Formulation Of Protein Drugs Research Articles

Injectable hyaluronic acid microhydrogels for controlled release formulation of erythropoietin

An injectable hyaluronic acid (HA) microhydrogel was successfully developed as a novel drug carrier for controlled release formulation of protein drugs. HA hydrogels were prepared by the disulfide bond formation of thiolated HA (HA-SH). EPO was loaded in situ during HA-SH hydrogel preparation using an accelerating agent of sodium tetrathionate. The gelation time was drastically reduced from a day to 30 min when sodium tetrathionate was added for HA-SH hydrogel preparation. In vitro release of EPO in PBS at 37 degrees C showed that EPO was rapidly released for 3 days with an initial burst and then slowly up to 9 days from HA-SH hydrogels. HA-SH microhydrogels were prepared by the reactive spray drying of diluted HA-SH precursor solution. The mean particle size was approximately 2.3 mum and the water content after spray drying was approximately 14%. Ellman's test showed that sodium tetrathionate contributed not only for rapid crosslinking reaction but also for the reduction of residual free thiol content in HA-SH microhydrogels after spray drying. EPO recovery from HA-SH microhydrogels after degradation with hyaluronidase SD was higher than 95%. The released EPO appeared to be intact from the analysis with RP-HPLC. According to in vivo release test of EPO from HA-SH microhydrogels in Sprague Dawley (SD) rats, elevated plasma concentration of EPO higher than 0.1 ng/mL, which is a critical minimal concentration for EPO efficacy, was maintained up to 7 days. There was no adverse effect during and after the in vivo tests.

Integrity and stability studies of precipitated rhBMP-2 microparticles with a focus on ATR-FTIR measurements

A major obstacle in the development of protein drug formulations is the need to maintain the native, active protein structure both during the formulation process and upon long time storage. Controlled precipitation was evaluated for its potential to supply stable microparticulate formulations of bone-regenerating recombinant human Bone Morphogenetic Protein-2 (rhBMP-2). Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) did provide insight into the protein formulation and stability. Temperature dependent ATR-FTIR measurements and DSC measurements allow for the study of changes in the protein structure during melting. To address the question of isomerization, peptide mapping was performed, and protein aggregation was monitored by size exclusion chromatography (SEC). It could be demonstrated by ATR-FTIR that controlled precipitation did not harm the protein and the process is fully reversible. DSC measurements further confirmed these findings. No changes in the transition temperature and process were observed after precipitation and redissolution. Upon storage, isomerization and aggregation could be detected, but to a lower extent in the precipitated formulation as compared to a solution reference. Thus, controlled precipitation of rhBMP-2 is fully reversible and has the potential as alternative formulation tool for the generation of a microparticulate drug delivery system.

Design of long-acting formulation of protein drugs with a double-layer structure and its application to rhG-CSF

In order to design a sustained-release formulation of protein drugs characterized by excellent long-acting properties without an initial burst, a new double-layer minipellet (DL-MP) in which the lateral side of a matrix-type sustained-release formulation ‘minipellet’ using collagen as a carrier was coated with collagen was designed, and its performance was evaluated. In a DL-MP using bovine serum albumin (BSA) as a model drug, the initial burst observed with a single-layer minipellet (SL-MP) was effectively inhibited in an in vitro release test, and the addition of additives such as chondroitin sulfate (CS) permitted control of release rate. This formulation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was then prepared, and its characteristics were determined in normal rats. It was found that blood rhG-CSF concentration was maintained for about 1 week after administration of a DL-MP with additional CS, with persistent increase in white cell count. The results of this study indicated that DL-MP was useful as a long-acting formulation of rhG-CSF characterized by excellent long acting properties without an initial burst.

Evaluation of in vivo release characteristics of protein-loaded biodegradable microspheres in rats and severe combined immunodeficiency disease mice

The in vivo release characteristics of protein-loaded biodegradable microspheres were examined in normal rats and severe combined immunodeficiency disease (SCID) mice. Bovine-derived superoxide dismutase (bSOD), encapsulated into microspheres comprised of poly( d,l-lactic-co-glycolic acid) and poly( d,l-lactic acid), was used as a model protein. Three types of bSOD-loaded microspheres with different release profiles were subcutaneously administered to normal rats. Anti-bSOD antibodies were first detected at day 9 after the administration of these microspheres, which was independent of their release profiles in vitro. A typical formulation with a sigmoidal release profile was subcutaneously administered to SCID mice. No immunoreaction was observed. The plasma bSOD concentration–time profile, determined by an enzyme-linked immunosorbent assay, well reflected the in vitro release profile of the formulation. The disappearance profile of active bSOD from the administration site also partly corresponded to both profiles. However, the relative bioavailability calculated with a profile on the subcutaneous injection of bSOD solution was 40.7%. These results indicated possible instability of bSOD released gradually at the administration site. The results of the present study using SCID mice would be suitable for discussing the in vitro–in vivo correlation of protein release from microspheres, and useful for designing long-term release formulations of protein drugs.

DepoFoam™ technology: a vehicle for controlled delivery of protein and peptide drugs

A major challenge in the development of sustained-release formulations for protein and peptide drugs is to achieve high drug loading sufficient for prolonged therapeutic effect coupled with a high recovery of the protein/peptide. This challenge has been successfully met in the formulation of several peptide and protein drugs using the DepoFoam™, multivesicular lipid-based drug delivery system. DepoFoam technology consists of novel multivesicular liposomes characterized by their unique structure of multiple non-concentric aqueous chambers surrounded by a network of lipid membranes. The objective of this paper is to demonstrate that DepoFoam technology can be used to develop sustained-release formulations of therapeutic proteins and peptides with high loading. DepoFoam formulations of a protein such as insulin, and peptides such as leuprolide, enkephalin and octreotide have been developed and characterized. The data show that these formulations have high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the drug caused by the formulation process, narrow particle size distribution, and spherical particle morphology. Drug release assays conducted in vitro in biological suspending media such as human plasma indicate that these formulations provide sustained release of encapsulated drug over a period from a few days to several weeks, and that the rate of release can be modulated. In vivo pharmacodynamic studies in rats also show a sustained therapeutic effect over a prolonged period. These results demonstrate that the DepoFoam system is capable of efficiently encapsulating therapeutic proteins and peptides and effectively providing controlled delivery of these biologically active macromolecules.

A central composite design to investigate the thermal stabilization of lysozyme.

The formulation and processing of protein drugs requires the stabilization of the native, biologically active structure. Our aim was to investigate the thermal stability of a model protein, lysozyme, in the presence of two model excipients, sucrose and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). We used high sensitivity differential scanning calorimetry (HSDSC) in combination with a central composite design (CCD). As indicators of protein thermal stability, the measured responses were the unfolding transition temperature (Tm), the onset temperature of the denaturation (To), and the extrapolated onset temperature (To,e). A highly significant (F probability <0.001) statistical model resulted from analysis of the data The largest effect was due to pH (over the range 3.2-7.2), and the pH value that maximized Tm was 4.8. Several minor but significant effects were detected that were useful for mechanistic understanding. In particular, the effects of protein concentration and cyclodextrin concentration on Tm and To,e were found to be pH-dependent This was indicative of the partially hydrophilic nature of protein-protein interactions and protein-cyclodextrin interactions, respectively. Response surface methodology (RSM) proved efficient for the modeling and optimization of lysozyme thermal stability as well as for the physical understanding of the protein-sugar-cyclodextrin system in aqueous solution.

Novel delivery system for proteins using collagen as a carrier material: the minipellet

It is critical to develop effective delivery systems for biologically active peptides and proteins since most of them are rapidly destroyed by the body. In this study, interferon (IFN) was used as a model protein drug and matrix type formulations were prepared with natural biodegradable polymers, albumin, gelatin and collagen. The release of IFN from albumin and gelatin was much faster than that of collagen in vitro. The release of IFN from the collagen matrix varied with various processing conditions and were explained based on the density of collagen matrix. IFN was constantly released with a cylindrical solid dosage form prepared by extrusion and air-drying of a 30% (w/w) collagen solution of a high concentration in vitro. Pharmacokinetic studies in mice showed that the serum IFN concentration was maintained for a long time with this solid formulation called minipellet. The advantages of the minipellet are ( 1 ) carrier material is a biodegradable natural protein; (2) it is manufactured under mild processing conditions without any organic solvent or heating process; and (3) it is easily administered in the same way as conventional injections. Therefore, it is applicable to various kinds of biologically active peptides and proteins and expected to facilitate their potential therapeutic use.

Pharmacological and pharmaceutical properties of freeze-dried formulations of egg albumin, indomethacin, olive oil, or fatty acids. II.

To confirm the increased bioavailability of indomethacin (IND) when incorporated in a preparation with egg albumin and olive oil, we studied the detailed pharmaceutical characteristics of a ternary formulation consisting of egg albumin, IND and olive oil. From the results of X-ray powder diffraction measurements, the drug in the formulation was found to be in an amorphous form. When orally administered to rats, the ternary formulation significantly increased the plasma concentration and cumulative biliary and urinary excretion of IND alone as well as the urinary excretion of its major metabolite, desmethylindomethacin, compared with the drug alone. In addition, the dissolution rate of IND from the formulation was higher than that of the drug alone. These results clearly suggest that the bioavailability of IND was markedly improved by incorporating it in a protein-drug formulation containing olive oil as an absorbefacient element, and this effect may be due to an increased absorption of IND.