Receptor-mediated endocytosis(RME) is now well recognized as a polypeptidc clearance system. In addition, a drug delivery system (DDS) via RME has been developed to deliver some drug specifically into the target cell expressing receplors on its plasma membrane. Therefore, kinetic analysis of RME process is important, both to clarify the pharinacokinetics of polypeptide itself and to estimate the efficiency of drug targeting. We have been studying kinetically both the hepatic and renal handling of epidermal growth factor (EGF) using in vivo, perfused organ, and isolated cell system. Such an analysis enables us to determine the kinetic parameters for the construction of a kinetic model for RME. Based on such a kinetic model, we can discuss the contribution of each process in RME on the efficiency of drug delivery into the cell.

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Recently, with the introduction of medical electronics, new technologies for measurement, communication and operation to achieve the adaptive control have been developed in many fields of science. As one of the most evolutional models of such achievement, the artificial endocrine pancreas has been developed. The artificial endocrine pancreas is a feedback control system regulating insulin delivery on a minute-by-minute basis according to the measured blood glucose levels. The bedside-type artificial endocrine pancreas has been proven to be useful not only as a therapeutic tool for diabetes mellitus, but also as a elegant research tool for investigating the pathophysiology of the disease. With significant advances in the development of a subcutaneous tissue glucose monitoring system, the wearable-type artificial endocrine pancreas has been applied to diabetic patients. With this system, perfect glycemic control can be obtained for longer periods in ambulatory diabetic patients. The trend in the development of artificial endocrine pancreas is now directed to implantable devices. Much efforts have been conducted to realize these devices.

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Novel stabilizing method for unmodified oligodeoxynucleotides (ODNs) has been investigated. Chemically modified ODNs, which carry complementary sequences of unmodified antisense ODN, can form a double-stranded complex with the ability to stabilize the unmodified oligomer. The modifications were made on phosphodiester back-bone (phosphorothioate, SO), thymine base (5-phenylethyl substitution), 5'-terminal(introduction of poly(ethylene glycol)). To determine the potential of this method, the in vitro stability in human plasma and in vivo retention in mouse after iv injection of the hemi-modified double-stranded ODNs were evaluated. Dissociation of unmodified antisensc ODN from the hemi modified double-strand and reconstitution of the double-strand with an unmodified sense ODN were also recognized.

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The aim of the present study was to develop the most favorable conjugation condition by evaluating the drug activity and the antibody activity of immunoconjugates consist of monoclonal antibodies and carboplatin in various ratios. The monoclonal antibodies used are 6D7, which recognizes cytokeratin-8, and 1B2, which recognizes CEA on the cell surface. Carboxymethyl dextran (MW=13, 000) was used as the carrier, which was added to the monoclonal antibody in the molar ratios from 1 : 1 to 3, 000 : 1, and then filtrated to remove uncoupled dextran molecules. And then carbopIatin was given to the mixture in the fixed molar ratio of 100 : 1, and again filtrated. In cases of both 6D7 and 1B2, the binding rate of dextran was almost stable, from 90 to 98 %, and that of carboplatin was increased from around 40 % to more than 80 %, in relation to the amount of the drug given to the antibody-dextran complex. The weight ratio of carboplatin to monoclonal antibodies were calculated, which also increased in relation to the amount of the drug given to the complex, and the maximal drug/antibody weight ratio was 626 : 1. The cell proliferation inhibition activity was evaluated by colony formation method using a human ovarian carcinoma cell line, HOC-21, whose culture medium was found to contain high amount of TPA and CEA. Immunoconjugates containing 6D7 or 1B2 showed dose-dependent inhibition activity, which was similar to unbound drug and nonspecific mouse IgG-drug conjugate. Thus, the drug activity was found not to be deranged by the conjugation procedure. The antibody activity was studied by an enzyme immunoassay using antigen-coated beads, and it was showed that, in 6D7-conjugates, the antibody activity was decreased when a large amount of drug was conjugated, while 1B2 conjugates showed a negligible change, although these changes seemed to be toretable. From the results obtained, as for the immunoconjugates we developed, the drug activity was preserved, and the decrease in the antibody activity seemed to he torelable, which should be preferable for the immunotargeting therapy.

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We examined the ability of polyethyleneglycol in avoiding reticuloendothelial system (RES) from colloid chemical view points. In this respects, we examined the effects of the dose on the distributions, in vivo. The thickness of the fixed aqueous layer(TAL) around liposomes modified with 1-(monomethoxy polyethyleneglycol)-2, 3-dipalmitoyl glycerol(PEG-DPG) containing adriamycin (ADR) (PEG-LADR : 5 mol%) was determined to be 1.8 nm, while those around conventional plain liposomal ADR (PLADR) and PEG (×2)-LADR (PEG : 10 mol%) were 0.3, 1.6 nm, respectively. The thickness of the fixed aqueous layer around PEG-DPG modified liposomes was not dependent on PEG-DPG mol content. The RES avoiding was dependent on TAL. In vivo study, the prolonged circulation time in the blood, decrease in the distribution to the heart and RES avoiding was confirmed both in high (ADR 7.5 mg/kg, i.v.) and low (ADR 2.5 mg/kg, i.v.) dose study. In the liver, in 7.5 mg/kg dose, the ADR concentrations by liposomes were higher than that of ADRsol while 2.5 mg/kg dose, the ADR concentrations by liposomes were lower than that of ADRsol. Thus, the RES uptakes were affected by ADR dose as well as TAL. Therefore, distribution study of liposomes must be investigated at doses as close to the clinical doses as possible.

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The transdermal delivery of theophylline was evaluated using various ethanol (EtOH)/water (60/40)-hydroxypropylmethylcellulose (HPMC) systems with or without lauric acid as a permeation enhancer. The in vitro skin permeability of theophylline across the excised hairless mouse skin and the in vivo skin absorption of the drug using abdominal rat skin were remarkably enhanced by the addition of lauric acid to the EtOH/water (60/40) binary vehicle or EtOH/water(60/40)-HPMC systems. The limited concentration of lauric acid for the maximal permeability of the drug was 4% (w/w). The in vivo skin absorptions of theophylline from EtOH/water (60/40)-HPMC systems with 4% (w/w) lauric acid and with various concentrations of three HPMCs(65SH-400, -1500, -4000) showed essentially same patterns as that from EtOH/water (60/40) binary vehicle with 4% (w/w) lauric acid. Furthermore, EtOH/water (60/40) -3% (w/w) HPMC 65SH-4000 system with 4% (w/w) lauric acid indicated the greatest bioavailability (92.7%) in the systems investigated and was considered as the most useful recipe for delivery of theophylline applicable to the skin.

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The molecular evolution by replicase (RNA-dependent RNA polymerase) was attempted for generating new nuclease-resistant RNAs, which realizes a cell free Darwinian molecular evolution. The Qβ replicase replicates genome RNA of Qβ virus, and additionally it can also synthesize other small RNAs using “minivaliant” as a template. With the aid of Spiegelman's serial trasfer method, which is a step by step molecular evolution system in a laboratory by RNA proliferation with frequent mutation using Qβ replicase, high concentration ribonuclease T2-resistant fragments of RNA could he obtained.

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Microspheres were prepared using a polymer consisting of glycolic acid-L-lactic acid copolymer (PGLA) with a molecular weight of 27, 000 and cisplatin (CDDP) for local therapy of pleuritis carcinomatosa. The CDDP-containing PGLA microspheres(CDDP-MS) were about 100 μm in diameter with a CDDP content of 5%, and all the CDDP was released in vitro within about 3 weeks. For administration, a drain was inserted into the thoracic cavity of the patients, and CDDP-MS, equivalent to 100 mg CDDP, were infused into the pleural cavity with 100 ml of physiologic saline, followed by clamping of the drain. The clamp was removed after 24 h. All the pleural effusion was recovered thereafter, and CDDP was assayed periodically in the effusion and serum. As a control, CDDP solution was administered into the pleural cavity. After administration of CDDP-MS, CDDP was detectable in serum until 5 days and in the effusion until 8 days, at the time of extubation. The concentration of CDDP in serum was lower than that in the CDDP solution. The total amount of drained CDDP was about 1, 000 μg, or about 1% of the total dose. In contrast, after treatment with CDDP solution, 3.4% of the CDDP dose was drained. Upon removal of the drain, the cytology of the pleural effusion was changed from Class V to Class III. No side effects of CDDP-MS were noted, but nausea and vomiting were observed after treatment with the CDDP solution. Since CDDP-MS release non-protein-binding CDDP, they would be expected to produce a prolonged antitumor effect at the tumor cell implantation site, are therefore considered to be useful for the treatment of malignant pleural effusions.

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