Yutaka Yamagata

Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique.

Biodegradable microcapsules for sustained release of recombinant human growth hormone (rhGH) were prepared by a solid-in-oil-in-water (S/O/W) emulsion solvent evaporation technique using lyophilized protein microparticles. The minimum mean particle size of rhGH in S/O dispersions was 2.8-3.0 microm when ammonium acetate was added at molar ratios of 10-20 times against rhGH. High entrapment of rhGH in microcapsules was achieved by incorporating rhGH powder with a smaller particle size obtained by lyophilizing with ammonium acetate. As the particle size of rhGH decreased, the in vivo initial release decreased, while subsequent serum levels of rhGH in sustained release phase were higher. Addition of zinc oxide to microcapsules resulted in higher serum levels than those prepared without zinc oxide, suggesting a stabilizing effect of zinc oxide after subcutaneous injection into rats. The release profile of rhGH from microcapsules was controllable by selecting the proper copoly(DL-lactic/glycolic)acid (PLGA) with L/G ratio and molecular weight. Utilization of rhGH powder with a smaller particle size obtained by lyophilizing with ammonium acetate is essential for preparation of microcapsules with high entrapment and well-controlled sustained release profile with small initial release.

Preparation of a copoly (dl-lactic/glycolic acid)-zinc oxide complex and its utilization to microcapsules containing recombinant human growth hormone

A procedure to prepare a complex of copoly (dl-lactic/glycolic acid) and zinc oxide (PLGA-zinc oxide complex) was developed. Out of sparingly water-soluble zinc compounds, zinc oxide was most remarkably soluble in a PLGA/dichloromethane solution and the dissolution rates became faster as the water contents in the PLGA/dichloromethane solutions increased. Since the solubility of zinc oxide was saturated at approximately 0.5-fold molar ratio to PLGA and water was generated with dissolution of zinc oxide in the PLGA/dichloromethane solutions, it is suggested that zinc oxide interacts with the terminal carboxyl group of PLGA. In addition, the glass-transition temperature of a solid material obtained by vacuum-drying the PLGA/dichloromethane solution dissolving zinc oxide became higher as the zinc content increased, suggesting that the formation of a PLGA-zinc oxide complex. Microcapsules were prepared with the PLGA-zinc oxide complex using recombinant human growth hormone (rhGH) in order to evaluate an effect of the complex on protein release and stability of protein in the microcapsules. Released rhGH amount from the microcapsules prepared with the PLGA-zinc oxide complex after subcutaneous administration in rats was significantly larger than that from microcapsules prepared with PLGA alone, indicating that rhGH molecules in the microcapsules was stabilized by the PLGA-zinc oxide complex.

Studies on quantitative levels of complement activation induced by immobilized and soluble forms of protein A: Relevance to extracorporeal immunoadsorption

Abstract Soluble staphylococcal protein A (SpA) has been shown to activate the complement system and complement activation has been demonstrated in patient plasma after extracorporeal immunoadsorption through SpA columns. Studies were performed to determine whether complement activation in human plasma is caused by immobilized SpA and/or minute quantities of soluble SpA which may “leach” from immunoadsorbent columns into plasma. Examination of several clinical plasma samples, after immunoadsorption through SpA columns, revealed minute quantities of soluble SpA which “leached” from the immunoadsorbent matrix ranging in concentration from 0.23 to 0.70 μg/mL. Additional studies indicated that the SpA/silica matrix utilized in immunoadsorbent columns activated the complement system and generated anaphylatoxins C3a, C4a and C5a. Moreover, soluble SpA, at concentrations approximating levels detected due to “leaching”, did not generate anaphylatoxins; higher concentrations of soluble SpA were observed to activate the complement system. Staphylococcal enterotoxin B (SEB), a potential contaminant of SpA preparations, did not activate the complement system. These results indicate that the complement activation observed in human plasma after extracorporeal immunoadsorption is due to the immobilized form of SpA rather than the minute quantities of SpA “leached” from the SpA/silica immunoadsorbent matrix.

A new animal model for evaluation of long-term growth rate over one month by rhGH/PLGA microcapsule formulations

A new animal model to evaluate the long‐term growth rate produced by a sustained‐release formulation of recombinant human growth hormone (rhGH) over one month was developed and the usefulness of our microcapsule formulations was demonstrated in this model. Long‐term pharmacological effects by subcutaneous injection of microcapsules for sustained release of rhGH were evaluated in hypophysectomized (Hpx) rats treated with immunosuppressive agent along with hormone supplement. Copoly(dl‐lactic/glycolic)acid (PLGA) microcapsules for sustained release of rhGH, a two‐week sustained‐release formulation (rhGH‐SR‐2W) and a one‐month sustained‐release formulation (rhGH‐SR‐1M), were prepared by a solid‐in‐oil‐in‐water emulsion solvent evaporation technique. Body‐weight gain, body‐length gain and serum levels of rat insulin‐like growth factor‐l (rlGF‐l) induced by subcutaneous injection of rhGH‐SR were compared with those by daily injections of rhGH solution in Hpx rats for 35 days. Serum IGF‐l levels in Hpx rats after the injection of rhGH‐SR‐2W microcapsules were higher than those after daily injections of rhGH solution. Body‐length gain, a new parameter, after single injection of rhGH‐SR‐1M microcapsules demonstrated the higher growth rate than that after daily injections of rhGH solution for 35 days. Thus, single injection of rhGH‐SR microcapsules demonstrated long‐term pharmacological effects greater than those by daily injections of rhGH solution in a newly developed model, immunosuppressed Hpx rats.