Mikiro Nakashima

Publications of Mikiro Nakashima

• Investigation of Protective Effects of Sodium Hyaluronate Eyedrop Against Corneal Epithelial Disorders Using an Electrophysiological Method.

  Authors: Ryosuke Araki, Mikiro Nakashima, Mugen Teshima, Yuichi Owaki, Mihoko N Nakashima, Masafumi Uematsu, Takashi Kitaoka, Tadahiro Nakamura, Takashi Kitahara, Hitoshi Sasaki

  Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics. 02/2012;

  Abstract Purpose: The purpose of this study was to investigate the protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders caused by antiglaucomatous eyedrops using anAbstract Purpose: The purpose of this study was to investigate the protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders caused by antiglaucomatous eyedrops using an electrophysiological method. Methods: Three kinds of antiglaucomatous eyedrops, including benzalkonium chloride (BAC) as an ophthalmic preservative, a BAC-free antiglaucomatous eyedrop, and a sodium hyaluronate eyedrop, were used in this study. Eyedrops were applied to excised rabbit corneas, and the electrophysiological property of the cornea was monitored using an Ussing chamber with a turnover system that mimics human tear turnover. With this system, changes in transepithelial electrical resistance (TER) in the corneal surface were recorded. Results: The corneal TER after applying antiglaucomatous eyedrops tended to decrease concomitantly with increasing the concentration of the BAC included as a preservative. On the other hand, there was no significant change in the corneal TER for the initial 60 min after applying sodium hyaluronate eyedrop compared with those of the control. Moreover, the pretreatment with a sodium hyaluronate eyedrop reduced the extent of decrease in the corneal TER observed after application of antiglaucomatous eyedrops alone. Conclusion: Those results indicate that a sodium hyaluronate eyedrop has the potential to protect the corneal surface from antiglaucomatous eyedrops, including BAC as an ophthalmic preservative.

• [Development of an advanced education program for community medicine by Nagasaki pharmacy and nursing science union consortium].

  Authors: Mugen Teshima, Mikiro Nakashima, Susumi Hatakeyama

  Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 01/2012; 132(1):11-5.

  The Nagasaki University School of Pharmaceutical Sciences has conducted a project concerning "development of an advanced education program for community medicine" for its students in collaborationThe Nagasaki University School of Pharmaceutical Sciences has conducted a project concerning "development of an advanced education program for community medicine" for its students in collaboration with the University's School of Nursing Sciences, the University of Nagasaki School of Nursing Sciences, and the Nagasaki International University School of Pharmaceutical Sciences. The project was named "formation of a strategic base for the integrated education of pharmacy and nursing science specially focused on home-healthcare and welfare", that has been adopted at "Strategic University Cooperative Support Program for Improving Graduate" by the Ministry of Education, Culture, Sports, Science and Technology, Japan from the 2009 academic year to the 2011 academic year. Our project is a novel education program about team medical care in collaboration with pharmacist and nurse. In order to perform this program smoothly, we established "Nagasaki pharmacy and nursing science union consortium (Nagasaki University, The University of Nagasaki, Nagasaki International University, Nagasaki Pharmaceutical Association, Nagasaki Society of Hospital Pharmacists, Nagasaki Nursing Association, Nagasaki Medical Association, Nagasaki Prefectural Government)". In this symposium, we introduce contents about university education program and life learning program of the project.

• Population pharmacokinetic investigation for optimization of amiodarone therapy in Japanese patients.

  Authors: Ryosuke Araki, Eiji Yukawa, Mihoko N Nakashima, Hiromitsu Fukuchi, Hitoshi Sasaki, Katsusuke Yano, Mikiro Nakashima

  Therapeutic drug monitoring. 12/2011; 33(6):750-6.

  Optimal use of amiodarone (AMD) requires information regarding the drug's pharmacokinetics and the influence of various factors on the drug's disposition. This study was conducted to establish theOptimal use of amiodarone (AMD) requires information regarding the drug's pharmacokinetics and the influence of various factors on the drug's disposition. This study was conducted to establish the role of patient characteristic in estimating doses of AMD using nonlinear mixed effects modeling in Japanese patients treated with oral therapy. Serum concentrations of AMD were determined by high-performance liquid chromatography. The 151 serum trough concentrations from 23 patients receiving repetitive oral AMD were collected. Analysis of the pharmacokinetics of AMD was accomplished using a 1-compartment open pharmacokinetic model. The effect of a variety of developmental and demographic factors on AMD disposition was investigated. Estimates generated by nonlinear mixed effects modeling indicated that the clearance of AMD was influenced by the demographic variables: total body weight (TBW), daily dosage of AMD (DD), body mass index (BMI), gender (GEN), duration of AMD dosing (DUR), and patient clearance factor (Conc(θ); Conc = serum trough concentration of AMD). The final pharmacokinetic parameters were CL/F (L/h) = 0.072·TBW·Conc(-1.01)·1.95(DD≥200)·0.931(BMI≥25)·1.37(GEN)·DUR(-0.016), and Vd/F (L) = 78.4·TBW, where CL is total body clearance and Vd is volume of distribution. As all doses were given orally, it was impossible to assess the bioavailability (F). DD ≧200 is an indicator variable that has a value of 1 if the patient is receiving more than 200 mg daily dosage of AMD, and 0 otherwise. BMI ≧25 is an indicator variable that has a value of 1 if the BMI is 25 kg/m(2) and over, and 0 otherwise. GEN is an indicator variable that has a value of 1 if the patient is woman, and 0 otherwise. The authors developed new population pharmacokinetic parameters. Clinical application of the findings in the present study to patient care may permit selection of an appropriate initial maintenance dose, thus enabling the clinician to achieve a desired therapeutic effect.

• 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.

• Donepezil significantly improves abilities in daily lives of female Down syndrome patients with severe cognitive impairment: a 24-week randomized, double-blind, placebo-controlled trial.

  Authors: Tatsuro Kondoh, Atsushi Kanno, Hiroshi Itoh, Mikiro Nakashima, Ryoko Honda, Michio Kojima, Mitsuru Noguchi, Hideyuki Nakane, Hidetsugu Nozaki, Hitoshi Sasaki, Toshiro Nagai, Rika Kosaki, Naoko Kakee, Torayuki Okuyama, Masafumi Fukuda, Masayuki Ikeda, Yoshisada Shibata, Hiroyuki Moriuchi

  International journal of psychiatry in medicine. 01/2011; 41(1):71-89.

  Down syndrome (DS) patients share certain neuropathological features with Alzheimer disease patients. A randomized, double-blind, placebo-controlled study was performed to investigate the efficacyDown syndrome (DS) patients share certain neuropathological features with Alzheimer disease patients. A randomized, double-blind, placebo-controlled study was performed to investigate the efficacy and safety of donepezil, an Alzheimer disease drug, for DS patients. Twenty-one DS patients with severe cognitive impairment were assigned to take donepezil (3 mg daily) or a placebo for 24 weeks, and evaluated for activities in daily lives by concisely modified International Classification of Functioning, Disability and Health (ICF) scaling system. ICF scores significantly increased without any adverse effects in the donepezil group in comparison to those in the placebo control. Among the individual functions tested, there was a dramatic improvement in the global mental functions and in specific mental functions. Donepezil may effectively and safely improve overall functioning of DS patients with severe cognitive impairment.

• 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.

• 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.

• 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.

• 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.

• Ocular pharmacokinetic/pharmacodynamic modeling for timolol in rabbits using a telemetry system.

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

  Biological & pharmaceutical bulletin. 06/2008; 31(5):970-5.

  We have established an ocular pharmacokinetic/pharmacodynamic (PK/PD) model for a beta-adrenergic antagonist, timolol, after instillation into rabbits. Timolol concentrations were determined by HPLCWe have established an ocular pharmacokinetic/pharmacodynamic (PK/PD) model for a beta-adrenergic antagonist, timolol, after instillation into rabbits. Timolol concentrations were determined by HPLC in the tear fluid, aqueous humor, cornea, and iris-ciliary body after instillation or ocular injection into the anterior chamber of the eye in rabbits. In addition, intraocular pressure (IOP) measurement was performed after instillation of timolol by a telemetry system, which was able to obtain detailed IOP data automatically. The PK/PD parameters were estimated by fitting the concentration-time profiles and the ocular hypotensive effect-time profiles using MULTI (RUNGE) program. The PK model consisted of six compartments and the PD model included aqueous humor dynamics based on an action mechanism of timolol, which causes lowering of IOP by suppressing aqueous humor production. The PK/PD model described well the concentration-time profiles and the ocular hypotensive effect-time profiles after instillation of timolol. This study is the first trial to develop an ocular PK/PD model for timolol after instillation. This model can predict both the drug concentrations in various ocular tissues and the ocular hypotensive effect after instillation of timolol.

• 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.

• Breakdown evaluation of corneal epithelial barrier caused by antiallergic eyedrops using an electrophysiologic method.

  Authors: Mikiro Nakashima, Tadahiro Nakamura, Mugen Teshima, Hideto To, Masafumi Uematsu, Takashi Kitaoka, Kotaro Taniyama, Koyo Nishida, Junzo Nakamura, Hitoshi Sasaki

  Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics. 03/2008; 24(1):43-51.

  AIM: The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those inAIM: The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. METHODS: Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. RESULTS: The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. CONCLUSIONS: Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.

• Exploitation of de novo helper-lipids for effective gene delivery.

  Authors: Tomoaki Kurosaki, Takashi Kitahara, Mugen Teshima, Koyo Nishida, Junzo Nakamura, Mikiro Nakashima, Hideto To, Hiromitsu Hukuchi, Tomoyuki Hamamoto, Hitoshi Sasaki

  Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Société canadienne des sciences pharmaceutiques. 02/2008; 11(4):56-67.

  Purpose: In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfectionPurpose: In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfection efficiency. We investigated the effect of penetration enhancers as alternative helper-lipids to DOPE. Methods: Transdermal penetraion enhancers such as N-lauroylsarcosine (LS), (R)-(+)-limonene (LM), vitamin E (VE), and phosphatidyl choline from eggs (EggPC) were used in this experiments as helper-lipids with N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethlylammonium chloride (DOTMA) and cholesterol (CHOL). We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of plasmid DNA/cationic liposomes complexes. Results: In transfection experiments in vitro, the cationic lipoplexes containing LS had highest transfection efficiency among the other lipoplexes independently of FBS. Furthermore, the lipoplexes containing LS had lowest cell toxicity among the other lipoplexes in the presence of FBS. As the results of erythrocytes interaction experiment, DOTMA/LS/CHOL, DOTMA/VE/CHOL, and DOTMA/EggPC/CHOL lipoplexes showed extremely lower hematotoxicity. On the basis of these results, the in vivo transfection efficiencies of the lipoplexes were examined. The lipoplexes containing LS had the highest transfection activity among the other lipoplexes. Conclusion: In conclusion, several transdermal penetration enhancers are available for alternative helper-lipids to DOPE in cationic liposomal vectors. Among them, DOTMA/LS/CHOL lipoplexes showed superior characteristics in in vitro transfection efficiency, cell toxicity, hematotoxicity, and in vivo transfection efficiency.

• 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.

• Electrophysiological characterization of tight junctional pathway of rabbit cornea treated with ophthalmic ingredients.

  Authors: Tadahiro Nakamura, Mikiko Yamada, Mugen Teshima, Mikiro Nakashima, Hideto To, Nobuhiro Ichikawa, Hitoshi Sasaki

  Biological & pharmaceutical bulletin. 01/2008; 30(12):2360-4.

  The purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctionalThe purpose of this study was to investigate the continuous and real-time influence of ophthalmic ingredients on rabbit cornea by monitoring electrophysiological characteristics. The tight junctional permeabilities of FITC-dextran 4,400 (FD-4) was also determined through the cornea in the presence of ophthalmic ingredients. Intact cornea showed approximately one k-ohmxcm(2) of transepithelial electrical resistance (TEER) and extremely low permeability of FD-4. The ophthalmic ingredients used in the present study were benzalkonium chloride (BK; 0.002%, 0.01%, 0.05%), ethylenediaminetetraacetic acid (EDTA; 0.5%), capric acid (C10; 0.25%), saponin (SP; 0.1%), taurocholic acid (TA; 1.0%) and sodium dodecyl sulfate (SDS; 0.01%). They were previously reported to be effective on corneal penetrations of various drugs at those concentrations without severe toxicity. These ingredients decreased TEER and increased corneal permeability of FD-4. BK reduced TEER in a concentration-dependent manner. There was a significant correlation (gamma=0.860) between the permeability coefficient (Papp) of FD-4 and conductance (Gm), which is the reciprocal value of TEER. It was also indicated that Papp and Gm have a relationship with the corneal cytotoxicity of the ingredients. In conclusion, an electrophysiological method using isolated cornea was very useful to determine the continuous and real-time influence of ophthalmic ingredients on the cornea. In this method, electrophysiological conductance must be able to predict corneal tight junction permeability and the corneal cytotoxicity of ingredients.