Adeno-associated virus (AAV) is a human parvovirus with a single-strand DNA genome of approximately 4.6 kb encapsulated in an icosahedral virion 20 to 26 nm in diameter. Recombinant AAV vectors have promising features as a vector for gene therapy, such as the lack of any apparent pathogenicity, low immunogenicity, and relatively high stability of transgene expression. Preclinical studies have been performed for the treatment of cystic fibrosis, Fanconi anemia, β-thalassemia, hemophilia, AIDS, Parkinson disease, and many kinds of neoplastic diseases such as malignant brain tumors. Here, we introduced gene therapy using AAV vectors containing herpes simplex virus-thymidine kinase (HS-tk) gene or cytokine genes. To produce gliorna in the brain of nude mice, we injected the cells suspended in PBS into the brain. Then we injected AAV-tk (9.6 × 10⁹ particles in 2 μl) was injected into the brain where glioma cells had been transplanted on day 7 (single injection) or on day 7, 10, and 13 (multiple injections). In the case of a single injection, 100 mg/kg ganciclovir (GCV) was administered ip, twice daily, in 0.5 ml saline, for 6 days after vector injection. Following multiple injections, the same volume of GCV was administered ip, twice daily, in 0.5 ml saline, for 2 days after vector injection and the procedure was repeated three times. When we injected AAV-tk three times, the tumors disappeared completely. Even when we injected the AAV vector once, the tumor size was much smaller than that of control. In the case of interferon-β gene, we got almost same antitumor effect. From these results, AAV vectors could be more efficiently transduced to congenital and acquired diseases than ordinary gene transfer techniques.

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Natural influenza virus infection has been shown to be superior to current viral protein vaccines, which are administered subcutaneously to induce serum antiviral IgG antibodies (Abs), for inducing cross-protection against variant virus infection. The cross-protection induced by natural infection seems to be largely due to the induction of cross-reacting IgA Abs in the upper respiratory tract. This fact suggests that the development of immunization procedures to stimulate mucosal IgA production would improve the protective efficacy of viral DNA vaccines, as well as protein vaccines. To stimulate the IgA production, the protein vaccine together with cholera toxin or Escherichia coli heat-labile enterotoxin as an adjuvant were administered intranasally into BALB/c mice. The vaccination provided cross-protection against variant viruses, in parallel with the production of cross-reacting IgA Abs. Next, field trials were conducted to evaluate the efficacy of the nasal vaccine. The adjuvant-combined vaccine could induce both IgA and IgG responses higher than do the vaccine alone, with the prevention of influenza. Although no serious adverse effects were observed in the human trials, further studies will be required to reduce the toxicity and the allergenicity of these toxin adjuvant. New non-proteinous adjuvants, which may be better than the toxin adjuvant, remain to be developed. To develop effective DNA vaccines, plasmid DNAs encoding hemagglutinin (HA), neuraminidase (NA). matrix protein, nucleoprotein and nonstructural protein from the virus were immunized in mice by particle-mediated DNA transfer to the epidermis (gene gun).The viral surface glycoprotein (HA and NA)-expressing DNAs were most protective among the five DNAs. However, the DNA vaccine failed to provide cross-protection against variant viruses without inducing IgA Abs. Thus, the protective efficacy of the DNA vaccine would be improved by the DNA transfer to the mucosal epithelial cells, whose procedure remains to be developed.

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Pharmacological responses of α-human atrial natriuretic peptide (α-hANP) and salmon calcitonin (s-CT) were estimated following intranasal (i. n.) administration with and without poly-L-arginine (poly-L-Arg) in rats. Maximum urine flow rate after i. n. administration of α-hANP (100 μg/kg) with poly-L-Arg was 2.5 times higher than that of α-hANP alone (control) or with bestatin, an enzymatic inhibitor. Surprisingly, the total urine volume (% volume against control) for the enhancer group was comparable or more than that after i. v. injection of α-hANP (100 μg/kg). On the other hand, maximum decrease of plasma calcium level after i. n. administration of s-CT (10 ID/kg) with poly-L-Arg was greater than that of s-CT alone and the decreased calcium level continued until at least 6h. The total calcium decreasing until 41h was about 23.5% and the same to that after i. v. injection of s-CT alone (13.4IU/kg), as reported previously. These results suggest that poly-L-Arg is very useful as a nasal absorption enhancer for improving pharmacological action of peptide drugs.

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Among various acylated β-cyclodextrins where all hydroxyl groups are substituted with different acyl groups, trivaIeryl β-cyclodextrin (TV-β-CyD) preferentially formed a transparent, adhesive thin-film. For example, when ethanol solution of TV-β-CyD was spread on the backing membranes such as polyethylene terephthalate film, polyethylene film and aluminium foil, a transparent film was formed, the film being tightly stuck on the membranes. The detaching force of TV-β-CyD film was higher and the decrease in the force by the addition of oleic acid was smaller than those of a commercial silicone pressure-sensitive adhesive which is used in transdermal drug delivery system. A vasodilator, isosorbide dinitrate (ISDN), was incorporated in TV-β-CyD film in molar ratios of 1 : 1 and 2 : 1 (ISDN : TV-β-CyD). The release rate of ISDN from TV-β-CyD film increased with increase in the film area, and slightly increased by the addition of oleic acid in the film. The plasma levels of ISDN after topical application of TV-β-CyD film containing ISDN to abdominal skin of rats were maintained 100 ng/ml for about 10 hours. The results suggests that TV-β-CyD film can serve as a drug reservoir for prolonged release of water-soluble drugs in transdermal preparations.

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Electro-responsive on-off switching controlled release devices using solenoid for the actuator were prepared and the functions were tested. The devices were constructed from solenoid, bias spring and drug reservoir with an iron plate in a wan and a hole gate in the bottom. It was found that the drug reservoir moved by magnetic interaction according to input of electric current through the solenoid so as to fit the gate holes to cause the drug release from the reservoir. Then, the effects of magnetic flux density, moved distance of reservoir and number of nichrom wire coil on the on-off switching function were examined. The competent and compactness of solenoid were also improved. U shape solenoid and permalloy for solenoid core was used to increase the megnetic power. It was proved that on-off switching release of methylene blue as a model drug occurred quickly and sensitively with on-off switching of electromagnetic field in the device using the solenoid as the actuator.

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We have previously developed a new medical material of hybrid liposomes. The hybrid liposomes can be prepared simply by applying sonication to the aqueous buffer solution which contains phospholipids and micellar surfactants ; they are free from any contamination of organic solvent, and more stable for a long time as compared with conventional liposomes. We have already demonstrated that the hybrid liposomes have an antitumor effect on lymphoma cells in vitro. Furthermore, the hybrid liposomes including lipid-soluble antitumor agent were found to markedly prolong the life span of mice model of gliorna. In this study, the uniform and stable hybrid liposomes composed of 90 mol% L-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(10)dodecyl ether (C₁₂(EO)₁₀) having a hydrodynamic diameter of 80 nm were prepared for the therapeutical application. The 50 mol% inhibitory concentration of the hybrid liposomes of DMPC/10 mol% C₁₂(EO)₁₀, ([DMPC]=0.383 mM) on the growth of Lewis lung carcinoma cells in vitro was smaller than that of DMPC liposomes (>1 mM). A mouse model of lung carcinoma was established by intraperitoneal inoculation of Lewis lung carcinoma cells. The survival times of mice model of lung carcinoma in the control groups were 25.0 and 45.0 days for median and maximum values, respectively. On the other hand, the survival times in the treated groups with the hybrid liposomes of DMPC/10 mol% C₁₂(EO)₁₀ were more than one year. These results suggest that the hybrid liposomes should be effective for the treatment of lung carcinoma.

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Hydrogels with water contents of 85, 90, 95, and 98 wt% were prepared through chemical crosslinking of acidic gelatin with varied concentrations of glutaraldehyde. In vivo release test of the gelatin hydrogels incorporating ¹²⁵I-labeled transforming growth factor (TGF)-β1 revealed that the period of TGF-β1 release in the mouse subcutis prolonged with the decreased hydrogel water content. When the skull defect of rabbits (6 mm diameter) was treated by the TGF-β1-incorporating gelatin hydrogels, significant bone regeneration at the defect was observed at TGF-β1 doses 0.1μg or higher per defect, in marked contrast to free TGF-β1. The most efficious bone regeneration was induced by the hydrogels with water contents of 90 and 95 wt%. This water content dependance is ascribed to balance of TGF-β1 release and hydrogel degradation. Shorter TGF-β1 release from fastest degraded hydrogel (98 wt%) will not be enough to induce its osteogenic effect, while the slowest degradation of hydrogel (85 wt%) will physically impair bone regeneration at the skull defect. It is concluded that our biodegradable gelatin hydrogel functioned not only as a release matrix of TGF-β1 but also as a barrier for making space to prevent ingrowth of soft tissues to the bone defect.

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Hollow-type suppositories, which have a hollow cavity to accomodate drugs in various forms such as powder and solution, have been given to a number of patients during the past ten years and their usefulness has been confirmed. For example, more than 13, 000 hollow-type suppositories containing morphine, a potent analgesic, have been administered to patients suffering from severe pain in the terminal stage of cancer in various clinical departments of Shinshu University Hospital. This type of suppository is very effective in relieving such pain. The feature that various doses of morphine can be placed within the hollow-type suppository is very useful, when a higher dose is required to extent the desired effect. The hollow-type suppository that contains valproic acid, an antiepileptic, is effective for prophylaxis and management of seizures. This suppository is particularly beneficial for epileptic patients who cannot take medicines orally. Vaginal administration of a hollow-type suppository containing ulinastatin, which decreases the activity of elastase in the cervix of the uterus, was found to be effective in the prevention of imminent abortion, in combination with oral administration of uterine-relaxing agents. Thus, rectal and vaginal administrations of hollow-type suppositories are clinically useful drug delivery systems.

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