We are exploring the use of ribozymes and novel dimeric minizymes as gene-inactivating agents for treatment of AIDS and CML desease, respectively. High-level expression under control of the pol III promoter would be advantageous not only for conventional ribozymes but also for minizymes since high-level expression should enhance the likelihood of the dimerization of minizymes. Three kinds of conventional ribozymes were designed, each targets against HIV-1 gene. The RNA polymerase III-based promoter we chose was the promoter of a human gene for tRNA^Val which has been used successfully in the suppression of target genes by ribozymes. The designed ribozyme was connected downstream of the tRNA^Val portion with different linker sequences, therefore, the efficiencies of the binding to substrate were different for each ribozyme transcript. Expression level of ribozymes was correlated with the half-lives of each ribozyme transcript, and the effect of the ribozyme-mediated repression of HIV-1 gene in living cells were in proportion to the stability of ribozymes. As a result, the most stable ribozyme in vivo could inhibit the proliferation of HIV-1 almost completely. We demonstrated previously that minimized-hammerhead ribozyme with low activity form very active dimeric structures with a common stem II. We are exploring the use of dimeric minizymes as gene-inactivating agents for treatment of CML desease. High-level expression under control of the pol III promoter would be advantageous for minizymes and enhance the likelihood of their dimerization. Therefore, we chose the promoter for a human gene for tRNA^Val. Although we feared initially that the tRNA^Val portion of the transcript might hinder the dimerization of tRNA^Val driven minizymes, the novel tRNA-embedded minizyme had strong activity, To our surprise, the cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA^Val-embedded conventional hammerhead ribozyme. Our results indicate that tRNA-emhedded minizymes should be considered as potential candidates for gene-inactivating agents.

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It is necessary to develop a more efficient gene expression system for gene therapy. A plasmid DNA, using eukaryoic or mammalian promoters, requires to localize into nuclear for gene expression. However, it is difficult to entry into nuclear, because nuclear pore size is not sufficient against the size of plasmid DNA. In this study, to develop a novel cytoplasmic gene expression system that dose not require nuclear localization of plasmid DNA to transcription, we examined the characterization of T7 cytoplasmic gene expression system. When co-transfected with pT7-IRES-L(luciferase expression plasmid containing T7 promoter) and T7 RNA polymerase into LLCMK₂ cells, the gene expression of pT7-IRES-L was observed rapidly within 6hr after transfection and significant level of luciferase activity was detected. In contrast, pRSVL, a common plasmid DNA consist of luciferase expression plasmid and Rous sarcoma virus promoter, required 24-48hr for induction of gene expression. The gene expression level of the T7 system was enhanced with an increase in the amount of T7 RNA polymerase. To increase and prolong the gene expression, a plasmid DNA(pT7 AUTO-2) which contained the T7 RNA polymerase gene driven by the T7 promoter was co-transfected with pT7-IRES-L and T7 RNA polymerase. The plasmid DNA(pT7 AUTO-2) dose-dependently enhanced the luciferase gene expression by pT7-IRES-L and T7 RNA polymerase. In addition, we attempted to optimize the cytoplasmic gene expression system. The optimal ratio for co-transfection of pT7-IRES-L and pT7 AUTO-2 was 1 to 3 (mole ratio). These results suggest that T7 gene expression system may be useful in many gene therapies where transient but rapid efficient gene expression is required.

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The living body is composed of numerous cells that function cleverly under various network to maintain homeostasis. It is especially surprising that bioactive molecules such as hormones and cytokines have temporal, quantitative, and local targets in living body. Regarding the bioactive molecules as drugs, biosynthesis function, sensor function and controlled release function of bioactive molecules which are inherent in living cells are ideal of drug delivery system. We have studied the development of cytomedicine, a concept which involves using intelligent particles, living cells as carriers of bioactive molecules. Previously we reported that this cytomedicine was very effective as an in vivo long-term delivery system for bioactive molecules in animal model experiment. However the cytomedicine we studied so far produced and secreted bioactive molecules and functioned only sustained release system. The biggest benefit of cytomedical therapy is that we can strictly control blood concentration of drugs and can avoid frequent administration of drugs by utilization of various functions of living cells such as sensor function. In this study, we examined the biocompativility of alginate-poly(L) lysine-alginate (APA) microcapsules as the immobilized devices of cells for cytomedicine. Moreover, to evaluate the possibility of cytomedical therapy for diabetes mellitus using microencapsulated pancreatic β cell lines (MIN6 cells and βTC6-F7 cells) with glucose sensor, viability and glucose responsiveness of microencapsulated MIN6 cells and βTC6-F7 cells were examined in vitro. These APA-microencapsulated cells maintained glucose sensor and should good viability at least three months. This results suggest that cytomedicine using microencapsulated pancreatic β cell lines with glucose sensor is useful for therapy for diabetes mellitus.

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We have produced hybrid liposomes(HL) which are effective to inhibit the growth of tumor cells in vitro. The properties of these liposomes, such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the composition. In this paper, inhibitory effects of hybrid liposomes composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenedodecyl ether (C₁₂(EO)₁₀) on the growth of melanoma cells in vitro and in vivo were examined. The 50% inhibitory concentration of the hybrid liposomes composed of DMPC and C₁₂(EO)₁₀ (0.045 mM) was one-twelfth of that of DMPC liposomes (0.55 mM). A mouse model of carcinoma was established by intraperitoneal inoculation of 0.04 ml suspension containing 1 × 10⁵ melanoma cells. The median survival times were 14.5 and 26.5 days in the control and treated groups, respectively. The significantly prolonged survival (183%) was obtained. These results suggest that the hybrid liposomes should be effective for the treatment of cancer.

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Proinflammatory mediators such as histamine, platelet activating factor and nitric oxide increase intestinal epithelial permeability and lead to disrupt its barrier function. ChemicalIy induced increase in the epithelial permeability could be mediated by stimulation of the release of these mediators. Nevertheless, the contribution of each mediators appears to be difficult to elucidate in vivo. Hence, an in vitro evaluation methodology for detecting the potential contribution of the mediators to the enhancement in mucosal permeability due to absorption enhancers has been established. Permeability of the rat colonic epithelial membrane in the presence or absence of the adjuvants was determined in vitro with a modified Ussing-type diffusion chambers. Both histamine and its releaser compound 48/80 significantly increased the transepithelial permeability that monitored by using carboxyfluorescein as a poorly permeable maker. The increase in permeability was found to be inhibited by the in vivo treatment of the intestine with the intraperitoneal dose of diphenhydramine, a H₁-blocker, or FPL-52694, a mast cell stabilizer, prior to the excision. The absorption enhancers, oleic acid, lauryl maltoside and sodium deoxycholate showed potent enhancement effects on the epithelial permeability. The enhanced permeability with sodium deoxycholate was found to be significantly suppressed by the H₁-blocker, indicating that the enhancing effect of sodium deoxycholate was partially mediated by histamine released within the mucosa. On the other hand, the treatment with H₁-blocker showed no effect on the mucosal permeability enhanced by oleic acid and lauryl maltoside. These results demonstrate that the methodology should be of potential usefulness in the evaluation of the possible contribution of inflammatory mediators to chemically induced hyperpermeability in intestinal mucosa especially by absorption enhancers. Investigation on the other inflammatory mediators is in progress.

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In an experimental pulmonary metastasis model employing Meth A cells, significant and dose-dependent inhibition was observed by intravenous pre-administration of PC-SOD. Unmodified SOD (U-SOD) was also effective, but was less potent than PC-SOD. The activity of endogenous SOD remained unchanged immediately after tumor cell implantation. In while, PC-SOD significantly increased the SOD activity compared with that of the control group. U-SOD also increased the activity, which was more quickly reduced. In vitro addition of PC-SOD dose dependently suppressed cell growth of Meth A, in while SOD had little effect. In addition, the combination of PC-SOD and S-nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide (NO) generating agent, had additive effect. It was also found that PC-SOD prevented the reduced levels of NO_x in the lung following tumor cell inoculation. Therefore, there might be a possibility that PC-SOD was more useful to prevent the excessive formation of superoxide anions (O₂^-) and peroxynitrite (ONOO^-) to elicit cell damage and facilitate metastatic incidence.

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Electro-responsive on-off switching controlled release devices using shape memory alloy (SMA) for the actuator were prepared and the functions were tested. The devices are constructed from SMA coil, bias spring and drug reservoir, and they are connected with each other. The reservoir and the outer wall of the device have small holes. The two holes overlap by SMA shrinkage and shifting of reservoir with switching on of electric current so as to cause the drug release, Then, the temperature change in SMA coil and the moving distance of reservoir was investigated under on-off states of electric current. It was observed that the reservoir movement occurred quickly with the shrinkage of SMA coil after on state of electric current and reached to about 12 mm at 35°C in SMA coil temperature. The reservoir returned to the original position correctly after off state of electric current. Then, a model drug is charged into the reservoir and the release function was tested. Very sensitive on-off switching release under on-off states of electric current was observed using the device with SMA actuator. Therefore, the possible mechanism can be explained by the opening and shutting of the reservoir gate with its movements owing to the repetition of shrinkage and expansion of SMA according to on-off switching of electric current. It was proved that electro-responsive on-off switching controlled release system is possible for the actuator using SMA.

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