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Biomedical subjects

Junzo Nakamura

Publications and source records attributed to Junzo Nakamura.

At least 19 recordsLinked to original sources
PubMed2006

Influence of disease stage on polyethylenimine-mediated plasmid DNA delivery in murine hepatitis.

In order to determine the influence of hepatic disease-stage on polyethylenimine-mediated gene delivery, we investigated branched and linear polyethylenimine (B-PEI, L-PEI)-mediated plasmid DNA delivery with time in murine hepatitis induced by a subcutaneous injection of tetrachloro carbon (CCl(4)). Plasmid DNA (pDNA) encoding firefly luciferase was used as the model reporter gene. We determined luciferase activity in various organs of CCl(4)-treated mice and control mice after an intravenous administration of B-PEI and L-PEI/pDNA complexes. Both B-PEI and L-PEI/pDNA complexes showed significantly lower gene expression in the liver, spleen, and lung at the stage of severe hepatitis (18 h after CCl(4) injection), whereas the complexes induced gene expression in the liver at the liver regeneration stage (48 h after CCl(4) injection). Significant differences in gene expressions between CCl(4)-treated mice and control mice vanished in most organs at the hepatitis subsidence stage (168 h after CCl(4) injection), indicating that the influence of hepatitis induced by CCl(4) was reversible with PEI-mediated gene delivery. Our findings demonstrated that murine hepatitis induced by CCl(4) could influence polyethylenimine-mediated plasmid DNA delivery according to the disease stage. These results indicate the necessity of considering the timing and dose of gene therapy according to the disease stage.

Animals↗
PubMed2006

Prolonged blood concentration of prednisolone after intravenous injection of liposomal palmitoyl prednisolone.

We compared the pharmacokinetic behavior of drugs after an intravenous administration of prednisolone (PLS), palmitoyl prednisolone (Pal-PLS), and liposomal Pal-PLS in rats. Pal-PLS showed higher lipophilicity and higher binding to plasma protein than PLS, and PLS regeneration in rat blood and liver homogenates. After the intravenous administration of Pal-PLS solution in polyethylene glycol (PEG) 400 to rats, Pal-PLS disappeared from the blood in a two-phase mode and PLS was rapidly regenerated. Pal-PLS showed a significantly higher accumulation than PLS in the liver and lung. The administration of Pal-PLS incorporated into egg yolk phosphatidylcholine (EggPC)/cholesterol (Chol) liposomes enhanced Pal-PLS concentrations in the blood, liver, and lung compared to that of Pal-PLS solution in PEG 400, suggesting the rapid removal of liposomes by the mononuclear phagocytic system. Pal-PLS incorporated into PEGylated liposomes constituted with EggPC/Chol/1% L-alpha-distearoylphosphatidylethanolamine (DSPE)-PEG 2000 and EggPC/Chol/10% DSPE-PEG 2000 decreased the initial distribution of Pal-PLS, and successfully maintained the blood concentrations of Pal-PLS and PLS. Thus, we could change the pharmacokinetics of PLS by introducing the palmitoyl function into the molecule and its liposomal formulation including PEGylation. This is the first study to evaluate liposomal PLS constituted with a lipophilic derivative and PEG lipids.

Animals↗
PubMed2006

PEGylated liposomes loading palmitoyl prednisolone for prolonged blood concentration of prednisolone.

We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-alpha-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (K(el)) and clearance (CL). The area under the concentration-time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.

Animals↗
PubMed2006

Partial hepatectomy enhances polyethylenimine-mediated plasmid DNA delivery.

Polyethylenimine (PEI) is widely used for non-viral transfection in vitro and in vivo. Hepatectomy is an interesting and considerable factor modifying PEI-mediated gene expression. We investigated the gene expression in mice over time following partial hepatectomy after an intravenous injection of PEI/plasmid DNA (pDNA) complex. pDNA encoding firefly luciferase was used as the model reporter gene. The hepatectomized liver was rapidly regenerated until 72 h. After 168 h, the liver weight of hepatectomized mice was similar to that of control mice. Slight liver function impairment was only observed at 1-24 h after hepatectomy in alanine aminotransferase and aspartate aminotransferase levels. Luciferase activity in the liver of partial hepatectomized mice at 48 h after partial hepatectomy increased by 70 times compared with that of control mice; however, luciferase activities did not significantly differ between hepatectomized mice and control mice in the spleen, lung, kidney, and heart. Among the lobes, luciferase activity by gram of tissue was not significantly different, indicating that gene expression enhancement by partial hepatectomy occurred equally throughout the liver. In conclusion, our findings demonstrate that liver resection is an influencing factor on PEI-mediated gene delivery in mice. These results indicate the necessity of considering cell division in PEI-mediated pDNA delivery.

Animals↗
PubMed2006

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

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 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.

Animals↗
PubMed2006

Transport of timolol and tilisolol in rabbit corneal epithelium.

The purpose of this study is to characterize the transport of tilisolol and timolol through the corneal epithelium, which is believed to be a tight barrier of ocular drug absorption. Cultured normal rabbit corneal epithelial cells (RCEC) were used to investigate drug transport. Primary RCEC were seeded on a filter membrane of Transwell-COL insert coated with fibronectin and grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with various supplements. Beta-blocker permeability through the RCEC layer was measured to assess the transcellular permeability coefficient (P(transcell)) in the absence or presence of inhibitors. The transcellular permeability of tilisolol was dependent on drug concentration although timolol showed no concentration dependency. Tilisolol flux from the apical to the basal side was larger than in the opposite direction although timolol showed no direction dependency. The transcellular permeability of tilisolol from the apical to the basal side was inhibited by sodium azide, tetraethylammonium, quinidine, taurocholic acid, guanidine and carnitine. Tilisolol had an active mechanism in uptake to the corneal epithelium, probably by the organic cation transporter family, although timolol predominantly permeated via passive diffusion. This RCEC system was useful to characterize the ocular permeation mechanism of drugs.

Adrenergic beta-Antagonists↗
PubMed2005

Regional delivery of model compounds and 5-Fluorouracil to the liver by their application to the liver surface in rats: its implication for clinical use.

PURPOSE: The purpose of this study was to examine drug distribution in the liver after drug application to the rat liver surface. METHODS: Phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextran (MW 4400, FD-4) as model compounds or 5-fluorouracil (5-FU) was applied to the rat liver surface by employing a cylindrical diffusion cell (i.d. 9 mm, 0.64 cm2). Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites: the region under the diffusion cell attachment site (site 1), the applied lobe except for site 1 (site 2), and non-applied lobes (site 3). RESULTS: In the case of i.v. administration, there were no differences in PSP concentrations among the three sites of the rat liver, and the concentrations rapidly decreased. On the other hand, the PSP concentration in site 1 after application to the rat liver surface was considerably higher than in site 2 and site 3. In addition, the area under the curve (AUC) value (AUCsite1), calculated from the PSP concentration profile in site 1, was about 10 times larger than that in site 3. A similar trend of regional delivery advantage by liver surface application was observed in the case of the macromolecule model FD-4, with a marked AUCsite1 of about 5 times larger than the other two sites. Moreover, we clarified that the anticancer drug 5-FU preferentially distributed in site 1 after application to the rat liver surface. CONCLUSION: These results demonstrate the possibility of regional delivery of drugs to the liver by application to the liver surface.

Absorption↗
PubMed2005

Delivery advantage to the unilateral kidney by direct drug application to the kidney surface in rats and pharmacokinetic verification based on a physiological model.

The objective of this study was to evaluate the drug delivery advantage to the unilateral kidney by direct drug application to the rat kidney surface based on a physiological pharmacokinetic model. Under anesthesia, a cylindrical diffusion cell (i.d. 6 mm, area 0.28 cm(2)) was attached to the right kidney surface in rats. Phenolsulfonphthalein (PSP), an organic anion chosen as a model compound, was added into the diffusion cell. The free PSP concentration in the right (applied) kidney after application to the right kidney surface at a dose of 1 mg was significantly higher than that of the left (non-applied) kidney until 60 min after application. Similarly, the urinary excretion rate of free PSP from the applied kidney was much faster than that from the non-applied kidney, with a 2.6 times larger excreted amount in 240 min. These results imply the possibility that a considerable drug delivery advantage to the unilateral kidney could be obtained after direct absorption from the kidney surface. This tendency was also observed at the other application doses of 0.3 and 1.5 mg. On the other hand, fluorescein isothiocyanate dextran (Mw 4400, FD-4) was equally excreted into the urine from each kidney and the renal concentrations in the applied and non-applied kidneys were almost the same, possibly due to the involvement of passive transport for the absorbed FD-4, i.e. glomerular filtration. The computer simulations of free PSP concentrations in the plasma and each kidney based on a physiological model after kidney surface application were consistent with the respective experimental data. Moreover, the delivery advantage of kidney surface application of PSP was verified by its comparison with other routes such as i.v. and i.a. administrations.

Absorption↗
PubMed2005

Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces.

We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4400; FD-10, MW 9500; FD-40, MW 40 500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1% of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient P(app) for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher P(app) values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the P(app) of the small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compound absorption from the peritoneal cavity, judging from absorption clearance, CL(a), calculated using the peritoneal organ surface area.

Animals↗
PubMed2005

Stomach- and site-selective delivery of 5-fluorouracil following its application on the gastric serosal surface in rats.

The present study was undertaken to elucidate the stomach- and site-selective delivery of 5-fluorouracil (5-FU) following its application on the gastric serosal surface in rats. An experimental system utilizing a cylindrical diffusion cell attached to the gastric serosal surface was established. To evaluate the gastric distribution of 5-FU, the stomach was separated into the site under the diffusion cell (site 1) and the site not under the diffusion cell (site 2). Furthermore, the mucosal side at site 1 was separated from the serosal side. After intravenous and oral administration of 5-FU, the 5-FU concentrations at sites 1 and 2 until 240 min were similar. After gastric serosal surface application of 5-FU, however, the concentration of 5-FU at site 1 until 240 min was approximately 10-fold higher than that at site 2, and was sustained. Furthermore, the 5-FU concentration on the mucosal side at site 1 and the serosal side at site 1 were comparable after gastric serosal surface application. The blood concentration of 5-FU was low (<4.4 microg/ml) until 240 min after gastric serosal surface application. The maximum blood concentration of 5-FU after gastric serosal surface application was significantly lower than after intravenous administration. Thus, the stomach- and site-selective delivery system following application on the gastric serosal surface could be applied with anticancer drugs for the treatment of gastric cancer.

Animals↗
PubMed2005

Unilateral kidney-selective gene transfer following the administration of naked plasmid DNA to the kidney surface in mice.

We developed a gene transfer following the administration of naked plasmid DNA (pDNA) to the kidney surface in mice, and found that the luciferase levels produced in the applied kidney were significantly higher than those produced in another kidney. In contrast, stable renal gene expression was not observed in the case of intraperitoneal or intravenous administration of pDNA. The level of gene expression after instillation of pDNA to the kidney surface reached maximum at 12 h and gradually diminished thereafter. The production of luciferase was saturated at 5 microg of pDNA, and was not affected by instillation volume. Furthermore, pDNA uptake from the kidney surface was proved by in situ experiments using a glass-made diffusion cell. We demonstrated a novel unilateral kidney-selective gene transfer following the administration of naked pDNA to the kidney surface in mice.

Animals↗
PubMed2005

Effect of solution composition of plasmid DNA on gene transfection following liver surface administration in mice.

We investigated the effect of plasmid DNA (pDNA) solution composition on gene transfection following liver surface administration in mice. Gene transfection experiments in situ and in vivo were performed using the following pDNA solutions: dextrose solution, NaCl solution, phosphate buffer, phosphate-buffered saline, Tris/HCl buffer with EDTA, Tris/HCl buffer with EDTA and Triton X-100, and water. In in situ experiments, we used a glass cylindrical diffusion cell that limited the contact area between the liver surface and the naked pDNA solution. The gene transfection at the site of diffusion cell attachment increased in hypotonic solution, and decreased in hypertonic solution, compared with isotonic solution. In in vivo experiments, instillation of naked pDNA solution onto the liver surface using a micropipette caused no significant differences in gene transfection in the applied lobe. These results suggest that it is important to select the optimal pDNA solution composition to control the gene transfection.

Administration, Topical↗
PubMed2004

In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits.

To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 microL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 microg (P < 0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 microg. Among these experiments, pDNA (85 microg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (- : +) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.

Animals↗
PubMed2004

Prednisolone retention in integrated liposomes by chemical approach and pharmaceutical approach.

The purpose of this study is to demonstrate a stable retention of prednisolone (PLS) in the unique liposomes integrated by lipophilic derivative approach and PEGylation approach. Palmitoyl prednisolone (Pal-PLS) was newly synthesized and used as a lipophilic derivative. The liposomes were composed of egg phosphatidylcholine (EggPC)/cholesterol (Chol) and L-alpha-distearoylphosphatidylcholine (DSPC)/Chol with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) or -PEG 5000 (DSPE-PEG 5000). The retentions of PLS and Pal-PLS in the various liposomes were examined by ultrafiltration and gel filtration. Although PLS showed high trapping efficiency by all liposomes after ultrafiltration, low incorporation efficiency was observed in gel filtration. It indicates that PLS was released from the liposomes by a dilution with elution medium in gel filtration. Pal-PLS showed high incorporation into all liposomes after both ultrafiltration and gel filtration. The high incorporation of Pal-PLS into EggPC/Chol liposomes, however, was reduced by incubation with rat plasma in gel filtration. The reducing effect of rat plasma on drug incorporation into liposomes was inhibited by using DSPC and DSPE-PEGs. Thus, we systemically examined the drug retention in various liposomes and demonstrated the high retention of PLS in the liposomes integrated by lipophilic derivative approach and pharmaceutical approach using special lipids.

Animals↗
PubMed2004

Absorption characteristics of compounds with different molecular weights after application to the unilateral kidney surface in rats.

The aim of the present study is to clarify the absorption mechanism of a drug from the kidney surface membrane in rats. We studied the absorption characteristics of phenolsulfonphthalein (PSP) and other compounds with different molecular weights after their application to the rat kidney surface in vivo, employing a cylindrical diffusion cell (i.d. 6 mm, area 0.28 cm(2)). The time course of free PSP amounts remaining in the diffusion cell obeyed first-order kinetics at a dose of 1 mg, and its rate constant k(a) was calculated to be 0.0137 min(-1). Absorption ratios of PSP in 4 h were calculated (from the amount recovered from the diffusion cell) to be 91.4, 96.4 and 97.7% at doses of 0.5, 1 and 1.5 mg, respectively. The area under the curve for the plasma concentration profile of free PSP was proportional to the application dose. It is thus suggested that the absorption process of PSP from the rat kidney surface does not approach saturation at a dose of 1.5 mg. Also, no significant difference was seen in the k(a) values within the dose range of 0.5-1.5 mg, which were estimated by curve-fitting the plasma concentration profiles of free PSP in a two-compartment model with first-order absorption. Furthermore, we examined the importance of molecular weight on the absorption from the kidney surface using fluorescein isothiocyanate-dextrans (FDs) with molecular weights of 4400 (FD-4), 11,000 (FD-10), 40,500 (FD-40) or 69,000 (FD-70), including the organic anions bromphenol blue and bromosulfonphthalein. The absorption ratios of FDs from the rat kidney surface in 6 h decreased with an increase in the molecular weight (76.1% for FD-4, 54.4% for FD-10, 11.5% for FD-40 and 3.9% for FD-70). A linear relationship was observed between k(a) and the reciprocal value of z the square root of the molecular weight of these compounds. The limit of absorption from the rat kidney surface was extrapolated to be at a molecular weight of approximately 130,000.

Absorption↗
PubMed2004

Postmortem changes in tissue concentrations of triazolam and diazepam in rats.

The aim of this study was to investigate the postmortem changes of concentrations of triazolam and diazepam in the rat model and to confirm the factor causing the phenomenon. We administered triazolam or diazepam orally to rats and then sacrificed them 1 h after administration. Abdominal tissues including stomach content and small intestine, thigh muscle and heart blood were collected at 0, 12, 24 h after death, and postmortem changes of the two drug concentrations were compared. Drug concentrations in the samples were analyzed by a selected ion monitoring of gas chromatography/mass spectrometry. Gastrointestinal postmortem concentrations of triazolam and diazepam decreased. On the other hand, the drug concentrations in the liver and kidney increased markedly, and those in lung and heart blood increased slightly during postmortem periods by diffusion from the gastrointestinal tract. The patterns of both drug concentration changes were similar. However, the extent of the triazolam increase tended to be larger than that of diazepam. This difference may be accounted for by lower triazolam concentrations before death. The postmortem drug concentrations of thigh muscle did not change when administered with triazolam and diazepam. For both triazolam and diazepam, a significant correlation was observed in the drug concentration between the small intestine and the kidney at 24 h after death, indicating that major cause of the change in the drug concentration in the kidney was diffusion from the small intestine up to 24 h after death. The results in this study indicate that diazepam as well as triazolam diffuses from the gastrointestinal tract into the surrounding tissues after death and suggest that the diffusion is influenced by their pharmaceutical properties before death.

Animals↗
PubMed2004

Ocular pharmacokinetic/ pharmacodynamic modeling for bunazosin after instillation into rabbits.

PURPOSE: To develop a pharmacokinetic/pharmacodynamic (PK/PD) model for an alpha1-blocker (bunazosin) after instillation. The PK/PD model can predict both the drug concentrations in various ocular tissues and the hypotensive effect. METHODS: Bunazosin concentrations were determined with High Performance Liquid Chromatography (HPLC) in tear fluid, the aqueous humor, cornea, and iris-ciliary body after instillation or ocular injection into the anterior chamber in rabbits. After instillation of bunazosin in rabbits, intraocular pressure (IOP) was also determined with a pneumatic tonometer. The PK/PD parameters were estimated by fitting the concentration-time profiles and the hypotensive effect-time profiles to the developed PK/PD models using the MULTI (RUNGE) program. RESULTS: On the basis of the concentration-time profiles of bunazosin, a PK model, including seven compartments, was developed for examining the behavior of bunazosin after instillation. Then, two PK/ PD models for hypotensive effect of bunazosin were developed using an indirect response (model A) and the relationship between IOP and aqueous humor flow (model B). These models well described the concentration-time profiles and hypotensive effect-time profiles of bunazosin after instillation. CONCLUSIONS: This study is the first trial to develop a PK/PD model for an antiglaucoma agent using an indirect response and the relationship between IOP and aqueous humor flow.

Adrenergic alpha-Antagonists↗