Investigation of protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders using an electrophysiological method.

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].

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 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.

Polyoxyethylene hydrogenated castor oil modulates benzalkonium chloride toxicity: comparison of acute corneal barrier dysfunction induced by travoprost Z and travoprost.

PURPOSE: To determine the element that modulates benzalkonium chloride (BAC) toxicity by using a new electrophysiological method to evaluate acute corneal barrier dysfunction induced by travoprost Z with sofZia (Travatan Z(®)), travoprost with 0.015% BAC (Travatan(®)), and its additives. METHODS: Corneal transepithelial electrical resistance (TER) was measured in live white Japanese rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea. We evaluated corneal TER changes after a 60-s exposure to travoprost Z, travoprost, and 0.015% BAC. Similarly, TER changes were evaluated after corneas were exposed for 60 s to the travoprost additives ethylenediaminetetraacetic acid disodium salt, boric acid, mannitol, trometamol, and polyoxyethylene hydrogenated castor oil 40 (HCO-40) with or without BAC. Corneal damage was examined after exposure to BAC with or without travoprost additives using scanning electron microscopy (SEM) and a cytotoxicity assay. RESULTS: Although no decreases of TER were noted after exposure to travoprost Z with sofZia and travoprost with 0.015% BAC, a significant decrease of corneal TER was observed after 0.015% BAC exposure. With the exception of BAC, no corneal TER decreases were observed for any travoprost additives. After corneal exposure to travoprost additives with BAC, HCO-40 was able to prevent the BAC-induced TER decrease. SEM observations and the cytotoxicity assay confirmed that there was a remarkable improvement of BAC-induced corneal epithelial toxicity after addition of HCO-40 to the BAC. CONCLUSIONS: Travoprost Z with sofZia and travoprost with BAC do not induce acute corneal barrier dysfunction. HCO-40 provides protection against BAC-induced corneal toxicity.

Injection site and pharmacokinetics after intravitreal injection of immunoglobulin G.

PURPOSE: The aim of this study was to investigate whether the pharmacokinetics differ after immunoglobulin G (IgG) intravitreal injections are done in the different sites of the vitreous cavity of rabbit eyes. METHODS: To examine the pharmacokinetics in the vitreous and the retina/choroid, fluorescein isothiocyanate-labeled IgG (5 µg/50 µL) was injected from the superior pars plana into the vitreous cavity of rabbit eyes. An original intravitreal injection guide was used to fix the tip of the injection needle, with the tip fixed in either the superior-anterior vitreous (superior group) or in the posterior vitreous (posterior group). At 1 h, 1, 4, and 7 days after injection, the eyes were enucleated and frozen. The vitreous was cut into superior, inferior, and posterior vitreous sections, whereas the retina/choroid was cut into superior, inferior, and posterior retina/choroid sections. The IgG concentrations in the vitreous and the retina/choroid sections were then determined. RESULTS: In the posterior vitreous, the IgG concentration in the posterior group was significantly higher than that in the superior group at 1 h after injection (P < 0.05). However, 1 day after injection, no significant differences were noted between the 2 groups. At 4 days after injection, the drug was diffusely spread in both groups. In the posterior retina/choroid, the IgG concentration was essentially the same regardless of the injection site or the amount of time after injection. The IgG concentration in the superior retina/choroid was significantly higher in the superior group than in the posterior group at 1 h and 1 day after injection (P < 0.05). There were no differences noted between the 2 groups for the IgG concentrations in the 3 sections at 4 and 7 days after injection. CONCLUSIONS: Intravitreally injected IgG remains in the area of the injection, with more than 1 day required for it to spread diffusely within the vitreous. In the posterior retina/choroid, results suggested that the concentration of IgG may be equal regardless of the injection site. In the superior retina/choroid area that was near the site of the injection, the concentration of drug tended to be higher.

Influence of alkyl chain length of benzalkonium chloride on acute corneal epithelial toxicity.

PURPOSE: To evaluate acute corneal epithelial toxicity induced by benzalkonium chloride (BAC) homologs with different alkyl chain lengths using an in vivo electrophysiological method. METHODS: BAC homologs with C12, C14, and C16 alkyl chain lengths were used at concentrations of 0.0025%, 0.005%, and 0.01%, respectively. Cytotoxicity of BAC homologs on the normal rabbit corneal epithelial cells was examined by using a WST-1 assay. Corneal transepithelial electrical resistance (TER) was measured in living Japanese white rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea. TER changes were then evaluated after a 60-second exposure to these BAC homologs. Morphological changes in corneal epithelium after exposure to the BAC homologs were examined using scanning electron microscopy. The antimicrobial activity of BAC homologs against Escherichia coli was also assessed. RESULTS: All BAC homologs caused cytotoxicity and corneal barrier dysfunction in a concentration-dependent manner. However, the degree of corneal toxicity differed among the BAC homologs. Based on cytotoxicity and TER measurement, C14-BAC caused the greatest corneal impairment followed in order of severity by mixed BAC/C16-BAC and C12-BAC. Scanning electron microscopy images indicated an intact corneal epithelium after exposure to 0.005% C12-BAC, whereas 0.005% C14-BAC damaged the epithelium. There were no remarkable differences noted in the antimicrobial activity among the BAC homologs. CONCLUSIONS: Acute corneal epithelial toxicity induced by BAC homologs depends on the alkyl chain length. Thus, the use of C12-BAC instead of commercially available BAC is potentially safer for patients undergoing ophthalmological pharmacotherapy.

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

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 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.

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

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. 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.

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

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 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.

Enhanced anti-inflammation of inhaled dexamethasone palmitate using mannosylated liposomes in an endotoxin-induced lung inflammation model.

Inhalation of bacterial endotoxin induces pulmonary inflammation by activation of nuclear factor kappaB (NFkappaB), production of cytokines and chemokines, and neutrophil activation. Although glucocorticoids are the drugs of choice, administration of free drugs results in adverse effects as a result of a lack of selectivity for the inflammatory effector cells. Because alveolar macrophages play a key role in the inflammatory response in the lung, selective targeting of glucocorticoids to alveolar macrophages offers efficacious pharmacological intervention with minimal side effects. We have demonstrated previously the selective targeting of mannosylated liposomes to alveolar macrophages via mannose receptor-mediated endocytosis after intratracheal administration. In this study, the anti-inflammatory effects of dexamethasone palmitate incorporated in mannosylated liposomes (DPML) at 0.5 mg/kg via intratracheal administration were investigated in lipopolysaccharide-induced lung inflammation in rats. DPML significantly inhibited tumor necrosis factor alpha, interleukin-1beta, and cytokine-induced neutrophil chemoattractant-1 levels, suppressed neutrophil infiltration and myeloperoxidase activity, and inhibited NFkappaB and p38 mitogen-activated protein kinase activation in the lung. These results prove the value of inhaled mannosylated liposomes as powerful targeting systems for the delivery of anti-inflammatory drugs to alveolar macrophages to improve their efficacy against lung inflammation.

Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis.

A simple capillary electrophoretic method was developed for simultaneous determination of mycophenolic acid (MPA) and its metabolites, acyl glucuronide (AcMPAG) and phenol glucuronide (MPAG), in human serum. The method utilized only running buffer in the separation, prior acidification of serum, and extraction with ethyl acetate. Separation was performed by capillary zone electrophoresis using 20 mM sodium acetate-acetic acid (pH 4.9) as running buffer, applied voltage of 15 kV, and UV detection at 217 nm. Each electrophoretic run was completed within 14 min. The optimized method demonstrated good performance concerning specificity, linearity (r>0.998), sensitivity (limit of detection: for MPA, 0.10 microg/ml; for AcMPAG, 0.10 microg/ml; MPAG, 0.42 microg/ml), accuracy (87-108%) and precision (<9.3%). This method was successfully applied to measurements of MPA, AcMPAG, and MPAG in renal transplant patient samples and could be a useful alternative to HPLC-based methods.

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

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 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.

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 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.

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

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 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.

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.

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.

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.