Takahiro Morita

Formation and isolation of spherical fine protein microparticles through lyophilization of protein-poly(ethylene glycol) aqueous mixture.

AbstractPurpose. Preparation of spherical fine protein microparticles by the lyophilization of a protein-poly(ethylene glycol) (PEG) aqueous mixture was investigated. The main objective was to establish a method for preparing protein microparticles suitable for pharmaceutical production. Methods. Aqueous solutions containing bovine serum albumin (BSA) and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. Analogous studies were performed using several model proteins. A phase diagram of the PEG-BSA aqueous system was obtained by the titration method. Results. The particle size of BSA decreased as the PEG-BSA ratio increased. A bending point was observed in this relationship, at which the PEG-BSA ratio coincided with that of the critical point on the phase diagram of the PEG-BSA system. These results were explained by the freezing-induced condensation, followed by phase separation in the PEG-BSA system. Conclusions. Spherical fine protein microparticles were successfully obtained at high yield and without any activity loss under optimum conditions. This new technology could be applicable to proteins with a wide range of molecular weights, and is expected to be developed for dry powder inhalations or long-term sustained release microsphere formulations.

Preparation of gelatin microparticles by co-lyophilization with poly(ethylene glycol): characterization and application to entrapment into biodegradable microspheres

Gelatin microparticles were prepared by co-lyophilization with poly(ethylene glycol) (PEG) as a protein micronization adjuvant. Aqueous solutions containing gelatin and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. The particle size decreased as the PEG/gelatin ratio increased. The microparticles isolated from the suspension had spherical microdomains with sizes ranging from 1 to 10 microm, which indicated that phase separation between PEG and gelatin during freezing was involved in the formation mechanism of gelatin microparticles. By using this technology, gelatin microparticles with an average size of less than 10 microm, with high purity of more than 90% and with good dispersibility could be obtained with high yield. The gelatin microparticles with average sizes from 5 to 20 microm were applied to encapsulation into biodegradable PLGA/PLA microspheres via a solid-in-oil-in-water emulsion process. The entrapment efficiency was highly dependent on the particle size and the size distribution, signifying that solid microparticles with an average diameter of less than 5 m and an maximal diameter of less than 10 microm would be required for effective encapsulation. These gelatin microparticles would be useful for studying and developing various drug delivery systems.

Improvement of the Pulmonary Absorption of (Asu1,7)-Eel Calcitonin by Various Protease Inhibitors in Rats

The effects of protease inhibitors, Na-glycocholate, bacitracin, bestatin, nafamostat mesilate and soybean trypsin inhibitor (STI) on the pulmonary absorption of (Asu1,7)-eel calcitonin (ECT, molecular weight 3363) were investigated in rats. The pulmonary absorption of ECT was estimated by measuring its hypocalcemic effect. When ECT alone was administered into the lung, the pharmacological availability of ECT was 2.7%. Co-administration with STI or bestatin did not change the pharmacological effect of ECT. However, Na-glycocholate, bacitracin and nafamostat mesilate caused a significant hypocalcemic effect following the pulmonary absorption of ECT and a maximal effect was noted in the presence of 20 mM bacitracin, approaching the effect after intravenous administration of ECT. Bacitracin and Na-glycocholate reduced the degradation of 111In-ECT in rat lung homogenate. Therefore, protease inhibitors effectively improved the pulmonary absorption of ECT.

Enhancement of nasal absorption of large molecular weight compounds by combination of mucolytic agent and nonionic surfactant

For improving the nasal absorption of poorly absorbable hydrophilic compounds, the suitability of a combination of a mucolytic agent, N-acetyl-L-cysteine (NAC), and a nonionic surfactant, polyoxyethylene (C25) lauryl ether (laureth-25), was examined. Rat studies with fluorescent isothiocyanate-labeled dextran (molecular weight ca. 4.4 kDa, FD-4) as a model hydrophilic compound revealed dramatic enhancement of nasal absorption when NAC and laureth-25 were simultaneously applied. The nasal bioavailability of FD-4 in saline solution was 8.2+/-0.6% but increased to 40.0+/-5.5% when 5% NAC and 5% laureth-25 were added. This synergistic enhancement could result from the mucolytic activity of NAC in reducing mucous viscosity by which the accessibilities of FD-4 and laureth-25 to the epithelial membrane were increased. Further rat studies proved that this formulation increased nasal absorption of salmon calcitonin. Absolute bioavailability from saline solution containing 5% NAC and 1% laureth-25 was 26.8+/-2.2%, 3.5 times that of the commercial calcitonin nasal spray Miacalcin (7.7+/-2.1%). The potential of the new formulation to cause tissue damage in terms of hemolytic activity and liberation of phospholipid from the nasal membranes was nil or slight. The combination of NAC and laureth-25 appears suitable for use in development of nasal products for poorly absorbable drugs, especially peptide and protein drugs.

Effect of absorption promoters on the nasal absorption of drugs with various molecular weights

Abstract The effect of absorption promoters on the nasal absorption of drugs with different average molecular weights was examined in rats. Phenol red and fluorescein isothiocyanate-dextrans (FITC-dextrans) with various molecular weights were chosen as model drugs while the absorption promoters used in this study were Na glycocholate, Na taurocholate, Na deoxycholate, Na salicylate, EDTA, Na caprylate and Na caprate, all at a concentration of 1%. Of the absorption promoters, bile salts such as Na glycocholate, Na taurocholate, Na deoxycholate, Na caprate and EDTA appeared to be more effective at enhancing the nasal absorption of drugs as compared to Na salicylate and Na caprylate. Na glycocholate, Na caprate and EDTA showed the strongest promoting effect of drugs with an approximate molecular weight 4000, while the maximal effect of Na taurocholate and Na deoxycholate was observed in the nasal absorption of drugs with an approximate molecular weight of 10000. In contrast, Na salicylate and Na caprylate did not affect the absorption of drugs having various molecular weights. These findings indicated that the efficacy of absorption promoters on the nasal absorption of drugs was dependent on their molecular weights.

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.

Characterization of LLC-PK1 kidney epithelial cells as an in vitro model for studying renal tubular reabsorption of protein drugs.

AbstractPurpose. The purpose of this study was to assess whether LLC-PK1 renal epithelial cells could serve as an in vitro model for studying the renal tubular reabsorption of protein drugs. Methods. The association of 111In-labeled model protein drugs, bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and [Asu1,7]-eel calcitonin (Asu-ECT), with the monolayers of LLC-PK1 renal epithelial cells was characterized under various conditions. Results. The cellular association of these proteins was temperature-dependent and varied according to the protein. Saturation kinetics were observed for STI association, with the apparent Km and Vmax values determined to be 66.3 µg/ml and 250 ng/mg protein/min, respectively. The association of STI decreased with increases in medium pH from 5.4 to 8.4 and was inhibited significantly by 2,4-dinitrophenol, sodium azide, cytochalasin B, and colchicine, suggesting that the cellular association involved endocytosis. Mutual inhibition was observed in competitive binding experiments with the four protein drugs, suggesting that they shared a common binding site on the luminal membrane of LLC-PK1 cells. Taken together, these findings show that a variety of protein drugs bind to LLC-PK1 cells in a non-specific manner and possibly undergo endocytosis, a phenomenon that is similar to in vivo proximal tubular reabsorption. Conclusions. LLC-PK1 renal epithelial cells would be a suitable model system for the study of the renal proximal tubular reabsorption of protein drugs.