Shintaro Fumoto

Publications of Shintaro Fumoto

• Evaluation of changes in hepatic disposition of phenolsulfonphthalein, indocyanine green and fluorescein isothiocyanate-dextran at low temperatures using a rat liver perfusion system.

  Authors: Hirotaka Miyamoto, Hideaki Miyake, Naoki Yoshikawa, Haruna Hirata, Yuichi Ohwaki, Shintaro Fumoto, Hitoshi Sasaki, Junzo Nakamura, Koyo Nishida

  The Journal of pharmacy and pharmacology. 06/2012; 64(6):848-54.

  Objectives  The aim of this study was to determine the factor changing the hepatic disposition of a drug during hypothermia using a rat liver perfusion system. Methods  The livers of male Wistar ratsObjectives  The aim of this study was to determine the factor changing the hepatic disposition of a drug during hypothermia using a rat liver perfusion system. Methods  The livers of male Wistar rats were perfused at 37, 32 or 28°C in the single-pass mode. Venous outflow dilution patterns and biliary excretion rate patterns of phenolsulfonphthalein (PSP), indocyanine green (ICG) and fluorescein isothiocyanate (FITC)-dextran (FD-4, MW 4400) after the injection of a bolus into the perfused rat liver were analysed based on statistical moment theory. Key findings  The first-pass extraction ratio (E(h) ) of PSP was significantly decreased at 32 and 28°C compared with 37°C. The biliary recovery of PSP and its conjugate was decreased and the biliary excretion was kept at a high concentration and was prolonged by low perfusion temperatures. ICG was almost extracted by a single-pass through the liver even at 32 and 28°C. The biliary recovery of ICG was significantly decreased at low temperature. Although the distribution volume of FD-4 as a vascular reference was not changed by perfusion temperature, the E(h) of FD-4 was decreased at 28°C although not markedly. Conclusion  The change in hepatic disposition of a drug at low perfusion temperatures differed according to disposition processes under hypothermia.

• Multiple components in serum contribute to hepatic transgene expression by lipoplex in mice.

  Authors: Naoki Yoshikawa, Keiko Sakamoto, Sachiyo Mizuno, Junichiro Sakaguchi, Hirotaka Miyamoto, Toyoharu Mine, Hitoshi Sasaki, Shintaro Fumoto, Koyo Nishida

  The journal of gene medicine. 11/2011; 13(11):632-43.

  Interaction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expressionInteraction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expression was enhanced by interaction with serum in mice. In the present study, we analyzed the mechanism of enhanced hepatic transgene expression of lipoplex by interaction with serum components. Lipoplexes were incubated with several serum components for 5  min at 37  ° C before administration. Transfection efficiency of lipoplexes was measured 6  h after intraportal injection of lipoplex in mice. Depletion of divalent cation from serum decreased hepatic transgene expression. The addition of calcium ion to divalent cation-depleted serum restored transgene expression. Heat-inactivated serum and bovine serum albumin diminished the enhancing effect of serum on hepatic transgene expression. On the other hand, removal of anionic proteins from serum using an anion-exchanging column was critical for the enhancing effect of serum on transgene expression. Among the serum components tested, fibronectin and complement component C3 enhanced hepatic transgene expression. Hepatic transgene expression by lipoplex was enhanced by interaction with multiple components in serum. Interaction of lipoplex with serum could be an important factor for successful in vivo gene transfer. Hence, the information obtained in the present study is valuable for the future development of effective gene carriers.

• Development of effective cancer vaccine using targeting system of antigen protein to APCs.

  Authors: Tomoaki Kurosaki, Takashi Kitahara, Tadahiro Nakamura, Koyo Nishida, Shintaro Fumoto, Yukinobu Kodama, Hiroo Nakagawa, Norihide Higuchi, Hitoshi Sasaki

  Pharmaceutical research. 09/2011; 29(2):483-9.

  To develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy. The novel delivery system was constructedTo develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy. The novel delivery system was constructed with antigen protein, benzalkonium chloride (BK), and γ-polyglutamic acid (γ-PGA), using ovalbumin (OVA) as a model antigen protein and evaluating its immune induction effects and utilities for cancer vaccine. BK and γ-PGA enabled encapsulation of OVA and formed stable anionic particles at nanoscale, OVA/BK/γ-PGA complex. Complex was taken up by dendritic cell line DC2.4 cells efficiently. We subcutaneously administered the complex to mice and examined induction of IgGs. The complex induced not only Th2-type immunoglobulins but also Th1-type immunoglobulins. OVA/BK/γ-PGA complex inhibited tumor growth of E.G7 cells expressing OVA regularly; administered OVA/BK/γ-PGA complex completely rejected tumor cells. The novel vaccine could be platform technology for a cancer vaccine.

• Relationship between lipophilicity and absorption from the liver surface of paraben derivatives and antipyrine in rats.

  Authors: Koyo Nishida, Masanori Kobayashi, Hirotaka Miyamoto, Naoki Yoshikawa, Shintaro Fumoto, Hitoshi Sasaki, Junzo Nakamura

  The Journal of pharmacy and pharmacology. 05/2011; 63(5):736-40.

  The importance of drug lipophilicity on absorption from the liver surface was examined in rats using paraben derivatives, antipyrine, Sudan III, and Sudan blue. The log partition coefficient (PC) ofThe importance of drug lipophilicity on absorption from the liver surface was examined in rats using paraben derivatives, antipyrine, Sudan III, and Sudan blue. The log partition coefficient (PC) of n-octanol/water ranged from -1.39 to 4.62. The compounds were applied to the rat liver surface using a cylindrical diffusion cell (i.d. 9 mm). The rate of absorption at 15 min was calculated to be 13.9% for paraben, much lower than that for its derivatives methylparaben, propylparaben and butylparaben (∼ 80%). The obtained first-order absorption rate constant (k(a) ) of paraben, methylparaben, propylparaben and antipyrine increased according to lipophilicity. Further lipophilicity resulted in a fall in k(a) , implying the importance of affinity for lipids and water in absorption from the liver surface. Thus, a compound with a log PC of around 2.5 is considered to have maximum absorbability from the rat liver surface. A good relationship (r(2) = 0.97) was recognized between the log k(a) and log reciprocal value with the square root of molecular weight of the compounds with a log PC below 2.5. The rate of absorption of a drug from the liver surface could be estimated from physicochemical properties such as lipophilicity and molecular weight.

• Rubbing gastric serosal surface enhances naked plasmid DNA transfer in rats and mice.

  Authors: Toyoharu Mine, Hiroki Ishii, Sayuri Nakajima, Naoki Yoshikawa, Hirotaka Miyamoto, Mikiro Nakashima, Junzo Nakamura, Shintaro Fumoto, Koyo Nishida

  Biological & pharmaceutical bulletin. 01/2011; 34(9):1514-7.

  We have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in miceWe have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in mice resulted in effective but transient transgene expression. Here, we developed a simple method to improve not only the transfection efficiency but also the duration of transgene expression. Rubbing the gastric serosal surface using a medical spoon immediately after instillation of naked plasmid DNA onto the gastric serosal surface resulted in 59-fold higher transgene expression 24 h after administration in rats. Without rubbing, transgene expression decreased under the detection limit 7 d after administration. On the other hand, rubbing the gastric serosal surface with a medical spoon after instillation of plasmid DNA prolonged transgene expression for one month. Mechanistic study in mice revealed that improved transfection should not be due to stimulation of cell function such as macropinocytosis by rubbing because rubbing before instillation of plasmid DNA did not improve transfection. Plasmid DNA should enter effectively into cells during rubbing. These findings are valuable to develop an effective method of in vivo gene transfer into peritoneal organs.

• Safety of liver surface instillation of plasmid DNA in normal and carbon tetrachloride-induced hepatitis mice.

  Authors: Shintaro Fumoto, Hiroyuki Furukawa, Junzo Nakamura, Koyo Nishida

  Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Société canadienne des sciences pharmaceutiques. 01/2011; 14(2):274-82.

  We previously demonstrated liver- and lobe-selective gene transfer following instillation of plasmid DNA (pDNA) onto the liver surface in mice. Safety concerns must be resolved prior to futureWe previously demonstrated liver- and lobe-selective gene transfer following instillation of plasmid DNA (pDNA) onto the liver surface in mice. Safety concerns must be resolved prior to future clinical use. Thus, we investigated safety of liver surface instillation of pDNA in normal and hepatitis mice. pDNA was instilled onto the liver surface in normal and carbon tetrachloride-induced hepatitis mice. Gene expression (luciferase activity) and serum transaminase activities were measured at appropriate time points. Transfection efficiency and target site selectivity were almost the same between the instillation of pDNA using a micropipette and a catheter. Serum transaminase activities 6 h after instillation of pDNA or a vehicle treatment using a micropipette were significantly higher than the no treatment mice, whereas instillation using a catheter did not raise serum transaminase activities throughout the tested time points, suggesting the safety of instillation using a catheter. This safety was also confirmed in hepatitis mice. A dose escalation study verified that liver surface instillation using a catheter did not raise serum transaminase at high doses with saturation of gene expression not only in normal mice, but also in hepatitis mice, supporting the safety of this method. We demonstrated that liver surface instillation of pDNA using a catheter did not raise serum transaminase activities. Information in this study will be useful for future clinical use of liver surface instillation of pDNA using a catheter.

• Chondroitin sulfate capsule system for efficient and secure gene delivery.

  Authors: Tomoaki Kurosaki, Takashi Kitahara, Shintaro Fumoto, Koyo Nishida, Kayo Yamamoto, Hiroo Nakagawa, Yukinobu Kodama, Norihide Higuchi, Tadahiro Nakamura, Hitoshi Sasaki

  Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Société canadienne des sciences pharmaceutiques. 01/2010; 13(3):351-61.

  In this study, we developed various ternary complexes of encapsulated polyplexes and lipoplexes using chondroitin sulfate (CS) and investigated their universal usefulness for gene delivery. ToIn this study, we developed various ternary complexes of encapsulated polyplexes and lipoplexes using chondroitin sulfate (CS) and investigated their universal usefulness for gene delivery. To prepare the cationic complexes, pDNA was mixed with some cationic vectors such as poly-L-arginine, poly-L-lysine, N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol liposomes, and DOTMA- dioleylphosphatidylethanolamine (DOPE) liposomes. CS was added to the cationic complexes for constructions of ternary complexes. We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of the ternary complexes. The cationic polymers and cationic liposomes bound to pDNA and formed stable cationic polyplexes and lipoplexes, respectively. Those cationic complexes showed high transgene efficiency in B16-F10 cells; however, they also had high cytotoxicity and strong agglutination with erythrocytes. CS could encapsulate the polyplexes and lipoplexes and form stable anionic particles without disrupting their structures. The ternary complexes encapsulated by CS showed high transgene efficiency in B16-F10 cells with low cytotoxicity and agglutination. As the result of animal experiments, the polyplexes had little transgene efficiency after intravenous administration in mice, whereas polyplexes encapsulated by CS showed specifically high transgene efficiency in the spleen. The capsulation of CS, however, reduced the high transgene efficiency of the lipoplexes. These results indicate that CS can contribute to polyplex-mediated gene delivery systems for effective and safe gene therapy.

• Organ-, region- and cell-selective gene transfer using non-viral vectors.

  Authors: Shintaro Fumoto

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 10/2009; 129(9):1055-61.

  Safety in gene therapy is an important issue since both viral and non-viral vectors have toxic side effects. Not only vectors themselves, but also distributions of produced proteins affect safety inSafety in gene therapy is an important issue since both viral and non-viral vectors have toxic side effects. Not only vectors themselves, but also distributions of produced proteins affect safety in gene therapy; thus, development of target-selective gene transfer methods is rational. We have developed organ-, region- and cell-selective gene transfer methods using non-viral vectors. To deliver foreign gene to liver parenchymal cells (hepatocytes), galactosylation of cationic liposome/plasmid DNA complex is useful strategy. Based on analyses for intrahepatic disposition characteristics and interaction with blood components, we formulated novel galactosylated lipoplex with regulated salt concentration to reduce particle size of lipoplex and to stabilize lipoplex simultaneously; as a consequence, we succeeded in improvement of hepatocyte-selective gene transfer after intraportal injection of the lipoplex in mice. On the other hand, administration routes are important for target-selective gene transfer. We discovered that simple instillation of naked plasmid DNA onto organ surface (the liver, kidney, spleen, stomach and lung) in mice and rats could result in effective and region-selective transgene expression. Neither physical force nor carriers are necessary for gene transfer onto organ surface mesothelial cells. To rationally improve transfection efficiency, mechanism of gene transfer should be elucidated. We clarified that Rac-mediated macropinocytosis was required for naked plasmid DNA transfer in gastric mesothelial cells.

• Liver- and Lobe-Specific Gene Transfer Following the Continuous Microinstillation of Plasmid DNA onto the Liver Surface in Mice: Effect of Instillation Speed.

  Authors: Shintaro Fumoto, Mayumi Tsuchimochi, Junya Nishi, Hiroki Ishii, Yukinobu Kodama, Mikiro Nakashima, Hitoshi Sasaki, Junzo Nakamura, Koyo Nishida

  Biological & pharmaceutical bulletin. 08/2009; 32(7):1298-302.

  Development of technology to deliver foreign gene(s) to a specific organ/tissue is one of the major challenges in gene therapy. Here, we show liver- and lobe-specific gene transfer following theDevelopment of technology to deliver foreign gene(s) to a specific organ/tissue is one of the major challenges in gene therapy. Here, we show liver- and lobe-specific gene transfer following the continuous microinstillation of plasmid DNA (pDNA) onto the liver surface in mice. Naked pDNA was continuously instilled onto the right medial liver lobe using syringe pump in male ddY mice. Our previous studies showed liver- and lobe-selective gene expression after instillation of 30 mul of pDNA solution onto the liver surface, but gene expression was also found in the other liver lobe, kidney and spleen. To improve target site selectivity of gene expression, the instillation volume was decreased; however, non-specific gene expression in the other liver lobe and diaphragm was still detected. To prevent immediate diffusion of the pDNA solution, we performed continuous microinstillation of pDNA using a syringe pump; as a result, target site selectivity was greatly improved. As for instillation speed, 5 min infusion was enough to prevent diffusion of pDNA solution. Furthermore, transfection efficiency in the target site was maintained when instillation speed was slowed. Wiping off residual pDNA solution from the applied liver lobe resulted in a further improvement in selectivity, suggesting not only immediate diffusion, but also gradual diffusion, are important factors for successful target site-specific gene transfer. Information in this study will be useful for further development of an effective gene delivery system targeted to a specific organ/tissue by use of other non-viral or viral vectors.

• Rac-Mediated Macropinocytosis Is a Critical Route for Naked Plasmid DNA Transfer in Mice.

  Authors: Shintaro Fumoto, Junya Nishi, Hiroki Ishii, Xuan Wang, Hirotaka Miyamoto, Naoki Yoshikawa, Mikiro Nakashima, Junzo Nakamura, Koyo Nishida

  Molecular pharmaceutics. 07/2009;

  We have recently discovered the potential for in vivo naked plasmid DNA (pDNA) transfer into gastric serosal surface cells in mice. As pDNA are huge molecules, the mechanism of gene transfer withoutWe have recently discovered the potential for in vivo naked plasmid DNA (pDNA) transfer into gastric serosal surface cells in mice. As pDNA are huge molecules, the mechanism of gene transfer without carriers and physical forces is of great biological interest. The endocytic route for naked pDNA transfer into gastric mesothelial cells was not clathrin- or caveolae-mediated endocytosis, but macropinocytosis. Naked pDNA transfer required both actin polymerization and myosin-based contraction. Upstream kinases of Rho family GTPases, Syk, Src family kinases and PI-3K were involved in naked pDNA transfer. Furthermore, the intracellular signaling pathway was not mediated via the Rho pathway, but by the Rac pathway. Downstream molecules of Rac, PAK and WAVE2 co-operated with naked pDNA transfer. Overall, it was demonstrated that the Rac signaling pathway regulated the macropinocytosis of naked pDNA. The information in this study would be helpful to clarify in vivo cell functions and to improve in vivo transfection efficiency.

• Ternary complexes of pDNA, polyethylenimine, and gamma-polyglutamic acid for gene delivery systems.

  Authors: Tomoaki Kurosaki, Takashi Kitahara, Shintaro Fumoto, Koyo Nishida, Junzo Nakamura, Takuro Niidome, Yukinobu Kodama, Hiroo Nakagawa, Hideto To, Hitoshi Sasaki

  Biomaterials. 03/2009;

  We discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexesWe discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, alpha-polyaspartic acid, alpha-polyglutamic acid, and gamma-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive zeta-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/gamma-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/gamma-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of gamma-PGA significantly inhibited the uptake of pDNA/PEI/gamma-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/gamma-PGA complex was taken up by gamma-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/gamma-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.

• Improved stomach selectivity of gene expression following microinstillation of plasmid DNA onto the gastric serosal surface in mice.

  Authors: Junya Nishi, Shintaro Fumoto, Hiroki Ishii, Yukinobu Kodama, Mikiro Nakashima, Hitoshi Sasaki, Junzo Nakamura, Koyo Nishida

  European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft für Pharmazeutische Verfahrenstechnik e.V. 07/2008; 69(2):633-9.

  Stomach-selective gene transfer is a promising approach as a therapeutic strategy for refractory gastric diseases. In this study, we improved the stomach selectivity of gene expression followingStomach-selective gene transfer is a promising approach as a therapeutic strategy for refractory gastric diseases. In this study, we improved the stomach selectivity of gene expression following microinstillation of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. pDNA encoding firefly luciferase was used as a reporter gene. It was confirmed that the gene expression level in the stomach 6h after gastric serosal surface microinstillation of pDNA was significantly higher than after intragastric, intraperitoneal and intravenous administration. Regarding selectivity of gene expression, the gene expression level in the stomach after gastric serosal surface microinstillation of 1 microg/1 microL (dose/volume) pDNA was 5.7 times higher than that in the spleen. In our previous study (30 microg/30 microL), the expression level in the stomach was 2.7 times higher than that in the spleen; therefore, the selectivity was 2.1 times higher in this study. When we investigated gene expression at various pDNA solution concentrations, the ratio of the gene expression level in the stomach to that in the spleen was the highest as 1 microg/1 microL of pDNA, which was considered the optimal concentration. Information in this study is useful for further development of target organ-selective gene delivery systems.

• Gene therapy for gastric diseases.

  Authors: Shintaro Fumoto, Junya Nishi, Junzo Nakamura, Koyo Nishida

  Current gene therapy. 07/2008; 8(3):187-200.

  Gene therapy for gastric cancer and gastric ulcer is a rationalized strategy since various genes correlate with these diseases. Since gene expressions in non-target tissues/cells cause side effects,Gene therapy for gastric cancer and gastric ulcer is a rationalized strategy since various genes correlate with these diseases. Since gene expressions in non-target tissues/cells cause side effects, a selective gene delivery system targeted to the stomach and/or cancer must be developed. The route of vector transfer (direct injection, systemic, intraperitoneal, gastric serosal surface and oral administration) is an important issue which can determine efficacy and safety. Strategies for cancer gene therapy can be categorized as suicide gene therapy, growth inhibition and apoptosis induction, immunotherapy, anti-angiogenesis, and others. Combination of the target gene with other genes and/or strategies such as chemotherapy and virotherapy is promising. Candidates for treatment of gastric ulcer are vascular endothelial growth factor, angiopoietin-1, serum response factor, and cationic host defense peptide cathelicidin. In this review, we discuss stomach- and cancer-targeted gene transfer methods and summarize gene therapy trials for gastric cancer and gastric ulcer.

• Absorption and distribution characteristics of 5-fluorouracil (5-FU) after an application to the liver surface in rats in order to reduce systemic side effects.

  Authors: Yukinobu Kodama, Shintaro Fumoto, Junya Nishi, Mikiro Nakashima, Hitoshi Sasaki, Junzo Nakamura, Koyo Nishida

  Biological & pharmaceutical bulletin. 05/2008; 31(5):1049-52.

  The present study was undertaken to elucidate the absorption and distribution characteristics of 5-fluorouracil (5-FU) after its application to the liver surface in rats to examine the possibility ofThe present study was undertaken to elucidate the absorption and distribution characteristics of 5-fluorouracil (5-FU) after its application to the liver surface in rats to examine the possibility of reducing the systemic side effects of this agent. 5-FU was applied to the surface of the liver by employing a cylindrical diffusion cell. Approximately 69% of the dose was absorbed in 360 min. The time course of the change in the amount of 5-FU remaining in the diffusion cell obeyed first-order kinetics. Also, a linear relationship was observed between the apparent permeability coefficient, P app, and the reciprocal of the square root of the molecular weight of several compounds including 5-FU. The estimated P app value of 5-FU was in good agreement with the experimental value. The plasma concentration of 5-FU was low (<1.2 microg/ml) until 360 min after the application. Following i.v. administration, 5-FU was rapidly eliminated from the plasma and could not be detected at 120 min. In the analysis of tissue distribution, the liver was divided into three sites; the region under the diffusion cell attachment site (site 1), the treated lobe excluding site 1 (site 2), and untreated lobes (site 3). After being administered i.v., 5-FU mainly distributed in the kidney, and the concentration in the liver was significantly lower than that in kidney, spleen, or heart. After its application to the liver surface, however, 5-FU preferentially distributed at site 1, and was not detected at the other sites or in other tissues. Thus, these results suggested the possibility of a reduction in the systemic side effect of 5-FU on its application to the liver surface.

• Highly stomach-selective gene transfer following gastric serosal surface instillation of naked plasmid DNA in rats.

  Authors: Junya Nishi, Shintaro Fumoto, Hiroki Ishii, Yukinobu Kodama, Mikiro Nakashima, Hitoshi Sasaki, Junzo Nakamura, Koyo Nishida

  Journal of gastroenterology. 02/2008; 43(12):912-9.

  BACKGROUND: The purpose of this study was to achieve stomach-selective gene transfer in rats by our simple and novel administration method, which is gastric serosal surface instillation of nakedBACKGROUND: The purpose of this study was to achieve stomach-selective gene transfer in rats by our simple and novel administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA). METHODS: Naked pDNA encoding firefly luciferase as a reporter gene was instilled onto the gastric serosal surface in male Wistar rats. As controls, we performed intraperitoneal, intragastric and intravenous administration of naked pDNA. At appropriate time intervals, we measured luciferase activities in the stomach and other tissues. RESULTS: Gene expression in the stomach 6 h after gastric serosal surface instillation of naked pDNA (5 mug) was significantly higher than that after using other administration methods. The present study is the first report on stomach-selective gene transfer following instillation of naked pDNA onto the gastric serosal surface in rats. Also, the gene expression level in the stomach 6 h after gastric serosal surface instillation of naked pDNA was markedly higher than that in other tissues. In a dose-dependent study, the gene expression level was saturated over 5 mug. Gene expression in the stomach was detected 3 h after gastric serosal surface instillation of naked pDNA. The gene expression level peaked 12-24 h after instillation of naked pDNA, then decreased to a level similar to 3 h at 48 h. CONCLUSIONS: Gastric serosal surface in stillation of naked pDNA can be a highly stomach-selective gene transfer method in rats.

• Evaluation of enhanced peritoneum permeability in methylglyoxal-treated rats as a diagnostic method for peritoneal damage.

  Authors: Shintaro Fumoto, Yukiko Nakashima, Koyo Nishida, Yukinobu Kodama, Junya Nishi, Mikiro Nakashima, Hitoshi Sasaki, Noboru Otsuka, Junzo Nakamura

  Pharmaceutical research. 11/2007; 24(10):1891-6.

  PURPOSE: As peritoneal damage in long-term peritoneal dialysis therapy is a major problem correlated to patient prognosis, diagnosis of peritoneal damage is important. To develop a diagnostic methodPURPOSE: As peritoneal damage in long-term peritoneal dialysis therapy is a major problem correlated to patient prognosis, diagnosis of peritoneal damage is important. To develop a diagnostic method for peritoneal damage, we focused on hyperpermeability across the peritoneum in a pathogenic peritoneal damage condition. In this study, disposition characteristics of an intraperitoneally injected marker substance in peritoneal damaged rats were analyzed. MATERIALS AND METHODS: Peritoneal damaged rats were prepared by intraperitoneal injection of a glucose degradation product, methylglyoxal (MGO), for five or ten consecutive days. Phenolsulfonphthalein (PSP), as a marker substance, was intraperitoneally or intravenously injected into MGO-treated rats. Subsequently, the PSP disposition characteristics were pharmacokinetically analyzed. RESULTS: In both cases of 5 and 10 days treatment of MGO, absorption of PSP after intraperitoneal injection was significantly enhanced. Plasma concentration and urinary excretion of PSP in MGO-treated rats were also higher than those in saline-treated rats in the early phase. On the contrary, there was no significant difference in terms of the pharmacokinetic parameters of intravenously injected PSP in saline- or MGO-treated rats. These results indicated that intraperitoneally injected MGO primarily acts on the peritoneal membrane; therefore, the peritoneal permeability of the marker substance was enhanced. CONCLUSION: We demonstrated that pharmacokinetic analysis of peritoneum permeability might be a potent diagnostic method for peritoneal damage in experimental animals and patients receiving peritoneal dialysis.

• Change in pharmacokinetics of model compounds with different elimination processes in rats during hypothermia.

  Authors: Koyo Nishida, Madoka Okazaki, Ryuichi Sakamoto, Natsuko Inaoka, Hideaki Miyake, Shintaro Fumoto, Junzo Nakamura, Mikiro Nakashima, Hitoshi Sasaki, Mikio Kakumoto, Toshiyuki Sakaeda

  Biological & pharmaceutical bulletin. 10/2007; 30(9):1763-7.

  We compared the pharmacokinetics of model compounds with different elimination processes between hypothermic and normothermic rats, to obtain basic information concerning drug therapy duringWe compared the pharmacokinetics of model compounds with different elimination processes between hypothermic and normothermic rats, to obtain basic information concerning drug therapy during hypothermia. Male Wistar rats were anesthetized with sodium pentobarbital and kept at temperatures of 37 degrees C (normothermic group) by heat lamp, and 32 degrees C or 28 degrees C (hypothermic group) by external cooling. We chose phenolsulfonphthalein (PSP), indocyanine green (ICG) and fluorescein isothiocyanate (FITC)-dextran (FD-4, Mw 4400) as model compounds to determine changes in clearance pathways during hypothermia therapy. The plasma concentrations of PSP as biliary, urinary and metabolic elimination type were increased significantly in the hypothermic group (32 degrees C, 28 degrees C) after i.v. administration at a dose of 1 mg, compared to the normothermic group (37 degrees C). Each PSP clearance (bile, urine and metabolites) in the hypothermic group was decreased, suggesting an influence of hypothermia on the active elimination process. The decreasing tendency was marked at a temperature of 28 degrees C. Moreover, the plasma concentrations of ICG as the biliary excretion type after i.v. administration to the hypothermic rats at a dose of 1 mg were higher with more than 50% decrease in the total body clearance compared to normothermic rats. On the other hand, there was almost no difference in the i.v. pharmacokinetics of FD-4 as the urinary excretion type between 37 degrees C and 32 degrees C. However, renal clearance of FD-4 was significantly decreased at a temperature of 28 degrees C. Accordingly, the change in pharmacokinetics of a drug in the hypothermic group could differ with the elimination processes.

• Spleen-selective gene transfer following the administration of naked plasmid DNA onto the spleen surface in mice.

  Authors: Junzo Nakamura, Shintaro Fumoto, Rie Kawanami, Yukinobu Kodama, Junya Nishi, Mikiro Nakashima, Hitoshi Sasaki, Koyo Nishida

  Biological & pharmaceutical bulletin. 06/2007; 30(5):941-5.

  The purpose of present study was to examine spleen-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the spleen surface in mice. Gene expression in the spleen andThe purpose of present study was to examine spleen-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the spleen surface in mice. Gene expression in the spleen and other tissues was evaluated based on firefly luciferase activity. Six hours after spleen surface instillation of naked pDNA, high gene expression in the spleen was observed. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the spleen surface, gene expression in the spleen was significantly higher than those in other tissues. Six hours after instillation of naked pDNA onto the spleen surface, gene expression in the spleen reached the peak value, and thereafter decreased gradually. By utilizing a glass-made diffusion cell that is able to limit the contact dimension between the spleen surface and naked pDNA solution administered, site-specific gene expression in the spleen was found. This novel gene transfer method is expected to be a safe and effective strategy for DNA vaccine against serious infectious diseases and cancers.

• Unilateral lung-selective gene transfer following the administration of naked plasmid DNA onto the pulmonary pleural surface in mice.

  Authors: Junzo Nakamura, Shintaro Fumoto, Kimiyo Ariyoshi, Yukinobu Kodama, Junya Nishi, Mikiro Nakashima, Hitoshi Sasaki, Koyo Nishida

  Biological & pharmaceutical bulletin. 05/2007; 30(4):729-32.

  The purpose of the present study was to examine unilateral lung-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the pulmonary pleural surface in mice. Naked pDNAThe purpose of the present study was to examine unilateral lung-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the pulmonary pleural surface in mice. Naked pDNA was administered intravenously, intraperitoneally, and instilled onto the right pulmonary pleural surface. Four hours later, right pulmonary pleural surface instillation of naked pDNA resulted in high gene expression in the right lung. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the right or left pulmonary pleural surface, gene expressions in the applied lung were significantly higher than those in the other lung and tissues. In addition, gene expressions were detected only in the intrathoracic tissues, not in the intraperitoneal tissues. Four hours after instillation of naked pDNA onto the right pulmonary pleural surface, gene expression in the right lung was the highest, and thereafter gene expression in the right lung decreased gradually. This novel gene transfer method is expected to be a safe and effective treatment against serious lung diseases.

• Stomach-selective gene transfer following the administration of naked plasmid DNA onto the gastric serosal surface in mice.

  Authors: Junzo Nakamura, Shintaro Fumoto, Keiko Shoji, Yukinobu Kodama, Junya Nishi, Mikiro Nakashima, Hitoshi Sasaki, Koyo Nishida

  Biological & pharmaceutical bulletin. 11/2006; 29(10):2082-6.

  The purpose of the present study was to achieve a stomach-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. Gene expression inThe purpose of the present study was to achieve a stomach-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. Gene expression in the stomach and other tissues was evaluated by firefly luciferase activity. Six hours after gastric serosal surface instillation of naked pDNA, high gene expression in the stomach was observed. On the contrary, intravenous and intraperitoneal injection of naked pDNA exhibited no detectable gene expression. Following instillation of naked pDNA onto the gastric serosal surface, gene expression in the stomach was significantly higher than in other tissues. Gene expression in the stomach was highest 12 h after the instillation and thereafter decreased gradually. Utilizing a glass-made diffusion cell that is able to limit the contact dimension between the gastric serosal surface and the naked pDNA solution administered, site-specific gene expression in the stomach was achieved. This novel gene transfer method is expected to be a safe and effective treatment against serious stomach diseases.