We reviewed the effectiveness of chitosan capsules for colon-specific delivery of insulin. The release of drug from the chitosan capsules was markedly increased in the presence of rat cecal contents, although no release from the capsules was observed in an artificial gastric juice and an artificial intestinal juice. The results of swelling and elongation tests using the chitosan film strongly suggested that chitosan capsules were degraded by the microorganisms in rat cecal contents. The intestinal absorption of insulin following oral administration of chitosan capsules containing insulin with or without additives was investigated by an in vivo rat absorption experiment. A marked decrease in plasma glucose level was observed following administration of the capsules containing 20 IU insulin and Na-glycocholate as compared with the capsules containing insulin only. These results suggested that chitosan capsule could be a useful carrier for colon-specific delivery of peptides including insulin.

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Divinyl ether and maleic anhydride (DIVEMA) is a synthetic polyanion known to stimulate antitumor activity in macrophages (Mφ) and natural killer cells (NK). This compound is used as a carrier of anticancer drugs because of its efficient covalent binding and release of anti-cancer drugs. Recently, the conjugate of DIVEMA with adriamycin (ADM) was reported to have higher tumor control activity in mice-bearing intraperitoneal dissemination of fibrosarcoma (FSaI) cells than either DIVEMA or ADM alone. We here report on the effects of intraperitoneal (ip) injection of this conjugate on the cytotoxicity of peritoneal Mφ and splenic NK against FSaI cells. The conjugate clearly enhanced Mφ cyotoxicity, the maximum cytotoxicity being obsereved 7 days after ip. This cytotxicity enhancement was similar to the activation by DIVEMA, but it was stronger than that by ADM alone. On the other hand no activation of splenic NK by administration of either the conjugate or ADM was observed, despite remarkable NK activation by DIVEMA. These findings indicate that a part of the immunostimulatory activity of DIVEMA remains in the conjugate when DIVEMA is used as a carrier for ADM. Consequently the data also suggest that the antitumor effects of the conjugate resulted from combined effects of the host-mediated activity and the direct cytotoxicity of ADM itself.

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Oxybutynin (OB) is widely used for the treatment of incontinence due to neurogenic bladder dysfunction. In order to reduce side effects and increase therapeutic efficiency, we investigated transdermal therapeutic system of OB. in vitro skin permeation study was performed by employing the vertical diffusion cell in which the excised rat abdominal skin was mounted, and menthol derivatives were selected as effective enhancers. The flux of OB was remarkably increased by the addition of several kind of menthol derivatives (e.g. O-ethylether (MET), O-allylether, O-2-propynylether, O-methylester, O-propylester, O-isopropylester, and O-cyclopropylester). Among these compounds, the skin irritancy of MET was significantly low compared with the other compounds. Therefore, MET is thought to be a promising compound as effective absorption enhancer for the transdermal drug delivery of OB. The data obtained from in vitro skin permeation study were analyzed by a membrane diffusion model derived from Fick's second law, and the diffusion and partition parameters of OB were estimated. The diffusion parameters were increased when the menthol derivatives were incorporated in hydrogel. On the other hand, partition parameters were almost constant. The flux of OB increased with increase of the concentration of MET. Maximum flux was observed when the hydrogel containing 0.5% MET was applied. No further increase of flux was obtained when the amount of MET was increased. In in vivo percutaneous absorption, the excretion of urine was restrained until 8h by the administration of OB hydrogel containing 0.5% MET. It was suggested that OB was delivered through the skin from hydrogel containing 0.5% MET. In conclusion, it was suggested that OB hydrogels containing menthol derivatives, especially MET, were available as a new dosage form for the treatment of incontinence due to neurogenic bladder dysfunction.

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Plasma kallikrein is a protease that releases bradykinin from high-molecular-weight kininogen in plasma. Bradykinin is thought to be involved in the pathogenesis of septic shock. Soybean trypsin inhibitor (SBTI, Kunitz type) is a potent inhibitor against plasma kallikrein. However, half-life (T_1/2) of SBTI in the circulating blood is too short after iv injection because of its small molecular size (Mr 21, 000), and hence the therapeutic application is limited. To enhance therapeutic value of SBTI, we conjugated SBTI with succinylated gelatin (suc-gel-SBTI), which exhibits about 6 times longer plasma T_1/2 and about 4 times larger AUC. We report here improved therapeutic effect against septic shock in rats and guinea pigs by treatment with suc-gel-SBTI. The conjugate prevented hypotension together with production of bradykinin in blood induced by Pseudomonas elastase and lipopolysaccharide from E. coli much more effectively and for a longer time period than that with the native SBTI. In conclusion, the therapeutic efficacy of SBTI is much improved by its polymer conjugation due to the better pharmacokinetics than the native SBTI.

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Superoxide dismutase (SOD) has been recognized as an attractive component not only for dermal anti-inflammation medicaments but also for cosmetics to scavenge and detoxicate superoxide (O₂^-), a very highly active oxygen species to the skin cells or its sebum layer when exposed to the sunshine (especially UV-A wave range). However, the wide application of SOD is still limited in development because of its cost for the large scale production and its unstable nature in formulation. Our recent study concerning the relationship between human skin and its resident bacterial flora revealed that the first abundant (10^3-4 cfu/cm²) aerobic bacteria group, Staphylococcus epiclermidis, possessed the highest SOD activity/cell among frequently detectable skin resident bacterial species including transient bacteria (Propionibacterium acnes, Micrococcus sp., Streptococcus sp., Staphylococcus aureus, Bacillus sp.). The first abundant anaerobic bacteria (10^4-5 cfu/cm²), P. acnes, showed SOD activity only 1/120 of that of S. epidermidis in cell extract but negligible extracellular excretion of it. The SOD production of S. epidermidis was found to be enhanced by the addition of Mn/Zn ions to the culture medium and its excretion as well. Furthermore, it was found that the secreted SOD suppressed the peroxidation of squalene in vitro under UV-A irradiation. These findings suggested that an unique tool to use skin resident bacteria as a skin care cosmetic machinery to produce and deliver bacterial SOD freshly and effectively toward the skin cell surface and lipid layer on the skin surface surrounding the bacterial cells simply by the application of cosmetic lotion containing Mn/Zn ions.

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We have previously demonstrated that oral administration of chitosan-coated liposomes containing insulin (CS-Lip Ins) lead to a long-term reduction of blood glucose level in rats. In this study, we investigated protective effects of chitosan and liposomal formulation on enzymatic degradation of insulin and promoting effect of chitosan in absorption of insulin in the gastrointestinal tract. A homogenized mucus layer including epithelial cells of rat intestine was used to evaluate the stability of insulin in the gastrointestinal tract. In assessing absorption promoting effects of chitosan, pulse-chaser test, in which the polymer solution was preadministered before insulin administration, was carried out. As the total protein concentration in mucous layer homogenate was increased, the degradation rate of insulin in the solution was accelerated. The enzymatic degradation of insulin was protected in the presence of chitosan or liposomal encapsulation of insulin. Free amino groups of chitosan were supposed to be important in protecting enzymatic degradation of insulin, because carboxymethyl chitin, which possesses a similar structure with chitosan but has no free amino groups, showed little protective effects. Absorption promoting effects of chitosan were not observed in simultaneous administration of chitosan solution with an insulin solution. This result suggested the protective effect of chitosan against enzymatic degradation of insulin is not feasible in vivo. However, co-administration of chitosan solution with liposomal insulin lead to more reduction of blood glucose levels. As the absorption promoting effect was decreased in the pulse-chaser test, the effect was considered to be reversible. In comparing the decreased blood glucose level after simultaneous administration of chitosan solution and liposomal insulin with that of CS-Lip Ins, more prolonged effect was observed for the administration of CS-Lip Ins. These results suggested that both mucoadhesive property of CS-Lip Ins and protective effect of liposomal encapsulation for enzymatic degradation of insulin are important factors in insulin absorption with oral administration of CS-Lip Ins.

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Transferrin receptor (TFR) levels in proliferating malignant cells have often been found to be far higher than in the corresponding normal cells. Cisplatin(cis-diamminedichloroplatinum (II) : CDDP) was complexed via an intermediate carboxymethyl dextran (CMD) to transferrin (TF) which recognizes TFR on the cell surface. CMD was first conjugated to TF by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. Conjugates of TF and CMD of differing molar ratios (TF/CMD 1 : 0.4, 1 : 1.4 and 1 : 1.6) were prepared by this method. Spectrophotometric titration of the conjugates with Fe³⁺ showed that the ferric ion binding activity of apo-TF-CMD was reduced as the molar ratio of CMD to TF increased. CDDP was complexed to the TF-CMD resulting in complexes (TF-CMD-CDDP) carrying up to 8 w/w % of the drug which was reversibly released from the carrier conjugate. Diferric-TF-CMD-CDDP consisted of 0.4 mol of CMD per 1 mol of TF and 3.6 w/w % of CDDP retained cytotoxic activity against human leukemia cell lines, HL60 and K562. The LD₅₀ values of the diferric-TF-CMD-CDDP and CMD-CDDP were 21.0 μM and 29.5 μM in HL60, and were 40.0 μM and 62.0 μM in K562, respectively, suggesting that the specific complex showed preferential cytotoxicity for the tumor cells in comparison to the nonspecific CMD-CDDP.

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This study was carried out to determine the possibility of estimating drug concentration profiles in skin via a diffusion model. In an in vitro permeation study using butylparaben as model drug, percent of drug permeated amounted to approximately 57% and 76% for intact and stripped skin respectively, suggesting that the drug itself highly permeated through the stratum carneum. On the other hand, in vivo absorption study revealed that concentration of butylparaben decreased with skin depth where the maximum was achieved at approximately 30 rain and thereafter decreased gradually with time. In order to analyze both in vitro and in vivo percutaneous drug absorptions, Laplace transformed equations were derived based on four different kinds of hypothetical diffusion models. Initially, diffusion and partition parameters for butylparaben was calculated by curve-fitting using nonlinear regression program combined with a fast Laplace transform algorithm (MULTI(FILT)). Using these parameters, further simulation studies of in vivo drug concentration profiles were carried out. When washout process due to blood flow was not taken into consideration, the decline of drug concentration profiles could hardly be stimulated. However, when model considering washout process to have occurred at the specific range in the dermis (50-200 μm from the stratum carneum/dermis boundary), this could fit the actual concentration profiles of butylparaben in skin well. Thus, this study demonstrated that the diffusion model enables us to evaluate the in vivo behavior of drug in skin.

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