RESUMO
OBJECTIVE: To examine the potential of insulin, in a sustained delivery system, as a treatment for arthritis. DESIGN: The effect of insulin on matrix synthesis, matrix breakdown, and nitric oxide production in primary cartilage explants was examined. The activity of insulin on diseased cartilage from Dunkin Hartley guinea pigs, diabetic mice, and osteoarthritic patients was measured. The specificity of insulin stimulation was compared to that of IGF-I using osteoblasts and fibroblasts. Finally, the stability of insulin in a biologically relevant system was tested, and a slow-release formulation of insulin was developed and characterized. RESULTS: In articular cartilage explants, insulin stimulated proteoglycan (PG) synthesis, inhibited PG release and nitric oxide production, and overcame the detrimental effects of interleukin 1 (IL-1). The mechanism whereby insulin decreased matrix breakdown was through inhibition of aggrecanase activity. Insulin was active on cartilage at concentrations at which insulin does not cross-react with insulin-like growth factor I (IGF-I) receptors nor stimulate proliferation of other cells types. The response of cartilage to insulin did not diminish with age or disease. Insulin stimulated matrix synthesis in osteoarthritic cartilage and local treatment with insulin overcame endogenous suppression of matrix synthesis in diabetic cartilage. Poly-lactic-coglycolic acid (PLGA) was found to be an effective carrier for delivery of insulin, and PLGA-Insulin was active on articular cartilage in vitro and in vivo. CONCLUSIONS: As the incidence of arthritis increases with the aging population, an effective therapy to induce repair of cartilage is needed. Based on its biological activities, insulin appears to be an attractive protein therapeutic candidate. Maximum insulin effectiveness may require a sustained delivery system.
Assuntos
Insulina/administração & dosagem , Osteoartrite do Joelho/tratamento farmacológico , Idoso , Animais , Disponibilidade Biológica , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Preparações de Ação Retardada , Diabetes Mellitus Experimental/metabolismo , Estabilidade de Medicamentos , Feminino , Cobaias , Humanos , Insulina/farmacocinética , Insulina/uso terapêutico , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoartrite do Joelho/metabolismo , Proteoglicanas/biossínteseRESUMO
The development of a stable sustained-release formulation of recombinant human nerve growth factor (rhNGF) for the treatment of neuronal diseases is described. The protein was encapsulated into poly(lactic-co-glycolic) acid (PLGA) microspheres using a spray freeze drying technique. Liquid nitrogen and cold ethanol were used to spray-freeze-dry solid rhNGF that had been suspended in a solution of PLGA dissolved in ethyl acetate. When excipients such as sugar (trehalose), surfactant (pluronic F68), and poly(ethylene glycol) (PEG) were added to the PLGA formulation to protect rhNGF from degradation during spray freeze drying, the protein degraded via aggregation during in vitro release. The formation of an insoluble rhNGF-zinc complex prior to encapsulation into PLGA microspheres stabilized the protein during both microencapsulation and release. In this study, we have demonstrated that the addition of zinc acetate in a 1:12 rhNGF-to-zinc acetate molar ratio in a solid rhNGF formulation (4 mM sodium bicarbonate at pH 7.4) improves stability of rhNGF during release at 37 degrees C (physiological temperature). The stabilization may be due to rhNGF complexation with zinc to form stable aggregates. The PLGA formulation consisting of 10% rhNGF encapsulated in 12 kDa PLGA (50:50 lactide/glycolide) provided a continuous release of 14 days. The low initial burst (approximately 1%) and controlled-release rate were achieved by the addition of 3 or 6% solid zinc carbonate to the polymer phase during microencapsulation.
Assuntos
Materiais Biocompatíveis/farmacocinética , Ácido Láctico/farmacocinética , Fator de Crescimento Neural/farmacocinética , Ácido Poliglicólico/farmacocinética , Polímeros/farmacocinética , Acetatos/farmacocinética , Animais , Células CHO , Química Farmacêutica , Cricetinae , Preparações de Ação Retardada/farmacocinética , Estabilidade de Medicamentos , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Acetato de Zinco/farmacocinéticaRESUMO
PURPOSE: The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. METHODS: A liquid formulation of rhVEGF was exposed to fluorescent light (2 x 10(4) lux for up to 4 weeks), hydrogen peroxide (H2O2), or t-butythydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. RESULTS: Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78.81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%. possibly due to the role of Met 18in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H2O2, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H2O2 and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. CONCLUSIONS: Methionine residues with exposed surface areas greater than 65 A2 and sulfur surface areas greater than 16 A2 were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H2O2 to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 A2, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.
Assuntos
Fatores de Crescimento Endotelial/química , Linfocinas/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cristalografia por Raios X , Fatores de Crescimento Endotelial/efeitos da radiação , Humanos , Hidrólise , Luz , Linfocinas/efeitos da radiação , Metionina/química , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação , Enxofre/química , Propriedades de Superfície , Tripsina , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Although preclinical animal studies have demonstrated the utility of recombinant human vascular endothelial growth factor (rhVEGF) in promoting neovascularization in regions of ischemia, rhVEGF systemic administration did not provide clinical benefit to patients in recent placebo-controlled Phase II clinical trials. The amount of rhVEGF localized in the ischemic region after systemic administration is minimal and does not persist for more than 1 day. A greater persistence of rhVEGF at the region of ischemia may provide an increased angiogenesis with the eventual formation of patent blood vessels to restore nourishment to the tissues. We sought to develop a formulation of rhVEGF in poly(D,L-lactide--co-glycolide) (PLG) microspheres that would provide a continuous local delivery of intact protein. A stable formulation of rhVEGF for encapsulation contained a small amount of a stabilizing sugar, trehalose. Addition of excess trehalose increased the rate of release from the PLG. In addition, PLG with free acid end groups appeared to retard the initial release of rhVEGF by associating with it through ionic interactions at the positively charged heparin binding domain. rhVEGF was released continuously for 21 days with a very low (less than 10%) initial burst. The released rhVEGF aggregated and hydrolyzed over time and lost heparin affinity but not receptor affinity. The compression molding of rhVEGF PLG microspheres into disks yielded formulations with a low initial release and a lag of 10 days followed by complete release. The PLG microsphere formulations were assessed in the corneal implant model of angiogenesis and generated a dose-dependent angiogenic response. These formulations were also administered intravitreally and subretinally, generating local neovascularization comparable to the human disease states, vitroretinopathy and age-related macular degeneration, respectively. The rhVEGF PLG formulations may increase local angiogenesis without systemic side effects and may also be useful in the development of ocular disease models.
Assuntos
Portadores de Fármacos/química , Fatores de Crescimento Endotelial/administração & dosagem , Fatores de Crescimento Endotelial/farmacologia , Ácido Láctico/química , Linfocinas/administração & dosagem , Linfocinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Ácido Poliglicólico/química , Polímeros/química , Animais , Cromatografia de Afinidade , Cromatografia em Gel , Córnea/irrigação sanguínea , Heparina/química , Microscopia Eletrônica de Varredura , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Solubilidade , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The equilibrium dissociation of recombinant human IFN-gamma was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was -209 +/- 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 +/- 1.6 nm2/molecule of dimer. The first-order aggregation rate of recombinant human IFN-gamma in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were -41 +/- 9 ml/mol of dimer and 3.5 +/- 0.2 nm2/molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values approximately 4-fold larger for both transitions.
Assuntos
Interferon gama/química , Dimerização , Guanidina , Humanos , Cinética , Modelos Teóricos , Pressão , Desnaturação Proteica , Proteínas Recombinantes , Espectrofotometria , Propriedades de Superfície , Termodinâmica , ÁguaRESUMO
The efficient and safe delivery of therapeutic proteins is the key to commercial success and, in some cases, the demonstration of efficacy in current and future biotechnology products. Numerous delivery technologies and companies have evolved over the past year. To critically evaluate the available options, each method must be assessed in terms of how easily it can be manufactured, impact on protein quality, bioavailability, and toxicity. Recent advances in depot delivery systems have, for the most part, overcome all of these obstacles except for complex and costly manufacturing. On the other hand, pulmonary delivery usually involves efficient manufacturing, but low protein bioavailability resulting in higher doses compared with injections. Although recent advances in transdermal and oral delivery have been significant, both of these delivery routes require logarithmic increases in bioavailability to make them viable candidates for commercialization. In the next few years, protein delivery for commercial products will probably be limited to injection devices, depot systems and pulmonary administration.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas/administração & dosagem , Disponibilidade Biológica , Vias de Administração de Medicamentos , Humanos , Infusões Parenterais , Proteínas/farmacocinética , Proteínas/uso terapêuticoRESUMO
The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.
Assuntos
Anticorpos Monoclonais/química , Proteínas/química , Cromatografia Líquida/métodos , Estabilidade de Medicamentos , Excipientes/química , Liofilização , Humanos , Concentração Osmolar , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
Metal-catalyzed oxidation (MCO) represents a prominent pathway of protein degradation. To evaluate the importance of the integrity of the metal-binding site on MCO, we subjected recombinant human growth hormone (rhGH), to MCO (ascorbate, Cu(2+), (3)O(2)) in the presence of various aliphatic alcohols (ethanol, ethylene glycol, trifluoroethanol, 1-propanol, 2-propanol, 1,2-propylene glycol, 1-butanol, 2-butanol, and tert-butanol). All alcohols inhibited MCO in a concentration-dependent and sigmoidal manner. Half-points, P(1/2), were dependent on the nature of the alcohol. Circular dichroism and fluorescence spectroscopy were used to monitor cosolvent-induced secondary and tertiary structural changes. The presence of alcohols increased the helical content of rhGH and induced a red shift in the tryptophan emission. The midpoints of the tertiary structural change correlated with the P(1/2) values. Solvent polarity at P(1/2) was determined according to the E(T)(30) scale. All alcohol/water mixtures at P(1/2) had rather similar solvent polarities between 54.5 to 56.4 kcal/mol, with the exception of ethylene glycol. On the other hand, no correlation was obtained between the protection against MCO and the hydroxyl radical-scavenging properties of the cosolvent. We conclude that the primary mechanism of MCO inhibition is a cosolvent-induced conformational perturbation of the metal-binding site as opposed to pure radical scavenging.
Assuntos
Hormônio do Crescimento Humano/metabolismo , Metais/química , Catálise , Hormônio do Crescimento Humano/química , Radical Hidroxila/química , Modelos Moleculares , Oxirredução , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solventes/químicaRESUMO
Metal-induced precipitation of protein therapeutics is being used and further developed as a processing step in protein formulation and may have utility in protein purification and bulk storage. In such processes, it is imperative that native protein structure is maintained and the metal complexation is reversible. In the current study, we investigated the effects of zinc-induced precipitation on recombinant human growth hormone (rhGH) and recombinant human nerve growth factor (rhNGF). On the addition of ethylenediaminetetraacetic acid (EDTA), the precipitates were dissolved, yielding complete recovery of native protein in both cases. Both proteins have specific metal binding sites and require specific molar ratios of zinc to protein to initiate precipitation (zinc:rhGH > 2:1; zinc:rhNGF > 18:1). Furthermore, the secondary structures of both proteins were unperturbed in soluble zinc complexes and zinc-induced precipitates, as measured by infrared and circular dichroism spectroscopies. The soluble zinc complex of rhGH had minor tertiary structural alterations, whereas zinc binding did not alter the tertiary structure of rhNGF. These studies indicated that metal-induced precipitation provides a method to maintain proteins in their native state in precipitates, which may be useful for purification, storage, and formulation.
Assuntos
Precipitação Química , Hormônio do Crescimento Humano/química , Fator de Crescimento Neural/química , Zinco/química , Sítios de Ligação , Hormônio do Crescimento Humano/efeitos dos fármacos , Humanos , Fator de Crescimento Neural/efeitos dos fármacos , Zinco/farmacologiaRESUMO
The effects of a histidine (His) residue located on the C-terminal side of an asparaginyl (Asn) residue on the rate of deamidation were studied using Gly-Gln-Asn-X-His pentapeptides. The rates of deamidation of the pentapeptides were determined at 37 degrees C (I = 0.5) as function of pH, buffer species, and buffer concentration. A capillary electrophoresis stability-indicating assay was developed to monitor simultaneously the disappearance of the starting peptides and the appearance of the degradation products. The rates of degradation of the peptides were pH dependent, increasing with pH, and followed apparent first-order kinetics. At pH values <6.5, Gly-Gln-Asn-His-His degraded faster than Gly-Gln-Asn-Gly-His, suggesting that the N+1 His residue is catalyzing the deamidation of the Asn residue. The His side chain at these pH values could function as a general acid catalyst, stabilizing the oxyanionic transition state of the cyclic imide intermediate formation. In contrast, at pH values >6.5, Gly-Gln-Asn-Gly-His deamidates more rapidly than Gly-Gln-Asn-His-His. The bulk of the side chain of the N+1 His residue versus the N+1 Gly residue apparently inhibits the flexibility of the peptide around the reaction site and, consequently, reduces the rate of the reaction. The significance of this steric hindrance effect of the N+1 His residue on the rate of deamidation was examined further. It was observed that at pH >6.0, Gly-Gln-Asn-His-His undergoes deamidation faster than Gly-Gln-Asn-Val-His. This observation indicated that, at the higher pH values, the N+1 His residue is also acting as a catalyst. Thus, at basic pH, the N+1 His residue influences the rate of deamidation via two opposing effects; that is, general base catalysis and steric interference. The pentapeptide Gly-Gln-Asn-His-His, in addition to undergoing the deamidation reaction, also undergoes bond cleavage at the Asn-His peptide bond. The enhanced rate of Asn-His peptide bond cleavage can be attributed to the general base behavior of the His residue, leading to increased nucleophilicity of the Asn side-chain amide group. Finally, we have shown that the His residue that is two amino acids removed from the Asn, the N+2 position, has little or no effect on the rate of deamidation.
Assuntos
Asparagina/química , Histidina/química , Oligopeptídeos/química , Amidas/química , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , CinéticaRESUMO
Recombinant human insulin-like growth factor-I (rhIGF-I) was found to improve glycemic control and enhance insulin sensitivity in patients with a syndrome of severe insulin resistance. Therefore, the protein may be considered as an alternative therapy in the treatment of diabetes when the patients become insensitive to insulin treatment. Because the protein was administered twice per day in the clinical trials, a sustained release polylactic-co-glycolic acid (PLGA) formulation for rhIGF-I with low initial burst (<20%), maximum possible protein loading (15-20%) and a continuous release of 1-2 weeks may provide greater patient convenience and compliance. The protein was encapsulated in PLGA for sustained release using a spray freeze-drying technique. Formulation parameters such as protein loading, polymer end group, and the presence of zinc carbonate were studied for their effects on in vitro release of rhIGF-I from PLGA microspheres. As the protein loading was increased, the initial burst increased. Due to the hydrophilic properties of the polymers, rhIGF-I encapsulated in unblocked PLGA (free acid end groups) gave a lower initial burst and a more steady-state release profile than the blocked PLGA (hydrocarbon end groups) with the same protein loading and PLGA molecular weight. At 15% w/w protein loading, the addition of 6% w/w zinc carbonate as a protein release modifier to the unblocked PLGA (12 kDa) decreased the initial burst of rhIGF-I. Therefore, a formulation consisting of 15% rhIGF-I and 6% zinc carbonate in 12 kDa, unblocked 50:50 PLGA can provide the required release characteristics in vitro. Rat studies revealed that rhIGF-I in this formulation was released in vivo at a rate which was comparable to that observed in vitro. These studies demonstrate the potential for a sustained release, 14-day formulation for rhIGF-I.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/uso terapêutico , Animais , Carbonatos/química , Preparações de Ação Retardada , Diabetes Mellitus/genética , Composição de Medicamentos , Feminino , Fator de Crescimento Insulin-Like I/metabolismo , Ácido Láctico , Microscopia Eletrônica de Varredura , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Compostos de Zinco/químicaRESUMO
In this work, we examine the way in which stability information obtained from studies on small model peptides correlates with similar information acquired from a protein. The rates of deamidation, oxidation, and diketopiperazine reactions in model peptide systems were compared to those of recombinant human vascular endothelial growth factor (rhVEGF). The N-terminal residues of rhVEGF, a potent mitogen in angiogenesis, are susceptible to the aforementioned reactions. The degradation of the peptides L-Ala-L-Pro-L-Met (APM) and Gly-L-Gln-L-Asn-L-His-L-His (GQNHH), residues 1-3 and 8-12 of rhVEGF, respectively, and rhVEGF were examined at pH 5 and 8 at 37 degrees C. Capillary electrophoresis and high-performance liquid chromatography (HPLC) stability-indicating assays were developed to monitor the degradation of the penta- and tripeptides, respectively. The degradation of rhVEGF was determined by tryptic mapping and quantified by RP-HPLC. The rates of degradation of both peptides and the protein followed apparent first-order kinetics and increased with increasing pH. The tripeptide APM underwent diketopiperazine formation (Ala-Pro-diketopiperazine) and oxidation of the Met residue, whereas the pentapeptide GQNHH degraded via the deamidation pathway. The results indicate that the rates of deamidation and oxidation of the protein are comparable to those observed in the model peptides at both pH values. However, the rate of the diketo-piperazine reaction was slower in the protein than in the model peptide, which may be the result of differences in the cis-trans equilibrium of the X-Pro peptide bonds in the 2 molecules.
Assuntos
Fatores de Crescimento Endotelial/química , Linfocinas/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Piperazinas/química , Amidas/química , Cromatografia Líquida de Alta Pressão , Dicetopiperazinas , Eletroforese Capilar , Humanos , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Proteínas Recombinantes/química , Temperatura , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
PURPOSE: Three different polymeric delivery systems, composed of either poly(ethylene-co-vinyl acetate) (EVAc) or poly(lactide-co-glycolide) (PLGA), were used to administer recombinant human nerve growth factor (rhNGF) intracranially in rats. METHODS: The delivery systems were characterized with respect to release kinetics, both in the brain and in well-stirred buffer solutions. RESULTS: During incubation in buffered saline, the delivery systems released rhNGF in distinct patterns: sustained (EVAc), immediate (PLGA1) and delayed (PLGA2). One 10-mg delivery system was implanted in each rat and an ELISA technique was used to determine the amount of rhNGF in 1-mm coronal brain slices produced immediately after removal of the delivery system. High levels of rhNGF (as high as 60,000 ng in a brain slice of approximately 50 microliters) were recovered from the brain tissue at 1, 2, and 4 weeks after implantation. With all three delivery systems, the amount of rhNGF in each brain slice decreased exponentially with distance from the implant site: the distance over which concentration decreased by 10-fold was 2-3 mm for all delivery systems. When rhNGF release was moderate (10 to 200 ng rhNGF/day), the total amount of rhNGF in the brain increased linearly with release rate, suggesting an overall rate of rhNGF elimination of 0.4 hr-1 or a half-life of 1.7 hr. With higher release rates (500 to 50,000 ng rhNGF/day), total amounts of rhNGF in the brain were considerably higher than anticipated based on this rate of elimination. CONCLUSIONS: Polymeric controlled release can provide high, localized doses of rhNGF in the brain. All of the experimental data were consistent with penetration of rhNGF through the brain tissue with a diffusion coefficient approximately 8 x 10(-7) cm2/s, which is approximately 50% of the diffusion coefficient in water.
Assuntos
Fatores de Crescimento Neural/administração & dosagem , Animais , Biodegradação Ambiental , Encéfalo/metabolismo , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Humanos , Cinética , Ácido Láctico/administração & dosagem , Masculino , Microesferas , Fatores de Crescimento Neural/sangue , Fatores de Crescimento Neural/farmacocinética , Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/administração & dosagem , Polivinil/administração & dosagem , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinética , Distribuição TecidualRESUMO
The most effective mechanism for the elimination of disease from society is the use of vaccinations, but these often require repeated administration. However, single-administration vaccine formulations provide the repeated administrations automatically. One approach is the development of injectable controlled-release microsphere formulations containing the vaccine antigen that is released as a pulse 1-6 months after injection. The time of the pulse is dependent upon the rate of polymer degradation, which is dictated by the polymer's composition and molecular weight. This controlled-release technology may provide complete protection against disease after a single administration.
Assuntos
Preparações de Ação Retardada , Vacinas/administração & dosagem , Complacência (Medida de Distensibilidade) , Sistemas de Liberação de Medicamentos , Proteína gp120 do Envelope de HIV/imunologia , Imunização , Microesferas , Ácido Poliglicólico/farmacologia , Fatores de TempoRESUMO
The purpose of this study was to test the utility of infrared (IR) spectroscopy to determine protein secondary structure in biodegradable microspheres. Encapsulation of proteins within biodegradable polymers, [e.g. poly(lactic-co-glycolic acid) (PLGA)] for controlled drug release has recently been the subject of intense research effort. The ability to assess protein integrity after microsphere production is necessary to successfully produce microspheres that release native proteins. We used IR spectroscopy, a noninvasive method-as opposed to conventional organic solvent extraction or in vitro release at elevated temperature-to assess the secondary structure of recombinant human growth hormone (rhGH) within dry and rehydrated microspheres. PLGA microspheres containing rhGH with different excipients were prepared by a conventional double-emulsion method. The protein IR spectra indicated that the encapsulation process could perturb the structure of rhGH and that excipients could inhibit this damage to varying degrees. A strong positive correlation was found between intensity of the dominant alpha-helical band in the spectra of rhGH in rehydrated microspheres and the percent monomer released from microspheres during incubation in buffer. We also studied microspheres prepared with zinc-precipitated rhGH. The addition of Zn2+ during microsphere processing partially inhibited protein unfolding and fostered complete refolding of rhGH upon rehydration. In conclusion, IR spectroscopy can serve as a valuable tool to assess protein structure within both dried and rehydrated microspheres.
Assuntos
Hormônio do Crescimento/química , Hormônio do Crescimento Humano/química , Preparações de Ação Retardada , Emulsões , Excipientes , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento Humano/administração & dosagem , Humanos , Ácido Láctico , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho , Zinco/químicaRESUMO
Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-gamma (rhIFN-gamma) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-gamma aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-gamma and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-gamma native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-gamma against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.
Assuntos
Interferon gama/química , Cromatografia em Gel , Escherichia coli , Guanidina , Humanos , Cinética , Luz , Modelos Químicos , Ligação Proteica , Proteínas Recombinantes , Espalhamento de Radiação , Sacarose , Tensão SuperficialRESUMO
Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.
Assuntos
Interferon gama/química , Conformação Proteica , Humanos , Dobramento de Proteína , Proteínas Recombinantes , Análise EspectralAssuntos
Composição de Medicamentos , Hormônio do Crescimento Humano/química , Microesferas , Animais , Hormônio do Crescimento Humano/sangue , Ácido Láctico/química , Macaca mulatta , Masculino , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/sangue , Proteínas Recombinantes/químicaRESUMO
Novel drug delivery technologies have now evolved to allow the clinical production of new dosage forms. One new form, biodegradable microspheres, may have utility as a second-generation formulation for parenterally administered proteins and peptides or may be required for the success of some new chemical entities. Here, we have focused on the use of poly(lactic-co-glycolic acid) (PLGA) microspheres to provide a continuous delivery of therapeutic proteins or a pulsatile delivery of protein-based vaccines. To date, our success with solvent evaporation processes has been primarily in the production of microspheres with a triphasic protein release pattern instead of a continuous release. However, with continued development efforts, this method may be used to produce continuous release protein formulations. While this review draws from our own work, there is a great deal of excellent research in this area at both universities and industrial laboratories. This work combined with our studies indicates that this technology holds much promise for new protein formulations that will provide improvements in patient care and, perhaps, increased efficacy.
Assuntos
Materiais Biocompatíveis , Ácido Láctico , Ácido Poliglicólico , Polímeros , Proteínas , Solventes , Vacinas , Biodegradação Ambiental , Preparações de Ação Retardada , Microesferas , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
The subunit vaccine for HIV-1, recombinant glycoprotein 120 (rgp120), was used as a model antigen to evaluate the potential for a pulsatile single immunization vaccine formulation consisting of poly(lactic-co-glycolic) acid (PLGA) microspheres. We designed rgp120 PLGA microsphere formulations that provide a pulse of rgp120 at 1 to 6 months (depending on the polymer) after administration, mimicking another immunization. In these studies, the in vitro pulse of rgp120 correlated well with the observed in vivo autoboost as measured by an increase in anti-gp120 antibodies in guinea pigs. The immune response to the rgp120 PLGA microsphere formulations was increased by adding the soluble form of the saponin-derived adjuvant, QS-21. The use of small microspheres, however, did not increase the humoral response to rgp120. A single immunization with rgp120 PLGA microspheres resuspended in soluble rgp120 and QS-21 elicited neutralizing antibody titers that were comparable to titers obtained from two immunizations of rgp120 and QS-21 at the same total dose. Administration of rgp120 PLGA microspheres in baboons resulted in high, long-lasting neutralizing antibody titers that were greater than repeated immunizations with soluble rgp120 and QS-21. These studies also indicated that a continuous release of QS-21 at the injection site may provide a greater immune response than a bolus injection. Overall, this work demonstrated that PLGA microsphere formulations may be designed to provide in vivo pulses of an antigen eliminating the need for repeated immunizations.