The critical point for us to develop therapeutic strategies for advanced cancer is how we can achieve cytotoxic effects which are specific for tumor cells. Genetic transduction technology provides us with various approaches to this goal. Specific antitumor cytocidal effect could be achieved by several strategies. (A) The use of tissue-specific promoters to drive suicide genes : We developed adenoviral vectors expressing suicide genes under the control of tissue-specific promoters (i. e., promoters for AFP, CEA, PSA). With these recombinant adenoviruses, we attained specific killing of the tumor cells from certain tissue origins (i. e., hepatocellular carcinoma, gastric and pancreas cancer, and prostate cancer). We characterized ganciclovir/HSV-TK and 5-FC/E. coli cytosine deaminase systems as prodrug/enzyme therapies. E. coli upp gene for uracil phosphoribosyl transferase, UPRTase, in combination with 5-FU is also an interesting system. We showed that adenovirus-mediated upp gene transduction resulted in 10 to 100-fold enhancement of the therapeutic effect of 5-FU toxicity for cancer cells. Upp gene transduction is a hopeful modality to overcome the 5-FU resistance which is often seen in patients with cancers from various tissue origins. (B) The use of tumor-specific mutations such as p53 tumor suppressor gene : Our recombinant adenoviruses with deficient E1B55K (AxE1AdB) was demonstrated to efficiently replicate in and kill tumor cells deficient in p53 gene activity. Since mutations in p53 are the most common type of genetic abnormality in cancer occurring in more than 50 percent of human cancer cases, this approach will have a wide applicability in specific tumor cell-targeting. (C) The use of targetable viral vectors : We have established a system of constructing recombinant adenoviruses with mutant fiber knob or penton base proteins, which are responsible for virus-host cell interaction. Now it is feasible to develop mutant viral vectors which could be targetable for specific cancer cells.

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In order to design an optimum liposome immunoadjuvant for tumor vaccines, we investigated the relationship between liposomal surface charge and adjuvant action. Multilamellar vesicles (MLV) showed substantial increase in both cell-association and uptake by macrophages following enhancement of the degree of liposomal positive charge. Consistent with this, MLVs containing soluble ovalbumin (OVA) functioned increasingly as a more potent inducer of antigen-specific cytotoxic T lymphocyte (CTL) responses and antibody production after increasing the degree of liposomal positive charge. Furthermore, we examined the in vivo anti-tumor effect of various charged liposomal antigens using OVA and OVA-expressing transfectant, EG7. Vaccinal effects of MLV containing OVA were found to be dependent on the degree of liposomal positive charge. These results indicate that the positive charge on the surface of liposomes represents an important factor in enhancing their immunoadjuvancy in the induction of antigen-specific immune responses and vaccinal effects against tumors.

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The development of an efficient and safe vector is the most important for effective gene therapy, because viral and nonviral vectors used in clinical studies showed various risks, such so insertion mutation or less efficiency. In this study, we attempted to design a novel hybrid gene transfer vector, VSV-liposomes, prepared by fusing simple liposomes and vesicular stomatitis virus (VSV) particles. VSV is an enveloped virus whose nucleocapsid finds its way to the cytoplasm of the host cell by receptor-mediated endocytosis, followed by fusion with the membrane of the endocytic vesicle. Therefore VSV-liposomes are not reduced by lysosomal enzyme, VSV-liposomes can introduce encapsulating valuable materials such as drugs, proteins, nucleic acids, and other biochemical reagents efficiently into cytoplasm, The uptake of fragment A of diphtheria (DTA) into cultured cells by VSV-liposomes is one hundred times higher than that by liposomes. VSV-liposomes and VSV did not show any hemolytic effect. In addition, no cytotoxicity was observed by even higher concentration of VSV-liposomes. When the cells were treated VSV-liposomes containing pCAL2, a firefly luciferase expression plasmid, the luciferase activity of cells had higher than that of these cells which were treated conventional liposomes. These results indicate VSV-liposomes are useful as a in vivo gene transfer vector.

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Ulcerative colitis and Chrohn's disease are recurrent, idiopathic inflammatory disorders involving the mucosa and sub-mucosa of the colon. The objective of this study was to estimate colon-specific delivery of anti-ulcerative colitis drugs with chitosan capsules. Release studies of 5-aminosalicylic acid (5-ASA) from chitosan capsules were carried out by the Japan Pharmacopoeia (JP) rotating basket method with some slight modifications. There is no release of 5-ASA from chitosan capsules in the phosphate buffered saline, however the release of 5-ASA was markedly increased in the presence of rat cecal contents. The mucosal tissue concentrations of 5-ASA in the large intestine after oral administration using chitosan capsules were higher than those in the CMC suspension. The concentrations of 5-ASA in the contents of the large intestine, large intestinal tissue mucosa, and the plasma were determined by HPLC. Furthermore, the anti-inflammatory drugs were orally administered using chitosan capsules or a carboxymethyl cellulose sodium salt(CMC) suspension to trinitrobenzenesulfonic acid sodium salt (TNBS)-induced rats. The colonic injury and inflammation were assessed by measuring the myeloperoxidase (MPO) activities, the ratios of distal colon wet weight to body weight (C/B ratio) and the damage score. When 5-ASA was orally administered using chitosan capsules in TNBS-induced colitis rats, we found better therapeutic effects with 5-ASA than with a 5-ASA CMC suspension. Therefore, chitosan capsules may be useful carriers for the colon-specific delivery of anti-inflammatory drugs.

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Brain-derived neurotrophic factor (BDNF) was modified by carboxyl-directed protein pegylation in order to retain biologic activity and reduce the systemic clearance of this cationic protein in vivo. Since the modification of the surface lysine residue of BDNF resulted in loss of biologic activity, the present study examine the feasibility of placing polyethylene glycol (PEG) polymer on the carboxyl residue of BDNF, PEG molecules with terminal hydrazide moiety of molecular weight 2 kDa (PEG²⁰⁰⁰-Hz) and 5 kDa (PEG⁵⁰⁰⁰-Hz) were coupled to BDNF caboxyls using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide. Polyacrylamide gel electrophoresis revealed that the molecular weight distributions of BDNF-PEG²⁰⁰⁰ and BDNF-PEG⁵⁰⁰⁰ were 20 k∼30 kDa and 60 k∼80 kDa, respectively. Cell survival studies using 3T3 cells transfected with the BDNF receptor gene showed that the biologic activity of BDNF was not changed following pegylation with PEG²⁰⁰⁰, and was minimally impaired following pegylation with PEG⁵⁰⁰⁰. The systemic clearances of the ¹²⁵I-BDNF-PEG²⁰⁰⁰ and ¹²⁵I-BDNF-PEG⁵⁰⁰⁰ following intravenous administration to rats were reduced 67% and 91%, respectively, compared to unconjugated BDNF. TCA soluble fraction of the radioactivity in the plasma 60 min following administration were reduced 72% and 94%, respectively, showing the significant reduction of the BDNF metabolism by pegylation. These experiments describe the first successful carboxyl-directed pegylation of a neuropeptide, and show that this formulation substantially reduces the systemic clerance and metabolism of this neurotrophic factor. In conclusion, the carboxyl-directed pegylation is beneficial to the modification of the protein whose surface lysine or arginine residue is essential to the biologic activity.

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Nasal transport of thymidine (Thd) was investigated using in vivo nasal absorption technique in rats. Radio active thymidine was administered and Thd concentrations in the cerebrospinal fluid (CSF) and plasma was determined by liquid scintillation counter. Thd concentrations in the fractionated CSF which located close to the nasal cavity following intranasal (i.n.) administration were higher than that following intravenous (i.v.) administration, thus these observation suggests Thd, like other compounds, transported from nasal cavity to CSF directly following i.n. administration. The fractionated CSF concentration and the area under plasma concentration-time curve (AUC) following i.n. administration of Thd at 1.338∼267.6 mM were determined. The relationships between both the fractionated CSF concentration and AUC, and dose of Thd seemed dose-independent, though saturation in the transport of Thd from nasal membrane to CSF and systemic circulation was observed. The CSF concentrations and AUC following i.n. administration of Thd at a dose of 13.38 mM with 2'-deoxyuridine (dUrd, 25, 50 and 100 mM) or 2'-deoxyinosine (dIno, 50 mM) were also determined. Decrease in the concentration of CSF and AUC was observed, and the inhibitory effect of dUrd was dose-dependent. In the case of coadministration of Thd and dIno, dIno also showed an inhibitory effect for Thd transport, while the effect was less significant than that of dUrd. These results suggests that the possibility of the presence of Thd sensitive transport systems on nasal membrane in rats, and the systems, if exist, may be useful for the delivery of nucleoside analogues for central nervous system and systemic circulation via nasal membrane.

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The effect of radiating area and frequency of pulsed output ultrasound with an on/off ratio of 1 : 2 on transdermal absorption of indomethacin from an ointment was studied in rats. Ultrasonication was produced by a commercially available ultrasound system approved for human use which has two different sizes of transducer, large head (4.4 cm²) and small head (0.7 cm²). We did not find any difference in transdermal absorption of indomethacin between two transducer (effective radiating areas). Ultrasound should be generated by a small head since a small head enalbes the rats to administer the treatment. The frequency of the pulsed ultrasound was suggested to play an important role in the transdermal phonophoretic delivery system of indomethacin ; 1 MHz appeared to be more effective than 3 MHz in improving the transdermal absorption.

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