Lipophilicity and molecular weight have long been believed to be determinants of the transfer of drugs into the central nervous system(CNS) across the blood-brain barrier(BBB), which is formed by the brain capillary endothelial cells (BCECs). However, recent advances in studies on the BBB transport of xenobiotics, as well as nutrients and neuroactive agents, have led to a change in our concept of the BBB. It is no longer regarded as a static lipoidal membrane harrier of endothelial cells which have tight junctions without fenestrations, but rather is censidered to be a dynamic interface which has physiological functions for the specific and selective membrane transport of many compounds, as well as degradative enzyme activities. It has carrier-mediated transport systems for relatively small molecules, an absorptive-mediated endocytosis system for positively charged peptides and a receptor-mediated endocytosis system specific to certain other peptides. The recent discovery of P-glycoprotein as a specific efflux pump in addition to the above influx systems confirmed the idea that the BCECs form a well organized barrier that by actively and selectively controls the influx and efflux of compounds. From the viewpoint of drug delivery, although an increase in the lipophilicity of compounds is useful to enhance the permeation across the BBB, such a strategy would also increase the permeation into tissues other than brain. Accordingly, for the development of brain-specific drug delivery systems for neuroactive compounds, it is important to understand and utilize the specific transport mechanisms of the BCECs. Here I describe the physiological and molecular characterization of the transport mechanisms of the BBB for natural compounds, as well as bioactive xenobiotics.

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At present, vectors widely used for gene transduction are retroviruses and adenoviruses. However, the transduction using these vectors are primarily conducted ex vivo. The direct in vivo gene delivery method to target tumor cells are required. Trials to augment specificity of gene targeting to tumor cells have been in progress including approaches to use modified virus particles by gene technology instead of wild type particles or to take advantage of ligand-receptor or antibody-antigen systems by allowing vector DNAs to bind these ligand and antibodies. Transferrin receptor(TfR) is widely distributed on actively proliferating cells and is therefore an appropriate target for in vivo gene delivery into those cells. We developed a simple method to conjugate double-stranded DNA(biotinylated β-galactosidase DNA after transamination) to biotinylated Tf via streptavidin and demonstrated successful transduction of the conjugate into TfR-positive human leukemia(K562) and human colon cancer(M7609) cells. We achieved a better transduction rate than the presently available results of Tf-polylysine-DNA complex. The present method may provide a novel technique for gene transduction, which may be used especially to transfer various DNA vectors into cells specifically expressing TfR, in vivo as well as in vitro.

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A design and construction of new type of drug delivery systems having signal responsive controlled release function was studied using semiconductor material and the fabrication technique. A silicone wafer was fabricated by a lithographyetching to make chips with 5×5 mm wells and a pitch to connect the chips, The chips were covered with a pyrex glass plate. Holes for drug release were penetrated in the cover glass lid (Type A) or in the bottom of silicone well (Type B). A crosslinked poly acrylic acid layer was formed on the holes as the pH-responsive sensor-gate by a monomer coating and the photo-polymerization. Release of methylene blue as a model drug by natural stream was investigated. The release was promoted with the increases of numbers of chip and hole. Then the pH change responsive releases of methylene blue were studied. The drug release was remarkably promoted under acidic conditions and retarded under alkaline conditions owing to reversible volume changes of polyacrylic acid layer between shrinkage and expansion. It was proved that the system having polyacrylic acid sensor-gate carried out the pulsatile releases of methylene blue in response to the on-off switching of pH changes between acidic and alkaline pHs. Further studies would be done on the independent release design from each indivisual chip and the networked and programmed release design from the integrated chip systems, using the silicone semiconductor and the fabrication techniques.

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It was found that the stratum corneum was completely disrupted to intact individual corneocytes only by immersion in an aqueous solution of lauryldimethylamine oxide (LDMAO) /d-limonene mixture. The enhancing effect of binary system containing LDMAO and d-limonene on the skin permeation of antipyrine, a water-soluble model drug, was evaluated by in vitro permeation technique using hairless rat abdominal skin mounted in Franz type diffusion cells. The skin permeability of antipyrine in LDMAO/d-limonene binary system was higher than that in water without enhancer and that in each one of the binary system. It was found that when d-limonene was added at a concentration of 10 mM in 20 mM LDMAO aqueous solution, the additive effect of d-limonene reduced the lag time period of antipyrine permeation. The effect of alkyl chain length of a series of alkyldimethylamine oxides on the skin permeation of antipyrine was evaluated. The maximum effect was observed in the case of LDMAO, a chain length of twelve carbons. We have found LDMAO/d-limonene binary system to be usefull as penetration enhancer.

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Amino acids are amphoteric electrolytes and water-soluble compounds. In general, a water-soluble drug such as amino acid shows low skin permeability due to the barrier effects of stratum corneum. We had previously reported that skin permeability of L-phenylalanine improved by approximately 71∼220 times upon the use of its ester derivatives. In this paper, we selected L-phenylalanine as a model compound and investigated skin permeability of its amide derivatives. And also we investigated the correlation between chemical modification of L-phenylalanine and its skin permeability. Excised hairless mouse skin was mounted between the donor and the receptor chamber of a skin-permeation apparatus. The compound that permeated through the skin was analyzed by HPLC. The permeability of L-phenylalanine and its derivatives was calculated from the accumulated amount of the sample in the receptor chamber for specific time period. The results showed that the amide permeated through the skin without its structural change and the permeation rate of amide derivatives were almost the same as that of L-phenylalanine. The correlation between chemical modification of L-phenylalanine and its skin permeability showed that the esterification of carboxyl group improved its skin permeability and the amidation of amino group or carboxyl group reduced the permeability. The results indicated that the amidation of L-phenylalanine did not improve its permeability through the skin and that its amide derivatives might not be a good candidate as a prodrug of L-phenylalanine. On the other hand, since the esterification of L-phenylalanine improved its permeability through the skin, its ester derivatives could be a prodrug of L-phenylalanine.

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The pharmacokinetics of IFN after subcutaneous (s. c.) administration of IFN-minipellet, a new sustained release formulation of IFN, were investigated in rabbits. The serum IFN level was elevated slowly and reached the maximal level 7 hours after the administration of IFN-minipellet, while s.c. administration of aqueous solution presented a rapid elevation of IFN level in sera, The serum IFN concentration decreased gradually thereafter. No sharp peak suggesting an initial burst was seen. IFN was still detectable in sera 7 days after the administration. Serum IFN levels after the administration of IFN-minipellets in different lengths (i. e. different doses) were proportionally dose-dependent, and it was indicated that the release property of IFN from IFN-minipellet was not affected by the length of minipellet. S. c. administration of IFN-minipellet in abdominal, femoral, and dorsal region provided similar time courses of IFN concentration in sera. These results showed that the release of IFN from IFN-minipellet in vivo was wellcontrolled and continued over 7 days. The local toxicity of subcutaneously administered IFN-minipellet in rabbits was slso investigated by observing the administration site for 1 week. No inflammatory reaction around IFN-minipellet was seen. IFN-minipellet was considered to be highly biocompatible. Clinical investigations revealed that IFN minipellet caused much less side effects than conventional IFN therapy while their therapeutic effects were comparable. The mechanism of this phenomenon was discussed from the viewpoint of pharmacokinetics. It was suggested that the slow release of IFN may be the key to the low pyrogenicity of IFN-minipellet. IFN-minipellet seems to be highly useful in the treatment of clonic hepatitis C to improve QOL of patients by reducing the frequency of administration and adverse effects.

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The highly porous Dacron graft fabricated from polyester filaments is generally considered to be one of the most suitable synthetic vascular prostheses in arterial reconstructive surgery. To prevent blood leakage through the pores during an operation, several sealing materials such as albumin (ALB), collagen and gelatin have been employed. As vascular graft infection is a disastrous complication on vascular surgery, great effort must be taken to avoid it. To reduce the systemic effect while maintaining an increase in local resistance to graft infection, it seems reasonable that an antibiotic-loaded graft prolongs the release of antibiotics from the graft at the operated site. Common skin microorganisms, such as Staphylococcus aureus and Staphylococcus epidermidis, have been associated with graft-related infection. Levofloxacin(LVFX) was used as a model drug since it shows a broad protective spectrum against Gram-positive and Gram-negative bacteria, particularly staphylococci. To control the release rate of LVFX, ALB was bonded to the Dacron graft. In vitro and in vivo studies demonstrated that the release rate of LVFX decreased in the presence of ALB. One LVFX-ALB disk (diameter : 1.2 cm) was implanted in the skin pocket made in a rat, and inoculated with Staphylococcus aureus or Staphylococcus epidermidis (0.1 ml of 10⁸ CFU/ml). While all control grafts were infected at the time of removal, all LVFX-ALB Dacron grafts resisted infection, thus demonstrating their effectiveness. These studies suggest that LVFX controlled-releasing Dacron delivery system can decrease graft infection at the operated site.

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Because the cytotoxic effect of hyperthermia in the 42-43°C range has been demonstrated, a transition temperature of the conventional liposomes have so far been 42°C to enhance the synergistic effect of hyperthermia and antitumor drugs. When considered this drug delivery system as clinical use, an important problem about this treatment is that the glioma cells invade wide portions of the brain and present technology has not yet been able to achieve the heating of the wide areas invaded by glioma cells. On the other hand, it has been remarked that the blood brain barrier(BBB) limits the ability of many antitumor drugs to penetrate the central nervous system. Despite of their greater cytotoxic effects on glioma cells in vitro, many drugs, such as adriamycin (ADM), have not so far been used mainly because they are regarded as being blocked by the BBB. In order overcome these roblems we prepared liposomes which brought a sharp release rate of ADM above 40°C. This liposome showed a significantly great antitumor effect against rat malignant gliomas which were transplanted into their legs at the temperature of 40°C. These results suggests that this procedure (1) contributes to the synergistic effect of hyperthermia and antitumor drugs in the regions heated above 42°C, (2) helps to deliver the antitumor drugs more widely with the temperature of 40°C, (3) gives a good chance to many antitumor drugs which have so far not been used from the problem of the BBB. We suggest that this advantage of our plan could outweigh the disadvantages of the conventional thermo-chemotherapy.

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Lipiodol and non-ionic contrast medium diluted anti-cancer agents are widely used for a micro embolizing material in transcatheter arterial embolization(TAE) therapy for hepatocellular carcinoma (HCC). Hepatic resection and TAE therapy with lipiodolization(CDDP-epirubicin-lipiodol multi-complex : CEL) are most frequent therapy in our University Medical Hospital because of tumor biological behaviour and hepatic functional reserve. Experiments as concerning with liver associated lymphocytes (LAL) in mice were exerted prior to clinical use. In the case of trans-portal injection of OK-432 (a king of biological response modifier) liposome (OK-L), CD3⁺/IL-2Rβ⁺ cells as LAL were increased in the liver. It was shown that CD8⁺ cells were predominant. CD3^-/IL-2Rβ⁺ cells as natural killer cell were increased in the liver for systemic intravenous injection. Patients with HCC were received intra-arterial injection of CEL+OK-L. Prominent infiltrations of lymphocytes in both tumor and non. tumorous area were observed in case 1 patient, who was operated. Compared with patients received by CEL only, the proportion of CD3⁺ cells was elevated in the liver and tumor. CD4⁺ cells were predominant in OK-L group. In all cases (OK-L), fever was not detected and rise in white cell count and rapid fall in lymphocyte count were observed after intra-arterial injection therapy. CEL and TAE combination therapy of HCC appears to be more effective. Therefore, this treatment is built up for lymphocyte activate kliller therapy with OK-432 in liposome.

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