RESUMEN
The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.
Asunto(s)
Ligando de CD40/química , Animales , Células CHO , Rastreo Diferencial de Calorimetría , Cromatografía , Dicroismo Circular , Cricetinae , Dimerización , Análisis de Fourier , Concentración de Iones de Hidrógeno , Luz , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Espectrometría de Fluorescencia , Temperatura , UltracentrifugaciónRESUMEN
We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Materiales Biocompatibles , Tampones (Química) , Quitina/análogos & derivados , Quitosano , Glicerol , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Concentración de Iones de Hidrógeno , Membranas Artificiales , Vehículos Farmacéuticos , Polietilenglicoles/química , Proteínas Recombinantes , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Interleucina-3/química , Focalización Isoeléctrica/métodos , Isoformas de Proteínas/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Electroforesis en Gel de Poliacrilamida , Glicosilación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Interleucina-3/aislamiento & purificación , Datos de Secuencia Molecular , Mapeo Peptídico , Polisacáridos/química , Isoformas de Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
This study elucidates the importance of thermal reversibility as it pertains to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the greatest long-term storage stability. Both thermal reversibility and Tm were evaluated as microcalorimetric parameters of stability within the range extending from pH 6 to 9, where the Tm was shown to plateau near 80 degrees C. Within this region, the reversibility was shown to decrease from 96. 6% to 15.2% while the pH was increased from 6 to 9, respectively. Accelerated stability studies conducted at 50 degrees C exhibited rates of aggregation augmented by pH that inversely correlated with the thermal reversibility data. Namely, high thermal reversibility at the Tm plateau correlated with slower rates of aggregation. Enthalpic calorimetric to van't Hoff ratios (DeltaH1/DeltaHv) yielded results close to unity within the plateau region, suggesting that the unfolding of rhFlt3 ligand was approximately two-state. Evidence that unfolding preceded the formation of the aggregate was provided by far-UV CD data of a soluble islolate of the aggregated product exhibiting a 28% loss of alpha-helix offset by a 31% gain in beta-sheet. This information combined with the thermal reversibility data provided compelling evidence that unfolding was a key event in the aggregation pathway at 50 degrees C. Minimization of aggregation was achieved at pH 6 and corroborated by evidence acquired from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion data. Correspondingly, the bioactivity was found to be optimal at pH 6. The findings link thermal reversibility to the propensity of Flt3 ligand to aggregate once unfolded in the Tm plateau region and provide a basis for relating the reversibility of thermal denaturation to the prediction of long-term storage stability in aqueous solution.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Animales , Rastreo Diferencial de Calorimetría , Línea Celular , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Calor , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Proteínas de la Membrana/metabolismo , Ratones , Oxidación-Reducción , Conformación Proteica , Desnaturalización Proteica , Dodecil Sulfato de SodioRESUMEN
The desire to deliver protein and peptide biopharmaceuticals conveniently and effectively has led to intense investigation of site-specific drug-delivery systems. Despite challenges, progress towards the convenient noninvasive delivery of proteins and peptides has been achieved through specific routes of administration. In addition, the delivery of proteins and peptides to specific sites of action has been utilized to lower the total delivered dose, to gain access to specific organs or body compartments and to concentrate a therapeutic dose at a specific site of pharmacological action.
Asunto(s)
Biotecnología/tendencias , Sistemas de Liberación de Medicamentos/tendencias , Péptidos/administración & dosificación , Proteínas/administración & dosificación , Biofarmacia , Humanos , Péptidos/farmacocinética , Proteínas/farmacocinéticaRESUMEN
PURPOSE: To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC). METHODS: Optimal pH conditions were determined by monitoring degradation products encountered during accelerated studies (at elevated temperatures) using SDS-PAGE. At the pH optimum, DSC screened for excipients that enhanced thermal stability by shifting the Tm to higher values. Using SEC the relationship between thermal unfolding and stability was investigated by considering if lower Tm's in the presence of preservatives correlated with degradation products at 37 degrees C over time. The degree of aggregation relative to that of a control determined the level of stability achieved. RESULTS: Circular dichroism (CD) measurements confirmed molecular modeling studies showing IL-1R to be about 39% beta-sheet. Two major transitions characterized the DSC data with Tm's observed near 47 degrees C and 66 degrees C. Among 21 excipients screened, NaCl exhibited the greatest stabilizing influences based on shifting the low temperature transition to 53 degrees C. The low temperature transition was later found to comprise two transitions, yielding a total of three melting transitions for IL-1R. High Tm's arising from the presence of preservatives correlated with the order of stability (i.e., 0.065% phenol > 0.1% m-Cresol > 0.9% benzyl alcohol). CONCLUSIONS: The three melting transitions are consistent in origin with the cooperative unfolding of three unique immunoglobulin-like domains of IL-1R. Optimal stability was achieved in 20 mM sodium citrate at pH 6 with sufficient NaCl to attain the tonicity of human serum. A correlation between the predicted ranking of stability and the extent of aggregation was demonstrated using DSC.
Asunto(s)
Soluciones Farmacéuticas/química , Receptores de Interleucina-1/química , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Estabilidad de Medicamentos , Excipientes , Calor , Humanos , Modelos Moleculares , Proteínas Recombinantes/química , Espectrofotometría UltravioletaRESUMEN
The stability of thiotepa in a new formulation of the drug was studied. Vials of Thioplex (Immunex), a relatively new lyophilized formulation of thiotepa, were reconstituted with sterile water and diluted with 0.9% sodium chloride injection in polyvinyl chloride infusion bags to thiotepa concentrations of 0.5, 1, and 3 mg/mL. The solutions were stored at 8 and 25 degrees C in ambient light and analyzed at 0, 8, 24, and in most cases 48 hours for thiotepa concentration and chloro-adduct formation by stability-indicating high-performance liquid chromatography. Thiotepa 1 and 3 mg/mL was stable for 48 hours at 8 degrees C and for 24 hours at 25 degrees C. Thiotepa 0.5 mg/mL was not stable at either temperature. Storage at 8 degrees C slowed but did not prevent chloro-adduct formation and loss of potency. The pH tended to increase with time; turbidity remained low. Thiotepa (lyophilized) 1 and 3 mg/mL in 0.9% sodium chloride injection was stable for 48 hours at 8 degrees C and for 24 hours at 25 degrees C; the drug was unstable when diluted to 0.5 mg/mL and stored under the same conditions.
Asunto(s)
Antineoplásicos Alquilantes/química , Tiotepa/química , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Concentración de Iones de Hidrógeno , Nefelometría y Turbidimetría , Concentración Osmolar , Cloruro de Sodio , TemperaturaRESUMEN
PURPOSE: This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres. METHODS: GM-CSF was encapsulated in PLGA/PLA microspheres by a novel silicone oil based phase separation process. Several different blends of PLGA and low molecular weight PLA were used to prepare the microspheres. The microspheres and the encapsulated GM-CSF were extensively characterized both in vitro and in vivo. RESULTS: Steady release of GM-CSF was achieved over a period of about one week without significant "burst" of protein from the microspheres. Analysis of microsphere degradation kinetics by gel permeation chromatography (GPC) indicated that low molecular weight PLA enhanced the degradation of the PLGA and thereby affected release kinetics. GM-CSF released from the microspheres was found to be biologically active and physically intact by bioassay and chromatographic analysis. Analysis of serum from mice receiving huGM-CSF indicated that the GM-CSF was biologically active and that a concentration of greater than 10 ng/mL was maintained for a period lasting at least nine days. MuGM-CSF was not detected following in vivo administration of muGM-CSF microspheres. The tissues of mice receiving muGM-CSF microspheres were characterized by infiltration of neutrophils, and macrophages which were in significant excess of those found in mice administered with placebo controls (i.e. microspheres without GM-CSF). CONCLUSIONS: This study demonstrates the influence of formulation parameters on the encapsulation of GM-CSF in PLGA/PLA microspheres and its controlled release in biologically active form. The intense local tissue reaction in mice to muGM-CSF microspheres demonstrates the importance of the mode of delivery on the pharmacologic activity of GM-CSF.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Animales , Preparaciones de Acción Retardada , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacocinética , Histocitoquímica , Humanos , Inyecciones Subcutáneas , Cinética , Ratones , Ratones Endogámicos C57BL , Microesferas , Poliésteres , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Proteínas RecombinantesRESUMEN
Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of IL-15 interferes with beta but not alpha receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block beta and gamma receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of beta receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.
Asunto(s)
Interleucina-15/química , Modelos Químicos , Polietilenglicoles/metabolismo , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Humanos , Interleucina-15/metabolismo , Interleucina-15/farmacocinética , Interleucina-2/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-ActividadAsunto(s)
Factor de Crecimiento Transformador beta/química , Secuencia de Aminoácidos , Animales , Estabilidad de Medicamentos , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Enhancement of bone ingrowth with transforming growth factor-beta was evaluated in a canine model. Ten dogs had bilateral implantation of a titanium-fiber-metal-coated rod in the proximal part of the humerus. A three-millimeter gap between the outer surface of the porous coating and the surrounding cancellous bone was created to impair bone ingrowth. All of the implants were plasma-flame-sprayed with hydroxyapatite and tricalcium phosphate. In each animal, one implant was also treated with recombinant transforming growth factor-beta 1 while the other implant, which was not so treated, served as a paired control. Two doses of transforming growth factor-beta 1 were used: 335 micrograms in five animals and 120 micrograms in the other five. At four weeks, the amount of bone ingrowth in the implants that had been treated with 120 micrograms of transforming growth factor-beta 1 was threefold higher than that in the paired controls (p = 0.009), but with the numbers available there was no significant increase in bone ingrowth with the higher dose. The amount of new-bone formation in the three-millimeter gaps adjacent to the treated implants was twice that in the gaps of the paired controls, regardless of the dose. The differences between the treated and control implants with regard to the architecture of the new bone in the gap indicate that the mechanism of action of transforming growth factor-beta 1 may include both proliferation of osteoprogenitor cells and production of matrix by committed osteoblasts. Compared with the findings in a previous study in which this canine model was used, the data from the present investigation indicate that enhancement of bone ingrowth in implants that have been treated with a combination of a hydroxyapatite-tricalcium phosphate coating and transforming growth factor-beta 1 may exceed that obtainable with grafting of the gap with autogenous cancellous bone.
Asunto(s)
Oseointegración/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Clavos Ortopédicos , Fosfatos de Calcio , Perros , Durapatita , Húmero/cirugía , Húmero/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Proteínas Recombinantes/farmacología , Factores de Tiempo , TitanioRESUMEN
We have reviewed a large cross-section of degradable polymeric delivery systems for protein and peptide pharmaceuticals. These systems include monolithic type devices in which the drug is dispersed throughout the polymer and protein-polymer conjugates where the drug is covalently bound to the polymer. These delivery systems have unique challenges associated with their development that are related to both protein stability and protein release kinetics. Despite numerous reports in the scientific literature which include many encouraging results in preclinical models, very few of these systems have been developed into viable products. The products that have made it to market, however, have proven to be very successful and demonstrate the significant advantages that these systems can provide. The continuous advances in biotechnology will produce more proteins and peptides that will be difficult to administer by conventional means, and an increased demand for controlled or site-specific delivery systems is anticipated.
Asunto(s)
Materiales Biocompatibles , Portadores de Fármacos , Péptidos/administración & dosificación , Polímeros , Proteínas/administración & dosificación , Alginatos , Animales , Biodegradación Ambiental , Cápsulas , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Datos de Secuencia Molecular , PolietilenglicolesRESUMEN
The impaired wound healing associated with aging may reflect inadequate secretion or delivery of cytokines. Transforming growth factor-beta(1) is a mitogenic polypeptide with beneficial effects on wound healing. In the present study we questioned whether topical administration of transforming growth factor-beta(1) could improve the wound healing process in aged rats in vivo. Wound repair (from 1 to 14 days) was analyzed in full-thickness incisional wounds from 2-year-old rats with or without a single topical application of transforming growth factor-beta(1) (1 microg/wound) at the time of wounding. Identical wounds from 3-month-old, untreated rats served as controls. Histologic analysis showed a marked delay in several aspects of wound repair in the aged rats in comparison with that noted in the younger animals. Immunostaining of the wounds for proliferating cell nuclear antigen showed a reduction in the number of cycling fibroblasts in old rats. In addition, the number of capillaries per unit area of the wound as determined by a stain for Griffonin (Bandeiraea) simplicifolia lectin, and the number of inflammatory cells as identified by an antibody specific for macrophages, were also reduced in the wound area in old rats. Treatment with transforming growth factor-beta(1) resulted in marked enhancement of the following parameters: cell proliferation, inflammatory cell and fibroblast influx, wound closure, and angiogenesis. As seen with in situ hybridization, a similar temporal pattern of expression of messenger RNAs corresponding to type I procollagen and Secreted Protein, Acidic and Rich in Cysteine (osteonectin), known to be prevalent in healing wounds, was observed in both young and aged rats. However, the levels of mRNA corresponding to these secreted proteins appeared to be reduced in wound tissue from aged rats. Treatment with transforming growth factor-beta(1) subsequently resulted in an increase in the expression of both type I procollagen and Secreted Protein, Acidic and Rich in Cysteine mRNA in the wound tissue from aged rats. In summary, a single topical application of transforming growth factor-beta(1) to the wounds of aged rats at the time of wounding was associated with a healing response that, in all the parameters of wound repair examined, was similar to that of young rats. Topical transforming growth factor-beta(1) might therefore be beneficial in the treatment of dermal wounds in the aged.
RESUMEN
Transforming growth factor-beta 1 (TGF-beta 1) has beneficial effects on wound healing. However, the ideal method for its administration to the wound site remains unknown. Our aim was to analyze the release of TGF-beta 1 from different formulations and to study whether the changes in wound healing by TGF-beta 1 depend on its topical delivery system. For the studies the TGF-beta 1 was incorporated into phosphate-buffered saline, into a polyoxamer gel, into DuoDERM hydroactive paste, and into a poly(ethylene oxide) hydrogel. The release of 125I-labeled TGF-beta 1 from carriers was measured in full-thickness wounds in rats and the healing of the wounds was analyzed by histology and wound area measurements. The TGF-beta 1 was released from all formulations at a different rate and in an active form as determined by growth inhibition assay. Wound size measurements and the analysis on the amount of cellular influx, fibroplasia, and granulation tissue showed that a single dose (1 microgram/wound) of locally administered TGF-beta 1 significantly (P < 0.01) enhanced the wound healing. This effect was most prominent with polyoxamer gel formulation, which provided the most sustained release of TGF-beta 1. Our finding that the enhancement in wound healing by TGF-beta 1 was significantly dependent on the carrier used for its topical delivery to the wound site is novel and shows the importance of using adequate delivery systems when growth factors are used to enhance wound repair.
Asunto(s)
Sistemas de Liberación de Medicamentos , Factor de Crecimiento Transformador beta/administración & dosificación , Cicatrización de Heridas , Administración Tópica , Animales , Vendas Hidrocoloidales , Tampones (Química) , Coloides , Portadores de Fármacos , Masculino , Apósitos Oclusivos , Fosfatos , Poloxaleno , Polietilenglicoles , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Piel/lesiones , Piel/patología , Factor de Crecimiento Transformador beta/uso terapéuticoRESUMEN
BACKGROUND/AIMS: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa. METHODS: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen. RESULTS: Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration. CONCLUSIONS: This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.
Asunto(s)
Sistemas de Liberación de Medicamentos , Mucosa Intestinal/citología , Factor de Crecimiento Transformador beta/administración & dosificación , Administración Oral , Alginatos , Animales , División Celular , Células Epiteliales , Epitelio/inmunología , Ácido Glucurónico , Ácidos Hexurónicos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Mucosa Intestinal/inmunología , Masculino , Índice Mitótico , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Células Madre/citología , Células Madre/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The ability to transforming growth factor-beta 1 (TGF-beta 1), to stimulate bone healing was evaluated in a rat critical calvarial defect model. Both a low dose and a high dose of TGF-beta 1 were incorporated into two different types of implants: one made from a composite of poly(lactic-co-glycolic acid) (PLPG) (50:50) and demineralized bone matrix (DBM), and the other from calcium sulfate (CaSO 4). Scanning electron microscopy showed that the CaSO 4 implants were more porous than the PLPG/DBM samples. Both types of implants released biologically active TGF-beta 1 for over 300 h in vitro. The samples were implanted in a 9-mm diameter rat calvarial defect for 6 weeks along with contralateral control implants containing no TGF-beta 1. Microradiography and histological analysis were used to assess the bone healing in the defects. Microradiography revealed that the greatest amount of calcified bone (67.5%) was present in in the CaSO 4 implants containing a high dose of TGF-beta 1 while minimal new bone formation occurred in the PLPG/DBM implants. Histologically, the PLPG/DBM implants exhibited an inflammatory response with little mineralization or bone formation. The defects containing the PLPG/DBM implants consisted of a connective tissue stroma with large void spaces. Giant cells and numerous polymorphonuclear leukocytes were present throughout the implants. In contrast, the CaSO 4 implants had only a few inflammatory cells and the presence of mineralization and true bone was a more consistent feature. These preliminary studies show that TGF-beta 1 is capable of inducing new bone formation. Furthermore, the materials used to deliver the growth factor can play a significant role in the bone healing process.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Sustancias de Crecimiento , Prótesis e Implantes , Cicatrización de Heridas , Animales , Materiales Biocompatibles , Biodegradación Ambiental , Regeneración Ósea/fisiología , Sulfato de Calcio/química , Sulfato de Calcio/farmacocinética , Sustancias de Crecimiento/administración & dosificación , Sustancias de Crecimiento/química , Sustancias de Crecimiento/farmacocinética , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Polímeros/química , Polímeros/farmacocinética , Porosidad , Conejos , Ratas Sprague-DawleyRESUMEN
An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein-protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina M/química , Povidona/química , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Excipientes , Calor , Luz , Peso Molecular , Dispersión de RadiaciónRESUMEN
Proteins are subject to a variety of physical and chemical reactions that lead to a loss of activity. These reactions are a particular problem in controlled-release devices, where temperatures and protein concentrations are high. Current approaches to increasing protein stability include the addition of saccharides, amino acids, or polymers. New synthetic polymers may be promising protein stabilizers because properties such as molecular weight and side-chain composition can be controlled. In this study, the stability of a murine monoclonal antibody, BR96, was evaluated in solution at 37 degrees C. The antibody was incubated in the presence of a series of synthetic polymers that included poly(glucosylethyl methacrylate) (GEMA) and copolymers of N-vinylpyrrolidone (NVP) and methyl methacrylate (MMA). Samples were taken periodically up to 30 days. The formation of precipitated antibody in particulate aggregates was measured with a Coulter counter, and the molecular-weight distribution of soluble antibody was measured by size-exclusion chromatography. Two trends were evident. First, with poly(GEMA) and copolymers of NVP and MMA, protein aggregation increased at higher polymer concentrations. Second, higher molecular weights of the poly(NVP) homopolymer also led to increases in protein aggregation. Effects of polymer hydrophobicity were more complex. A copolymer containing 9 mol% MMA caused immediate protein precipitation, while a copolymer containing 21 mol% MMA did not. The effects of the copolymer containing 21% MMA were strongly concentration dependent. At 1 wt%, the polymer reduced aggregation, but aggregation increased strongly between concentrations of 2 and 3 wt%.
Asunto(s)
Anticuerpos Monoclonales/química , Metilmetacrilatos , Povidona , Animales , Fenómenos Químicos , Química Física , Cromatografía en Gel , Estabilidad de Medicamentos , Etilenodiaminas , Glucósidos/química , Metilmetacrilatos/química , Ratones , Peso Molecular , Polímeros/química , Povidona/química , Desnaturalización Proteica , Solubilidad , AguaRESUMEN
Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-beta 1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-beta 1 to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-beta 1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-beta 1 released was determined using several methods including 125I-labeled TGF-beta 1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-beta 1 released was directly proportional to both the TGF-beta 1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-beta 1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.