15 days, and further demonstrated that insulin levels were controlled by the amount of insulin loaded into the copolymer and the copolymer concentration in the hydrogel. The effect of the insulin-gel complex was further investigated in the streptozotocin (STZ) diabetic rat model. After subcutaneously injecting complex mixtures into STZ-induced diabetic rats, blood glucose and plasma insulin levels were measured. The results showed that the diabetic rats could be treated for more than 1 week with a single injection of the complex mixture containing 10 mg/mL insulin in a 30 wt.% copolymer solution, suggesting that this pH/temperature-sensitive insulin-hydrogel complex system may have therapeutic potential.

Asunto(s)

Sistemas de Liberación de Medicamentos , Hidrogel de Polietilenoglicol-Dimetacrilato/uso terapéutico , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Poliésteres/uso terapéutico , Polietilenglicoles/uso terapéutico , Animales , Glucemia/análisis , Diabetes Mellitus Experimental/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Hipoglucemiantes/sangre , Hipoglucemiantes/uso terapéutico , Inyecciones Subcutáneas , Insulina/sangre , Insulina/uso terapéutico , Masculino , Ratas , Ratas Sprague-Dawley , Estreptozocina/toxicidad , Temperatura