Masafumi Misaki

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.

Antitumor Effect of Arterial Administration of a Medium-Chain Triglyceride Solution of an Angiogenesis Inhibitor, TNP-470, in Rabbits Bearing VX-2 Carcinoma

Using rabbits bearing VX-2 carcinoma on the inner side of the leg, we examined the antitumor activity of a medium-chain triglyceride (MCT) solution of an angiogenesis inhibitor, TNP-470 (AGM-1470, 6-O-(N-chloroacetylcarbamoyl)-fumagillol), following administration into the femoral artery feeding the tumor. The MCT solution of TNP-470 (1 and 5 mg) strongly suppressed tumor growth following a single intra-arterial (i. a.) injection 2 or 3 weeks after tumor inoculation. Moreover, remarkable regression of well-developed tumors, those 4 weeks after inoculation, was obtained by i.a. injection of the MCT solution containing 20 mg of TNP-470 without any influence on body weight. The antitumor effects were potentiated by coad-ministration of doxorubicin or mitomycin C (MMC) in the solution or microspheres containing MMC. In a shell-less chorioallantoic membrane (CAM) assay, angiogenesis was inhibited when a droplet of the MCT solution containing 25 µg of TNP-470 was placed on the CAM for 2 days, suggesting that the prolonged antitumor effect resulted from the inhibition of tumor neovascularization by sustained drug release from the preparation. These results indicate that i. a. injection of the MCT solution of TNP-470 is promising for treating well-developed tumors.

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.

Receptor-binding, in vitro cytotoxicity, and in vivo distribution of transferrin-bound cis-platinum (II) of differing molar ratios

Abstract Complexes of cis-diamminedichloroplatinum(II) (cisplatin) and transferrin (Tf(Fe)2) of differing molar ratios ( Pt Tf 3:1, 7:1, and 15:1 mol/mol) were prepared. Tf-receptor binding studies showed that the reactivity of Tf(Fe)2 to Tf receptors on A431 cells was reduced as the binding ratio of Pt to Tf(Fe)2 increased. The complexes of Pt Tf 3:1 and Pt Tf 7:1 inhibited the binding of 125I-Tf(Fe)2 to A431 cells with Ki values of 26 nM and 73 nM, respectively, while the complex of Pt Tf 15:1 did not show any inhibitory activity. The in vitro cytotoxicity of Pt against A431 cells also reduced as the ratio of Pt to Tf(Fe)2 increased, and IC50 of Pt were 15 μM, 13 μM, and 44 μM, for the complex of Pt Tf 3:1, Pt Tf 7:1, and Pt Tf 15:1, respectively. The pharmacokinetic analysis showed that the Pt-elimination from blood was delayed when Pt was administered in the form of the complex, while the Pt-elimination rate became higher in proportion as the ratio of Pt to Tf(Fe)2 increased. Thus, it was concluded that the receptor-binding activity, the cytotoxicity, and in vivo distribution of the complex could be optimized by altering the binding ratio of Pt to Tf(Fe)2, and the complex of Pt Tf 3:1 seemed to be most appropriate for the tumor-specific delivery of Pt.

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.