Modern biotechnology has realized to produce polypeptide drugs on a large scale. Although some of them need controlled release because of the too short half-times in the blood, any good systems for their sustained release are not yet available. The major reason is a high susceptibility of polypeptides to denaturation. To develop an effective system for the sustained release of polypeptides, we take advantage of polyion complexation that is defined as complex formation between two oppositely charged polyelectrolytes. Polypeptides are either positively or negatively charged in aqueous solution unless the solution pH is equal to the isoelectric point. Because the polymeric carrier being used for injectable drugs should be resorbable, we chosed gelatin as a carrier of polypeptide drugs. Both negatively and positively-charged gelatins are commercially available. As an example we describe here a combination of negatively-charged gelatin with basic fibroblast growth factor (bFGF) that is a cell growth factor having an isoelectric point of 9.6. Upon mixing in aqueous solution, these two biomacromolecules form a polyion complex, allowing the sustained release of bFGF when injected in animals. In vivo studies revealed that the bFGF molecules released in the body still maintained the biological activities as a cell growth factor. This new drug delivery system was applied to vascularization and regeneration of cartilage and bone for mice and rabbits.

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In order to reveal quantitatively interaction of complement (C) system with liposomes, we determined C3 fragments associated with the liposomes having different sizes after incubation in human plasma. The amount of C3 fragments per unit surface area on the unstable liposomes (Man-liposomes) which modified with synthesized glycolipid (cetylmannoside, Man) increased with the increase in the liposome size, whereas that of C3 fragments on the stable ones (PC-liposomes) was little found. In addition, the instability of Man-liposomes also increased with the increase in the liposome size and there was linear correlation. On the other hand, the amount of bound plasma proteins per unit surface area of liposomes was approximately constant regardless of differences of lipid composition and size, and showed no correlation with instability of the liposomes in the plasma. These in vitro results indicate that the instability of Man-liposomes is governed by the affinity of C system and the C system can recognize not only liposome surface characteristics but also liposome sizes. To demonstrate clearly the reason for the linear correlation between the affinity of C system and liposome size, we discussed the underlying mechanism based on the previous finding that the activation of C system by Man-MLVs was enhanced through classical C pathway and presented the hypothesized osculating model on the assumption that extent of C activation is governed by attachment of a C activator.

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The toxicity of lipiodol (LPD) w/o/w emulsions encapsulating epirubicin hydrochloride (FARM) after iv administration to rat was investigated compared to the conventional o/w emulsions used for transcathether arterial embolization (TAE) therapy for hepatocellular carcinoma. Survival time of the rat administered w/o/w emulsion was longer than that of the o/w emulsion. The rats administered o/w emulsion (≥1.5 ml/kg) were deceased within 2 days by pulmonary edema caused by embolism with fine oil droplets. The edema was not found in the lungs of the rats administered w/o/w emulsion. The toxicity of the FARM, ie, size reduction of thymus gland and ulceration in the stomach, was reduced by encapsulating the drug in the w/o/w emulsion. The present w/o/w emulsion prepared by us proved a desirable formulation for the TAE therapy because of reducing toxicities of oil droplets and drug.

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Prodrug of diclofenac (DF-C₂-P) was synthesized by introducing phospholipid-like compound into carboxyl group in the mother molecule. DF-C₂-P was rapidly decomposed in neutral and alkaline solution while stable in acidic medium. A considerable increase of hydrolysis was observed in the PBS containing enzyme, porcine liver esterase, and diclofenac (DF) was produced as a result of enzymatic degradation of DF-C₂-P. Plasma concentration of DF-C₂-P and DF were analyzed after i v bolus injection in male Wistar rats, and four-compartment open model was applied to estimate pharmacokinetic parameters. Pharmacological activities of DF-C₂-P was investigated with a tail-flick test and an acetic acid-induced writhing test in ddY mice. A significant enhancement in the antinociceptic action was observed by the administration of DF-C₂-P, compared with that of DF. Gastric mucosal irritation was hardly observed when orally administered DF-C₂-P in rats, suggesting that the prodrug prepared by introducing the phospholipid-like compound to the DF molecule may reduce ulcerogenic properties of DF.

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We used the possibility of α-tricalcium phosphate (α-TCP) particles incorporated with carboplatin (CBDCA) as a drug carrier using a rat experimental model of AH-130 abdominal carcinomatosis. α-TCP is known to have excellent biocompatibility and biodegradable and its chemical properties are similar to hydroxyapatite. The α-TCP paticles used in this study was 50-150 μm in diameter, which seemed to offer adequate space to incorporate drug. We studied the biodegradation behavior of the particles in the abdominal cavity and the distribution kinetics of platinum (Pt) in serum, ascites, and various organs following ip administration. The α-TCP paticles were predominantly taken up in the milky spot of greater omentum in a few days, and dissolved gradually in six months, during which no adhesion and side effects were observed. The Pt levels in ascites and the greater omentum were higher in the α-TCP-CBDCA ip group from 0.5 to 168 hours than those in the free-CBDCA ip and iv groups. The Pt levels in serum were almost the same both in the α-TCP-CBDCA ip and the free-CBDCA ip, but those in organs were lower in the α-TCP-CBDCA ip. These results suggest that the localization of CBDCA may be enhanced by using porous α-TCP particles as a drug delivery carrier and may be allow for the drug to contact with cancer cells for extended times.

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Recombinant human interleukin-2 (IL-2) was found to be strongly adsorbed onto small liposomes (20-50 mm) containing distearoyl phosphatidylcholine (DSPC) or hydrogenated soy bean phosphatidylcholine (HSPC) as a major lipid component. Under optimal conditions most of added IL-2 was attached to the liposomes. Pharmacokinetic studies for IL-2 merely mixed with such liposomes after intravenous administration in BDF1 female mice demonstrated more prolonged blood circulation and higher AUC in various organs except for kidneys in comparison with IL-2 alone. A mixture of IL-2 with DSPC/DSPG (10/1) liposome (Lipo 1-IL2) gave 13- or 18-times higher AUC in liver and spleen, respectively. Lipo 1-IL2 showed enhanced inhibition against hepatic metastases of M5076 intravenously inoculated in mice, when compared with IL-2 alone. This therapeutic effects against metastases in other organs, lung and ovary, were weaker than that in liver. The mixture did not affect liver function GOT or GPT when normal mice were treated with twice the dose showing excellent antitumor effect. This simple preparation with easy use is expected to have potential for increasing therapeutic efficacy against hepatic metastases.

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In the past decade, many researchers have been keen to apply the antisense oligonucleotides as therapeutic agents. Several antisense molecules are now going on the clinical trials. However, unappropriated targeting efficacy hamper to get suffifcient biological activities. The effort to overcome the nuclease instability of antisense molecules leads to developement of various stable analogues. Since antisense molecules distribute in lysosomes, sufficient biological functions can not be expected. If antisnese molecules can be delivered to appropriate site with high efficiency, application of antisense strategy would be widened. In this study, we synthesized antisense phosphorothioate oligonucleotides (S-oligo) toward mRNA of vascular endothelial growth factor (VEGF) and 80 S-oligos candidates were tested in in vitro translation system. From this selection, 4 compounds were tested as the inhibitory effect on tube formation of human umbilical vascular endothelial cell (HUVEC). Since inhibitory effect of S-oligo on bute formation of HUVEC was not sufficient enough, we used transfectam to enhance the biological activity of S-oligo. Transfectam enhanced the inhibitory activity of S-oligo. While S-oligo itself were distributed in the cytoplasm punctately, S-oligo and transfectam complex localized in the cytoplasm and some fractions were in the nucleus. Localization of S-oligo in whole cells would contribute to enhance the biological activity. However, further study is required to obtain the enhancement of particular antisense activity in a sequence specific manner.

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The lipid composition, particle diameter and dose have been reported to affect the liposomal uptake in the tumor. However, effects of encapsulation amount of drugs in liposomes on the uptakes have scarecely been reported either in vitro or in vivo. In this study, we examined the uptake of liposomes containing CPT-11 by Ehrlich ascites carcinoma in vitro changing the encapsulation amount of CPT-11, and also the tissue distribution of liposomal CPT-11 in vivo with mice. After the addition of high concentration as CPT-11, the uptakes in the tumor cell by liposomes was about 1/3 of that by the solution in vitro. However, after the addition of same level as tissue distribution of CPT-11 in vivo, there is no difference on CPT-11 uptakes between liposome and solution. Thus lipid satulation in the tumor cells was observed by increasing liposomes in the medium. For a definite dose, the decrease of CPT-11 amount in the liposomes reduced the uptake in the tumor cell. Therefore, we thought that the uptake of liposomes in the tumor cell depended on lipid amount of liposomes. The increase of CPT-11 amount in the liposomes enhanced CPT-11 concentration in the serum, liver and tumor after administration of liposomal CPT-11 to the mice. An enhanced antitumor activity was expected from the result of SN-38 concentration in the tumor.

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