Monoclonal antibodies against human α-fetoprotein (AFP) or carcinoembryonic antigen (CEA) were conjugated to liposomes containing adriamycin (ADM), and their therapeutic effects were experimentally studied in vivo on AFP or CEA positive human solid tumors maintained in BALB/c nu/nu mice. The anti-tumor effect of antibody-conjugated liposomes containing ADM was immunologically specific and was greater than that of unconjugated liposomes containing ADM or that of ADM alone. Furthermore, tissue distribution studies revealed the selective delivery of ADM to tumors occurred with immunoliposomes. However, it was also revealed the liposomes are preferentially accumulated to the reticuloendothelial cells. To improve the therapeutic effects, we further prepared the following three types of liposomes and their therapeutic activities were assessed. (1) Fab′ fragment was introduced instead of whole molecule of antibody. These liposomes are expected to keep the antigen-binding activity of antibody intact. Actually, the therapeutic effect of liposomes with Fab′ fragment was greater than that of liposomes with whole molecule. (2) GMI gangliosides were incorporated into liposomes and with these liposomes the accumulation of ADM to reticuloendothelial cells was reduced. (3) Temperature-sensitive liposomes were prepared with dipalmitoylphosphatidylcholine. Combination of temperature sensitivity and monoclonal antibodies for targeting therapy is now in progress.

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The drug delivery system using transferrin receptor mediated endocytosis is described. Transferrin is a serum glycoprotein which carries iron to cells possessing its receptor. The expression of transferrin receptor has been well known to be increased in cells with malignant transformation. Our aim is to utilize the transferrin-transferrin receptor system for the specific delivery of anticancer drugs and foreign genes. Human transferrin was conjugated with anticancer polypeptide, neocarzinostatin (NCS) by using (SPDP) and purified by ion exchange chromatography. The obtained Tf-NCS conjugate is capable binding to transferrin receptor and shows higher cytotoxicity. In vivo study using tumor inoculated mice also showed the significant growth suppression. The cytotoxicity of the conjugate was the sum of the receptor binding and intracellular degradation. Similar strategy was adopted to foreign gene transfer to tumor cells and IL-2 activated lymphocytes both of which express transferrin receptors. Transferrin was conjugated with vector containing β-galactosidase by transamination. The conjugate was effectively transferred to cells by receptor-mediated fashion and the transfected gene was expressed. This procedure would be useful for gene therapy.

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We have been attempting local injection of OK-432 in a lymph node or its surrounding areas in patients with lymph node metastases when we confirmed that the patients had relapse. We mixed OK-432 solution and lipiodol. After injection of this mixture, we examined the extent of infiltration of this mixture with fluoroscopy, We checked whether this mixture was actually in the state of emulsion. Immediately after mixing them, a single water phase was observed, and three hours later, two phases were already separated as determined by a visual inspection. However, at this stage, majority of the cases were in the state of emulsification. These findings may indicate that the mixture was in emulsion at the time of injection, and that it gradually degraded successively in the body. However, we speculate that it might be possible to achieve more stable emulsion by the use of surfactants or by adjusting differences in the specific gravities of two phases.

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Using albumin as the intermediate drug carrier, adriamycin (ADM) was conjugated with a monoclonal antibody (MoAb) S1 (IgG2a), which recognizes the antigenic determinant of the carbohydrate moiety expressed on human hepatocellular carcinoma cells. Selective cytotoxicity of MoAb S1-ADM conjugate was observed against S1 antigen positive c-Hc-4 hepatoma cells in vitro. In vivo experiments demonstrated that the conjugate significantly suppressed the human hepatoma line transplanted s. c. into nude mice. Autoradiographic examination of S1-¹⁴C-ADM conjugate revealed that the ¹⁴C-ADM accumulated in the tumor mass. Increase of the uptake of ¹²⁵I-labeled conjugate by c-Hc-4 hepatoma cells was observed after incubation of the cells with the conjugate at 37°C, but not at 0°C, suggesting the cells actively internalize the conjugate in the course of time. Flow cytometric analysis also suggested that the cells internalize the conjugate 15 to 30 min. after incubation.

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Multidrug resistant (MDR) cell line, AH66DR, derived from rat ascites hepatoma AH66 parental cell, AH66P, was established and overcoming effect of conjugate of bovine serum albumin (BSA) with doxorubicin (DXR) on the cell line was examined in vitro and in vivo. The conjugate (BSA : DXR molar ratio =1 : 3.28) was accumulated in the cytoplasm of the AH66DR by reduction of efflux of the drug. The conjugate expressed the excellent antitumor activity against AH66DR (IC50=50nM) and this value was almost similar cytotoxic titer of DXR against AH66P. The conjugate also showed the most efficacious life-prolonging effect against the rats bearing AH66P as well as AH66DR cell line.

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Effects of various protease inhibitors on the intestinal absorption of insulin were studied by in situ closed small and large intestinal loops of rats. Sodium glycocholate, bacitracin, aprotinin, soybean trypsin inhibitor and camostat mesilate were used as protease inhibitor in this study. The absorption insulin from the gastrointestinal tract was evaluated by its hypoglycemic effect. When insulin without these protease inhibitors was administered into small or large intestinal loops, no marked hypoglycemic response was noted in both regions. However, a significant hypoglycemic effect was obtained after large intestinal administration of insulin containing various protease inhibitors, especially, 20mM Na-glycocholate, 20mM camostat mesilate when compared with controls. In contrast, we found little hypoglycemic effect following small intestinal administration of insulin with these protease inhibitors. These findings suggest that the coadministration of the protease inhibitors could be useful to improve the large intestinal absorption of insulin.

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The in vitro skin permeation using hairless mouse and in vivo skin absorption using rat of alclofenac from ethanol/panasate 800 (tricaprylin) binary vehicle were investigated. The in vitro skin permeability of alclofenac was remarkably enhanced by the combination of ethanol and panasate 800. The greatest enhancement was obtained in the ethanol/panasate 800 (40/60) system. The results of in vivo skin absorption of alclofenac were shown almost the same tendency as that of the in vitro permeation. The skin permeation enhancement mechanism of alclofenac from the ethanol/panasate 800 (40/60) binary vehicle was evaluated by clarifying the different behavior of ethanol and panasate 800 for the diffusion process in the skin. It is suggested that ethanol increases permeation flux by increasing the uptake of alclofenac in the skin tissue and by gradually reducing the barrier function of stratum corneum. On the other hand, panasate 800 reduces lag time by increasing the uptake rate and diffusion of alclofenac in the stratum corneum and viable skin. Therefore, the enhancement effect of the ethanol/panasate 800 (40/60)binary vehicle is considered to be due to the mutual effect which is obtained by the effective interaction of the two vehicles.

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The releasing rate of poly(3-hydroxybutyrate)-P(3HB)-microapheres containing Lastet, an anticancer agent, was investigated both in vitro and in vivo. The rate of the release of Lastet from P(3HB) microspheres was very small and only 20% was released during a 15-day period. The addition of acylglycerols to P(3HB) increased the releasing rate of Lastet. P(3HB) microspheres which contained 25% glycerol tristearate (GTS) and 25% glycerol monostearate (MTS) released 100% and 50% of the Lastet during a 10-day period, respectively. In the surface of P (3HB) microspheres without acylglycerol, a number of small holes of 1 μm diameter were observed. And large holes of 5 μm diameter were observed in the surface of P(3HB) microspheres with GTS. There was no significant difference in surface property between P(3HB) microspheres with MTS and those without acylglycerol. P (3HB) microspheres which contained 25% MTS and 10% Lastet were administered intraperitoneally to rats and the change of Lastet concentration in blood and tissue was investigated. Although in low levels, Lastet was detected in the blood up to 24hr after administration. And Lastet was detected in high levels in the mediastinal tissue up to day 7. The change of the surface property of microspheres confirmed by scanning electron micrographs suggested that the decomposition of P(3HB) in vivo may be accelerated by some enzyme to increase the release of drugs.

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