Koyo Nishida

Publications of Koyo Nishida

• 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.

• Case-controlled study on risk factors for the development of constipation in hospitalized patients.

  Authors: Tetsuya Ueki, Keiko Nagai, Nobuharu Ooe, Mihoko N Nakashima, Koyo Nishida, Junzo Nakamura, Mikiro Nakashima

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 03/2011; 131(3):469-76.

  Constipation is a common problem in hospitalized patients; however, the relative risks of its development with various factors have not been clarified. To clarify the risk factors associated withConstipation is a common problem in hospitalized patients; however, the relative risks of its development with various factors have not been clarified. To clarify the risk factors associated with constipation, we performed a case-controlled study of 165 hospitalized patients who were not laxative users on admission. They were divided into case (n=35) and control (n=130) groups according to laxative administration during hospitalization. Comparison of the patient backgrounds in the two groups revealed significant differences in the activities of daily living, length of fasting, rest level on admission, cerebrovascular disease, and administration of hypnotics. Multiple logistic regression analysis using these five factors as autonomous variables showed that administration of hypnotics (odds ratio, 2.79; 95% confidence interval, 1.10-7.06; p=0.031) was significantly related to laxative use. Therefore, the administration of hypnotics may be the principal cause of constipation development in hospitalized patients and they should be used with caution.

• Cross-sectional study on relationship between constipation and medication in consideration of sleep disorder.

  Authors: Tetsuya Ueki, Keiko Nagai, Yasuko Mizukami, Akihiro Takahashi, Nobuharu Ooe, Mihoko N Nakashima, Koyo Nishida, Junzo Nakamura, Mikiro Nakashima

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 01/2011; 131(8):1225-32.

  Constipation can be caused by adverse drug reactions as a result of many drugs and might be induced by sleep disorders; however, the relative risk of its occurrence with individual drugs and theConstipation can be caused by adverse drug reactions as a result of many drugs and might be induced by sleep disorders; however, the relative risk of its occurrence with individual drugs and the influence of sleep conditions have not been clarified. To clarify the relationship between constipation and various drugs in consideration of sleep disorders, we investigated the self-reported bowel habits, use of laxatives, and the Athens Insomnia Scale (AIS, a self-administered psychometric instrument to measure insomnia) in 344 inpatients on admission. They were divided into a constipation group (self-reported bowel habits of "Constipation" or "Occasional constipation" and/or use of laxatives, n=161) and a non-constipation group (both "Normal" and the non-use of laxatives, n=183). A comparison of the backgrounds of the two patient groups revealed significant differences in age, gender, number of used drugs, AIS score, hypothyroidism, chronic obstructive pulmonary disease, use of diuretics, coronary vasodilators, thyroid hormones, non-steroidal anti-inflammatory drugs, proton pump inhibitors, antidepressants, anti-anxiety drugs, and hypnotics. Multiple logistic regression analysis using these fourteen factors as autonomous variables showed that age (odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01-1.04; p=0.007), female gender (OR, 1.96; 95% CI, 1.21-3.18; p=0.006), the AIS score (OR, 1.10; 95% CI, 1.02-1.18; p=0.010), and the use of hypnotics (OR, 2.33; 95% CI, 1.30-4.16; p=0.004) were significantly related to constipation; therefore, as hypnotics appear more likely to cause constipation than other drugs, they should be used with caution.

• 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.

• Sensitive and real-time method for evaluating corneal barrier considering tear flow.

  Authors: Tadahiro Nakamura, Mugen Teshima, Takashi Kitahara, Hitoshi Sasaki, Masafumi Uematsu, Takashi Kitaoka, Mikiro Nakashima, Koyo Nishida, Junzo Nakamura, Shun Higuchi

  Biological & pharmaceutical bulletin. 01/2010; 33(1):107-10.

  We developed a new electrophysiological method mimicking tear flow to evaluate the epithelial tight junction of rabbit cornea quantitatively. We investigated the effect of tear flow on the cornealWe developed a new electrophysiological method mimicking tear flow to evaluate the epithelial tight junction of rabbit cornea quantitatively. We investigated the effect of tear flow on the corneal damage induced by ophthalmic preservatives using this method. An Ussing chamber system with Ag/AgCl electrodes was used in the electrophysiological experiment. The excised rabbit cornea was mounted in the Ussing chamber and the precorneal solution in the chamber was perfused with a peristaltic pump at the rate of human tear flow. Corneal transepithelial electrical resistance (TEER) was monitored as corneal barrier ability. In the electrophysiological method mimicking tear flow, we observed stable TEER, which rapidly decreased with benzalkonium chloride (BAC), an eye drop preservative. Using this system, we first found that 0.004% BAC decreased corneal TEER reversibly. A high concentration of BAC showed strong irreversible damage to the tight junction. The influence of BAC on corneal TEER was not only concentration-dependent but also tear flow rate-dependent. The electrophysiological method mimicking tear flow was useful to evaluate the corneal barrier quantitatively. Using this method, we clarified that the tear flow was important to protect the corneal damage induced by preservatives.

• Development of drug delivery system by utilizing absorption from liver surface and its application.

  Authors: Koyo Nishida

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 09/2009; 129(8):925-32.

  Because it is difficult to achieve local drug activity following administration by the conventional intravenous and oral routes, I sought to develop a new route of administration utilizing drugBecause it is difficult to achieve local drug activity following administration by the conventional intravenous and oral routes, I sought to develop a new route of administration utilizing drug absorption from the liver surface in order to target that organ. Although direct application to the liver surface should yield local drug distribution, drug absorption from the liver surface has not been reported in the literature. Therefore, we analyzed, as a model, the efficiency of absorption of several organic anions and dextrans of various molecular weights following application to the rat liver surface in vivo using a cylindrical diffusion cell. Each compound appeared gradually in the plasma, followed by excretion into the bile and/or urine, indicating the possibility of drug absorption from the liver surface. The absorption process from the liver surface may not involve a specific transport system because dose and transport inhibitors had no detectable effect. In addition, molecular weight was found to be a determinant of absorption through the liver surface. The efficiency of targeting desired region in the liver was enhanced considerably by application to the liver surface, compared to intravenous administration. Moreover, I have obtained several promising results from the application of this new drug delivery system to anticancer drugs and gene therapy. On the other hand, I have also clarified the characteristics of drug absorption from the surfaces of the kidney, stomach, cecum and small intestine, and plan to apply the physiological findings to other fields.

• 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.

• The development of a gene vector electrostatically assembled with a polysaccharide capsule.

  Authors: Tomoaki Kurosaki, Takashi Kitahara, Shigeru Kawakami, Koyo Nishida, Junzo Nakamura, Mugen Teshima, Hiroo Nakagawa, Yukinobu Kodama, Hideto To, Hitoshi Sasaki

  Biomaterials. 06/2009;

  The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. TheThe purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, lambda-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive zeta-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative zeta-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.

• 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.

• Cationic liposomes-mediated plasmid DNA delivery in murine hepatitis induced by carbon tetrachloride.

  Authors: Takashi Yoshioka, Shohei Yoshida, Tomoaki Kurosaki, Mugen Teshima, Koyo Nishida, Junzo Nakamura, Mikiro Nakashima, Hideto To, Takashi Kitahara, Hitoshi Sasaki

  Journal of liposome research. 03/2009;

  In order to elucidate the influence of hepatic disease stage on cationic liposomes-mediated gene delivery, we investigated the cationic liposomes-mediated plasmid DNA delivery with time in murineIn order to elucidate the influence of hepatic disease stage on cationic liposomes-mediated gene delivery, we investigated the cationic liposomes-mediated plasmid DNA delivery with time in murine hepatitis induced by subcutaneous injection of CCl(4). Liver injury after injection of CCl(4) was confirmed by the determination of serum aspartate aminotransferase and alanine aminotransferase activities. Two kinds of liposomes constructed with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammoniumchloride and dioleylphosphatidylethanolamine (DOTMA-DOPE) or DOTMA and cholesterol (DOTMA-CHOL) were used for the gene-delivery vector. We determined luciferase activities in various organs after the intravenous administration of the lipoplexes. The CCl(4)-treated mice administered with DOTMA-DOPE lipoplexes showed the more significant decreases of transgene expression in the liver and spleen at 18 hours after CCl(4) injection. On the other hand, the CCl(4)-treated mice administered with DOTMA-CHOL lipoplexes showed a significant increase in the liver at 48 hours. In conclusion, our findings demonstrate that murine hepatitis induced by CCl(4) can influence cationic liposomes-mediated plasmid DNA delivery. The extent of influences was also affected by lipid contents. These results indicate the necessity of considering the timing and the formulation for gene therapy according to the disease stage.

• Influence of murine hepatitis induced by D-(+)-galactosamine hydrochloride and lipopolysaccharide on gene expression of polyethylenimine/plasmid DNA polyplex.

  Authors: Kei Miyanaga, Takashi Yoshioka, Hiroo Nakagawa, Takashi Kitahara, Hideto To, Nobuhiro Ichikawa, Mikiro Nakashima, Koyo Nishida, Junzo Nakamura, Hitoshi Sasaki

  Biological & pharmaceutical bulletin. 09/2008; 31(8):1585-9.

  We investigated the influence of murine hepatitis induced by D-(+)-galactosamine and lipopolysaccharide (D-GalN/LPS) on polyethylenimine (PEI)-mediated plasmid DNA (pDNA) delivery. pDNA encodingWe investigated the influence of murine hepatitis induced by D-(+)-galactosamine and lipopolysaccharide (D-GalN/LPS) on polyethylenimine (PEI)-mediated plasmid DNA (pDNA) delivery. pDNA encoding firefly luciferase was used as the model reporter gene. PEI was used as the non-viral vector because of its high gene expression and low toxicity. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice indicated the highest peaks at 12 h after D-GalN/LPS injection, then the activities of serum ALT and AST rapidly decreased. We determined luciferase activity in various organs of D-GalN/LPS-treated mice and control mice after an intravenous administration of PEI/pDNA complexes. High transgene expression was observed in the liver, spleen, and lung of both mice. Compared to the control mice, a significant increase of transgene expression was observed in the liver of D-GalN/LPS-treated mice after D-GalN/LPS injection. The transgene expression in the spleen and lung decreased at 6 and 12 h after D-GalN/LPS injection. In conclusion, we found that murine hepatitis induced by D-GalN/LPS injection can influence PEI-mediated pDNA delivery and its influence was different from that induced by CCl(4) injection which was reported previously. These results demonstrated the necessity of considering the timing and dose of gene therapy according to the disease and its stage.

• Ocular pharmacokinetic/pharmacodynamic modeling for multiple anti-glaucoma drugs.

  Authors: Koji Sakanaka, Kouichi Kawazu, Masahide Tomonari, Takashi Kitahara, Mikiro Nakashima, Koyo Nishida, Junzo Nakamura, Hitoshi Sasaki, Shun Higuchi

  Biological & pharmaceutical bulletin. 09/2008; 31(8):1590-5.

  We have constructed a new ocular pharmacokinetic pharmacodynamic (PK/PD) model for anti-glaucoma drugs to describe ocular hypotensive effects on intraocular pressure (IOP) after instillation of aWe have constructed a new ocular pharmacokinetic pharmacodynamic (PK/PD) model for anti-glaucoma drugs to describe ocular hypotensive effects on intraocular pressure (IOP) after instillation of a combination of an alpha(1)-adrenergic antagonist, bunazosin, and a beta-adrenergic antagonist, timolol, into rabbits. This model was constructed by the combination of two ocular PK/PD models for bunazosin and timolol by including aqueous humor dynamics based on both action mechanisms. We also verified the reliability of this model by confirming the drug concentrations in aqueous humor and ocular hypotensive effects after instillation of the drug combination. The aqueous humor concentrations of timolol and bunazosin were determined by an HPLC, and ocular hypotensive effect-time profiles were measured using a telemetry system, which was able to record automatically detailed effects. The combined model could simulate the aqueous humor concentrations of both drugs and the additive IOP-lowering effect after instillation of the combination using the MULTI (RUNGE) program and PK/PD parameters which were obtained from ocular hypotensive effects after instillation of bunazosin alone or timolol alone. The theoretical concentration curves of both drugs in the aqueous humor and the theoretical ocular hypotensive effect curves almost agreed with both the observed concentrations and ocular hypotensive effects after instillation of the drug combination. These results indicate the reliability and usefulness of PK/PD modeling considering aqueous humor dynamics to predict IOP in multidrug therapy. This is the first study to develop a PK/PD model for multidrug therapy for the eye.

• 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.