Recently gene therapy is raising up as a new strategy of cancer treatment. Many kinds of strategy for gene therapy of cancer were reported. HSV-TK gene, wild-type p53 gene, HLA gene and cytokine genes such as IL-2, IL-4, IL-7, TNF, IFN, GM-CSF etc. are used in gene therapy experiments and trials. However, many kind of problems still exist. Final end point of gene therapy of cancer is the repair of altered genes in cancer cells. However, technology of repair for altered genes in cancer cells is still not available. In the most of on going gene therapy experiments and trials, cancer cells are treated by gene or gene modified cells via immune systems or other unknown bystander effects. The transfection of gene dramatically change the phenotype of cancer cells, especially the growth pattern in vivo. On the other hand, therapeutic effects of gene or gene modified cells via immune systems or other unknown bystander effects on cancer cells without gene modification are not satisfied. Thus, the development of not only efficient vector systems but also new strategy using new genes are essential for the further application of gene therapy to clinical trials. Clinical trials of gene therapy have been started in USA, France, Italy, UK, Netherlands and China. In Japan, guide-lines for clinical trials of gene therapy were established from Ministry of Health and Welfare and Ministry of Education. However, no-body know when it could be started in Japan. Vectors supply and safety check and establishment of social consensus are critical for the introduction of gene therapy trials in Japan.

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In vivo biodistribution of lipophilic drugs, injected with various lipid carrier systems, was systemically studied and the possibility and affectable factors of biodistribution control were examined. It was clarified that drug lipophilicity is a determinant factor to control in vivo behavior of lipophilic drug utilizing lipid carriers. Briefly, sufficient lipophilicity, PCoct more than 10⁶ for liposome, 10⁹ for emulsion, not less than 10¹¹ for micelles, is required for biodistribution control. In situ single-pass rat liver perfusion experiment was also carried out to evaluate hepatic disposition of lipophilic drugs injected with lipid carriers, and to analyze retention ability of lipid carriers for lipophilic drugs, that is, drug release from lipid carriers. There was a large difference between release rates of lipophilic drug from lipid carriers such as micelle, liposome, and emulsions, demonstrating that the drug retention ability is also a determinant factor for biodistribution control of incorporated lipophilic drugs. In addition, on the basis of these pharmacokinetic findings, the feasibility of pharmacological control of highly lipophilic drug probucol, plasma cholesterol-lowering agent, was studied. Different pharmacological effect was observed between lipid carriers depending on the pharmacokinetic properties and retention ability of utilized lipid carriers.

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Oral and implant preparations for controlled release of drugs were developed by the use of porous calcium silicate (Florite^® RE(FLR), Eizai co.) as a drug carrier. Usefulness of the preparations was evaluated in in vitro and in vivo release studies, In oral preparation, sulfamethizole(SMZ) was used as a model compound. SMZ was incorporated into pores of FLR by grinding SMZ acetone solution with FLR. For controlling the release of SMZ, the pores were sealed off by adding different amounts of tristearin (TS) to SMZ-incorporating FLR. SMZ preparations sealed with TS administered orally at a dose of 20mg SMZ/kg in rabbits showed a remarkable sustained release of SMZ over 30 hrs, compared to SMZ powder(MRT : 10.5 vs 4.6hr). In implant preparations, FITC-dextrans with different molecular weights and human epidermal growth factor (hEGF) were used as model macromolecular compounds. The release of these compounds was controlled by sealing the pores with a mixture of TS and polyethylene glycol 6000 (PEG) at different weight ratios, where PEG was used as a water channel forming agent. Subcutaneously implanted FITC-dextran preparations at a dose of 10 μmol/kg showed a sustained release in rats over more than 1 month, although a small amount of burst release was observed in the early phase. A sustained and constant release in vivo was also observed in hEGF prepartion. Above results indicate that the release of relatively hydrophobic compounds, which can penetrate the intestinal mucosal membranes after oral administration, can be relatively easily controlled by changing the amount of TS for sealling of FLR pores, and the release of hydrophilic compounds for implant use can be controlled by mixing a water channel forming agent with TS after incorporating the drug into FLR pores.

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Distearoyl-N-(3-carboxypropionoyl polyethyleneglycol succinyl) phosphatidylethanolamine (PEG-COOH) was newly synthesized and used to prepare novel immunoliposomes carrying monoclonal antibodies at the distal ends of the PEG chains (Type C). Liposomes were prepared from egg phosphatidylcholine and cholesterol (2 : 1, m/m) containing 6 mol% of PEG-COOH, and a monoclonal antibody, 34A, which is highly specific to pulmonary endothelial cells, was conjugated to the carboxyl groups of PEG-COOH to give various amounts of antibody molecules per liposome. Other immunoliposomes with PEG coating(Type B) or without PEG coating(an earlier type of immunoliposome, Type A) were prepared for comparison. Type B and Type C liposomes without antibodies showed prolonged circulation time and reduced reticulo-endothelial system(RES) uptake owing to the presence of PEG. These three different types of 34A-immunoliposomes with 30-35 antibody molecules per vesicle and 2000 in the average molecular weight of PEG in Type B or C immunoliposomes were injected into mice to test the immuno targetability to the lung. The efficiency of lung binding of Type B-was one half of that of 34A-Type A, though a large amount of Type B remained in the blood circulation for a long time, suggesting that the steric hindrance of PEG chains reduced not only the immunospecific antibody-antigen binding, but also the RES uptake. The degree of lung binding of Type C was about 1. 3-fold higher than that of Type A, indicating that recognition by the antibodies attached to the PEG terminal was not sterically hindered and that the free PEG (i.e., that not carrying antibody) was effective in increasing the blood concentration of immunoliposomes by enabling them to evade RES uptake. It was shown that antibody density is an important factor for target binding of Type C. However, their immunotagetability was not affected by the molecular weight of PEG-COOH. Our approach provides a simple means of conjugating antibodies directly to the distal end of PEG which is already bound to the liposome membrane, and should contribute to the development of superior targetable drug delivery vehicles for use in diagnostics and therapy.

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The chemical modification of human recombinant interleukin-6(IL-6) with polyethylene glycol (PEG) was conducted to increase its thrombopoietic activity. PEG-modified IL-6(PEG-IL-6) was separated into several molecular weight fractions and characterized individualy. The specific activity of PEG-IL-6 was reduced with increase in its degree of PEG-modification, that is, whole molecular weight. On the other hand, the in vivo thrombopoietic activity of PEG-IL-6 was markedly increased. Especialy, PEG-IL-6, in which 54% of the 14 lysine amino groups of IL-6 were coupled with PEG, showed 10 times greater thrombopoietic effect than native IL-6. By the pharmacokinetic analysis, PEG-IL-6 greately enlarged the area under the time concentration curve. These findings suggesed that PEG-IL-6 may be a useful agent for the treatment of thrombocytopenia after chemotherapy.

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We examined the effects of polyethyleneglycol (PEG)-coated liposome containing adriamycin (PEG-LADR)on the antitumor activity and toxic side effect of adriamycin (ADR), using Ehrlich ascites carcinoma bearing CDF₁, male mice. PEG-LADR significantly enhanced the antitumor activity of ADR against the solid tumor. This increment of antitumor activity induced by PEG-coated liposomalization supported by the elevation of ADR concentration in the tumor. Next, we examined two biochemical parameters, lipid peroxide (LPO) level and glutathione peroxidase (GSHpx) activity, as indices of ADR induced side effects. There were the increase of LPO level and the decrease of GSHpx activity in the heart of mice after ADR solution administration. ADR concentration in the heart on the 24th hour after PEG-LADR administration showed higher than that after ADR solution treatment. However, PEG-LADR administration were not changed LPO level and GSHpx activity in the heart, compared to normal level. In the liver and the pancreas, same results were shown. Furthermore, regarding the lethal effect of ADR, the acute toxicity after PEG-LADR treatment significantly reduced, compared to that after ADR solution treatment. Therefore, these results suggested that PEG-LADR can significantly increase the antitumor activity of ADR solution with decreasing its side effects, and the side effects of liposomal ADR should not evaluated by ADR concentration in each tissues.

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Using carboxymethyl dextran (CMdex) as the intermediate drugcarrier, carboplatin (2 cis-diammin platin II, CBDCA : Bristol-Myers-Squibb) was conjugated to anti-CEA monoclonal antibody (MoAb : 1B2)with bridging agents 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-succinimide. MoAb and CMdex were mixed in the molar ratios of from 1 : 1 through 1 : 3000, but CMdex/CBDCA ratios were fixed at 1 : 100 in molar. The conjugation efficiency by this method was that when initially mixed molar ratio of carboplatin to MoAb was more than 10000, 80% of given carboplatin was found to be coupled to MoAb-CMdex, and maximally 626 mg of carboplatin could be conjugated to 1 mg of MoAb. The cytotoxicity and immunoreactivity of the immunoconjugates were evaluated by colony formation method, and by an enzyme immunoassay using Ag-coated beads, respec tively. The cytotoxicity of the immuno-conjugates was dependent only on the drug concentration, equal to that of unconjugated drug, and irrespective to the CBDCA/MoAb ratio. The immuno-reactivity of the conjugates was found no remarkable decrease in comparison with unconjugated antibody. Then we conclude that this conjugation method might be of use, at least significant on the conjugation of carboplatin to monoclonal antibodies.

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Human CEM cells were incubated with liposomes containing 15 mer oligodeoxyribonucleotide sequences. When the sequence was complementary to the 5'end of c-myc proto-oncogene mRNA, proliferation of the cells was blocked. Inhibition was not seen for empty lipsomes or lipsomes containing sequences irrelevant to c-myc mRNA. Nor was it seen when non-encapsulated oligonucleotides were incubated with cells, probably because non-encapsulated oligonucleotides are sensitive to nucleases. Oligodeoxyribonucleotides encapsulated in liposomes resists DNAse, and inhibit cell proliferation specifically.

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Internalization of albumin with synthetic glycolipid into blood cell was investigated by flowcytemeter. 1, 5-Dioctyl-N-glucono-L-glutamate (2C₈-glc) and dodecylmaltoside (DDM) enhanced the uptake of albumin into monocytes, but not into neutrophiles and granulocytes. Cationic lipids and anionic lipids showed no enhancment of cell uptake of albumin. Hemolysis was occured in the presence of DDM, but not in the presence of 2C₈-glc. The present study suggest that synthetic glycolipid 2C₈-glc is expected as a candidate for the delivery of proteins into blood monocytes.

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