Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury.

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca2+-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues. Herein, we investigated whether TG2 promotes fibrin deposition and mediates the adhesion of platelets to ECs in histone-induced acute lung injury (ALI). We evaluated the lung histology and the adhesion of platelets to endothelial cells (ECs) after injecting histones to wild-type (WT) C57BL/6J and TG2 knockout (TG2-/-) mice, and administered a TG2 inhibitor (NC9) to WT mice. Pulmonary haemorrhage was more severe in TG2-/- mice than that in WT mice. The area of fibrin deposition and the proportion of CD41+CD31+ cells were lower in TG2-/- mice than in WT mice. Pre-treatment of NC9 decreased the area of fibrin deposition and the proportion of CD41+CD31+ cells in WT mice. These results suggest that TG2 prevents from pulmonary haemorrhage in ALI by promoting the adhesion of platelets to ECs and the fibrin deposition.

Anti-seizure efficacy of perampanel in two established rodent models of early-life epilepsy.

Early-life seizures can be refractory to conventional antiseizure medications (ASMs) and can also result in chronic epilepsy and long-term behavioral and cognitive deficits. Treatments targeting age-specific mechanisms contributing to epilepsy would be of clinical benefit. One such target is the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subtype of excitatory glutamate receptor, which is upregulated in the developing brain. Perampanel is a non-competitive, selective AMPAR antagonist that is FDA-approved for focal onset seizures (FOS) or primary generalized tonic-clonic seizures (PGTC) in children and adults. However, the efficacy of perampanel treatment in epilepsy patients younger than 4 years has been less documented. We thus tested the efficacy of perampanel in two early-life seizure models: (1) a rat model of hypoxia-induced neonatal seizures and (2) a mouse model of Dravet syndrome with hyperthermia-induced seizures. Pretreatment with perampanel conferred dose-dependent protection against early-life seizures in both experimental models. These findings suggest that AMPAR-mediated hyperexcitability could be involved in the pathophysiology of early-life seizures, which may be amenable to treatment with perampanel.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

Frequency of Immune Checkpoint Inhibitor-Induced Vasculitides: An Observational Study Using Data From the Japanese Adverse Drug Event Report Database.

Information on immune checkpoint inhibitor-induced vasculitides is limited, and predictors for this condition have not been identified. Therefore, we have examined the frequency of immune checkpoint inhibitor-induced vasculitides by analyzing the data recorded in the Japanese Adverse Drug Event Report database. Data from April 2004 to March 2020 were extracted, and vasculitides as an immune-related adverse event was defined according to the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Adverse event signals were recognized as significant when the reporting odds ratio estimates and lower limits of the corresponding 95% confidence intervals exceeded 1. The use of nivolumab showed a significant signal for vasculitides. Furthermore, significant signals of polymyalgia rheumatica were found when the patients were treated with nivolumab, pembrolizumab, and ipilimumab. In addition, the frequencies of nivolumab- and pembrolizumab-induced polymyalgia rheumatica were higher in patients aged ≥70 years and female patients, respectively. Polymyalgia rheumatica was reported in 38 patients treated with nivolumab; 31 (82%) of these were either in recovery or in remission. Further, polymyalgia rheumatica was reported in 17 patients treated with pembrolizumab; 13 (76%) of these were in recovery or remission, while three (18%) were not. Polymyalgia rheumatica was reported in 12 patients treated with ipilimumab; seven (58%) of these were in recovery or remission. Our study highlights that careful monitoring for the symptom of PMR (e.g., bilateral pain in shoulder and pelvic girdles) is required when the patients are aged >70 years and have been treated with nivolumab and when the patients are women and have been treated with pembrolizumab.

Concomitant Proton Pump Inhibitors and Immune Checkpoint Inhibitors Increase Nephritis Frequency.

BACKGROUND/AIM: Concomitant proton pump inhibitor (PPI) and immune checkpoint inhibitor (ICPI) were determined as risk factors of acute kidney injury. To identify the type of PPI associated with ICPI-induced nephritis, we used the Japanese Adverse Drug Event Report database. PATIENTS AND METHODS: ICPIs (nivolumab, pembrolizumab, ipilimumab, atezolizumab, durvalumab, and avelumab) and PPIs (esomeprazole, omeprazole, vonoprazan, rabeprazole, and lansoprazole) were selected as suspected nephritis-inducing drugs. RESULTS: The cases of concomitant use of atezolizumab and rabeprazole, ipilimumab and omeprazole, ipilimumab and lansoprazole, nivolumab and esomeprazole, nivolumab and omeprazole, nivolumab and rabeprazole, nivolumab and lansoprazole, pembrolizumab and esomeprazole, as well as pembrolizumab and lansoprazole had a significantly higher reported odds ratio than monotherapy cases. CONCLUSION: Male patients or patients using ICPIs and PPIs (excluded vonoprazan) concomitantly should be monitored for renal function after chemotherapy.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Enhanced percutaneous absorption of cilostazol nanocrystals using aqueous gel patch systems and clarification of the absorption mechanism.

Cilostazol (CLZ), an anti-platelet agent, is primarily used following the onset of cerebral infarction. However, as CLZ is only marginally soluble in water, a strategy for patients with serious secondary conditions, such as impaired consciousness or aphagia, is required. In the present study, topical formulations containing CLZ nanocrystals (CLZnano) were designed to enhance percutaneous absorption. In addition, the mechanism of penetration of CLZnano through rat skin was investigated. A topical formulation containing CLZ nanoparticles (CLZnano gel patch) was prepared using a combination of recrystallization and ball milling of an aqueous gel. The particle size of CLZnano was 74.5±6.2 nm (mean ± standard deviation). The concentration of permeated CLZnano and penetration mechanism of the nanocrystals were measured in a percutaneous absorption experiment. The amount of penetrated CLZ, the penetration rate (Jc), the penetration coefficient through the skin (Kp) and the skin/preparation partition coefficient (Km) for the CLZnano gel patch were all significantly higher than those of the CLZ powder (CLZmicro) gel patch, the CLZnano ointment and the CLZmicro ointment. In in vitro percutaneous penetration experiments on the CLZnano gel patches, there was a positive correlation between the number of CLZnano. Following the application of the CLZnano gel patch on rat skin, 98% of penetrated CLZ was observed in nanoparticle form; for the CLZmicro gel patch, this figure was 9%. In addition, the CLZ concentrations in the plasma of rats administered the CLZnano gel patches were significantly higher than those of rats administered the CLZnano CP gel and PEG ointments. It was suggested that CLZnano (diameter <100 nm) were transferred through the intracellular spaces in the skin and then into peripheral blood vessels. To the best of our knowledge, this is the first report to elucidate the mechanism of the percutaneous penetration of nanocrystal medicines.

An oral formulation of cilostazol nanoparticles enhances intestinal drug absorption in rats.

Cilostazol (CLZ) is an anti-platelet agent that is generally used after the onset of cerebral infarction. However, CLZ is a poorly water-soluble drug and a strategy for increasing its bioavailability is required. In the present study, novel oral formulations were designed containing CLZ solid nanoparticles to improve bioavailability. The present study investigated the therapeutic effect of the oral formulations containing CLZ nanoparticles on ischemic stroke using a cerebral ischemia/reperfusion-induced injury model (MCAO/reperfusion mice). The oral formulation containing CLZ nanoparticles (CLZ/Rnano tablet) was prepared using a combination of recrystallization and ball milling with the following ingredients: CLZ, docusate sodium, methylcellulose, 2-hydoxypropyl-ß-cyclodextrin, gum arabic, polyvinylpyrrolidone, and mannitol. The particle size after re-dispersion of the CLZ/Rnano tablet was 64±47 nm (mean ± standard deviation). The CLZ areas under the concentration-time curve (AUC) and mean residence time (MRT) in rats that were administered CLZ/Rnano tablets were significantly greater compared with those in rats that were administered CLZ/Rmicro tablets. Results indicated, the AUC after administration of CLZ/Rnano tablets was 3.1-fold higher compared with that after administration of the commercially available CLZ OD tablet. In addition, oral administration with CLZ/Rnano tablets ameliorated neurological deficits caused by ischemic stroke in MCAO/reperfusion mice. It is possible that the oral formulation containing CLZ nanoparticles will be useful for the treatment of patients with ischemic stroke and that these findings will provide significant information that can be used to improve the drug with low bioavailability.

Diagnostic utility of automated SureFISH (Dako Omnis) in the diagnosis of musculoskeletal translocation-related sarcomas.

Fluorescence in situ hybridization (FISH) is an essential tool for genetic diagnosis in daily pathological work. Almost full automation of FISH can be achieved with the recently released automated SureFISH platform (Dako Omnis, Agilent Technologies, Santa Clara, CA, USA). Its utility has been reported in HER2 amplification of breast and gastric carcinoma and ALK-rearranged lung cancer. Here, we examined the utility of automated SureFISH for the identification of rearrangement signals in translocation-related sarcomas (TRSs), including 11 EWSR1-rearranged and 10 synovial sarcoma cases, compared with non-automated conventional FISH using the same specimens. The percentages of EWSR1 or SS18 split signals were higher in automated SureFISH than in conventional FISH in 13 of the 21 cases. On the other hand, 8 of the 21 cases showed the same or lower percentage of split signals in automated SureFISH. Both FISH approaches detected EWSR1 and SS18 split signals in more than 10% of tumor cells in all cases. The strongest advantage of automated SureFISH is its ability to reduce running time without sacrificing quality. Other advantages include improved signal sharpness with oligo probes and reduced ecological toxicity by avoiding formamide use. Automated SureFISH is an excellent tool for the genetic diagnosis of TRSs and contributes to their rapid definitive diagnosis.

A Nanoparticle-Based Ophthalmic Formulation of Dexamethasone Enhances Corneal Permeability of the Drug and Prolongs Its Corneal Residence Time.

We designed ophthalmic formulations containing dexamethasone-loaded solid nanoparticles (DEXnano dispersion), and investigated corneal permeability and toxicity. 0.1% dexamethasone (DEX) powder (DEX microparticles), 0.026% methyl p-hydroxybenzoate (MP), 0.014% propyl p-hydroxybenzoate (PP), and 0.5% methylcellulose were used, and the DEXnano dispersion was prepared by the bead mill method. The mean particle size of DEXnano dispersion was 78 nm. Antimicrobial activity of the DEXnano dispersion were measured by using Escherichia coli, and the corneal epithelium-debrided rat model and HCE-T cells (immortalized human corneal epithelial cell line) were used to estimate the corneal toxicity. The transcorneal penetration of the DEXnano dispersion were evaluated in the corneas of rabbit. The DEXnano dispersion was found to be highly stable until 14 d after its preparation. Although DEX itself did not exhibit antimicrobial activity, the DEXnano dispersion containing parabens (MP and PP) showed high antimicrobial activity, approximately equal to that of the solution containing parabens without DEX. The corneal penetration rate (Jc) and mean residence time (MRT) of DEX from the DEXnano dispersion were approximately 5.1- and 1.3-fold higher, respectively, than those of a dispersion containing DEX microparticles (mean particle size, 11.3 µm). In addition, no significant difference was found in corneal stimulation between the vehicle and DEXnano dispersion. In conclusion, we successfully prepared high quality dispersion containing DEX solid nanoparticles, and the nanoparticle-based ophthalmic formulation of DEX enhanced the corneal permeability and residence time of the drug. It is possible that DEXnano dispersion will show increased effectiveness in treating ocular inflammation.

Co-administration of Magnesium Ion Prevents Indomethacin-Induced Intestinal Ulcerogenic Lesions in Adjuvant-Induced Arthritis Rats.

In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.

Improvement of Dissolution Rate and Stability in a Pirenoxine Ophthalmic Suspension by the Bead Mill Methods.

Foreign matter sensation and blurred vision following instillation of ophthalmic suspension are often observed, and remaining of solid particle on cornea is related these side effects. In addition, low dispersion stability in the ophthalmic suspension affects the therapeutic effect. In this study, we have attempted to enhance the dissolution rate and stability of commercially available pirenoxine ophthalmic suspension (CA-pirenoxine eye drops), anti-cataract eye drops, by changes in particle size. Methylcellulose, zirconia beads (0.1 mm) and Micro Smash were used to mill the pirenoxine (bead mill method), and the distribution of particle size was changed to approximately 60-900 nm (nanodispersions) from 70 nm-3 µm (CA-pirenoxine eye drops). The dissolution rate of pirenoxine increased by the bead mill, and the dissolution rate constant in pirenoxine nanodispersions was 2.1-fold than that in CA-pirenoxine eye drops. Moreover, the dispersion stability in nanodispersions also significant higher in comparison with the CA-pirenoxine eye drops. The dispersion ratio in CA-pirenoxine eye drops and pirenoxine nanodispersions at 2 d after suspension was 48%, 99%, respectively. In conclusion, we showed that the dissolution rate and dispersion stability of CA-pirenoxine eye drops were enhanced by the bead mill method. These findings provide significant information that can be used in the design of ophthalmic suspension.

Ferulic Acid Suppresses Amyloid ß Production in the Human Lens Epithelial Cell Stimulated with Hydrogen Peroxide.

It is well known that oxidative stresses induce the production of amyloid ß (Aß) in the brain, lens, and retina, leading to age-related diseases. In the present study, we investigated the effects of ferulic acid on the Aß levels in H2O2-stimulated human lens epithelial (HLE) SRA 01/04 cells. Three types of Aß peptides (Aß1-40, Aß1-42, and Aß1-43) were measured by ELISA, and the levels of mRNA for the expressed proteins related to Aß production (APP, BACE1, and PS proteins) and degradation (ADAM10, NEP, and ECE1 proteins) were determined by quantitative real-time RT-PCR. H2O2 stimulation augmented gene expression of the proteins related to Aß production, resulting in the production of three types of Aß peptides. Treatment with 0.1 µM ferulic acid attenuated the augmentations of gene expression and production of the proteins related to the secretion of three types of Aß peptides in the H2O2-stimulated HLE cells. These results provided evidence of antioxidative functions of ferulic acid for lens epithelial cells.

Combination Ointment Containing Solid Tranilast Nanoparticles and Dissolved Sericin Is Efficacious for Treating Skin Wound-Healing Deficits and Redness in Diabetic Rats.

We attempted to design a combination ointment containing solid tranilast nanoparticles and dissolved sericin as a wound-healing drug (TS-combination ointment), and evaluated its usefulness as therapy for wound-healing deficits in streptozotocin-induced diabetic rat (STZ rat) using kinetic analyses as an index. Solid tranilast nanoparticles were prepared by bead mill methods with low-substituted methylcellulose; the mean particle size of the tranilast nanoparticles was 70 nm. The ointment was designed to contain the tranilast nanoparticles plus sericin powder and/or Carbopol® 934. Skin wound healing in STZ rats begins significantly later than in normal rats. Although the skin wound healing rate in STZ rats treated with an ointment containing tranilast nanoparticles was lower than in STZ rats treated with vehicle, the ointment was effective in reducing redness. An ointment containing sericin enhanced the skin-healing rate, but the preventive effect on redness was weak. On the other hand, the combination of tranilast and sericin increased both the skin healing rate and reduction in redness. In conclusion, we have adapted kinetic analyses to skin wound healing in rats, and found these analyses to be useful as an index of wound healing ability by a wound-healing drug. In addition, we show that treatment with the TS-combination ointment enhances the skin wound healing rate and reduces redness. These findings provide information significant to the search for new wound-healing therapies and for the design of wound-healing drugs.

A positive feedback loop between nitric oxide and amyloid ß (1-42) accelerates mitochondrial damage in human lens epithelial cells.

We have reported that excessive nitric oxide (NO), like other reactive oxygen species (ROS), causes a decrease in cytochrome c oxidase (CCO) activity and ATP levels (mitochondrial damage) resulting in lens opacity. In addition, previous reports have shown that oxidative stress caused by ROS enhances amyloid ß (Aß) production in mammalian lenses, and that Aß1-42 stimulates inducible nitric oxide synthase (iNOS) promoter activity. Based on these reports, we investigated the relationship between NO and Aß1-42 production in human lens epithelial (HLE) cells. iNOS was induced by the co-incubation of HLE cells with 1000 IU interferon-γ (IFN-γ) and 100ng/ml lipopolysaccharide (LPS) for 48h. This led to enhanced NO release, an increase in the gene expression levels of proteins related to Aß production, and the cellular accumulation of Aß1-42. Moreover, both aminoguanidine (AG, a selective inhibitor of iNOS) and diethyldithiocarbamate (DDC, a nuclear factor-kappa B (NFκB) inhibitor) attenuated these changes in IFN-γ and LPS stimulated HLE cells. Based on our finding that Aß1-42 accumulation is induced by co-incubation of HLE cells with both IFN-γ and LPS, we prepared a HLE cell model with Aß1-42 accumulation (Aß-accumulated-HLE cell model) by pre-stimulating cells with IFN-γ and LPS for 48h. Aß1-42 accumulation caused NO production via iNOS, resulting in an enhancement in the mRNA levels for enzymes necessary for the proteolysis of amyloid precursor protein (APP) to Aß in HLE cells. In addition, excessive NO produced in response to Aß1-42 accumulation led to a decrease in CCO activity and ATP levels. Taken together, we hypothesize that excessive NO production in the lens epithelium enhances Aß1-42 production, and that this enhancement accelerates NO release. The enhancement in NO production in the lens epithelium based on positive feedback (NO-Aß positive feedback loop, a vicious cycle) may promote the onset of cataracts (lens opacification) via the decrease in CCO activity and ATP levels. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

Pharmacokinetic Studies of Gel System Containing Ibuprofen Solid Nanoparticles.

In the therapy of rheumatoid arthritis, ibuprofen (IBU) is widely used; however, it has been limited the clinical use by its systemic side effect, such as gastrointestinal lesions. Therefore, we prepared topical gel ointment used IBU solid nanoparticles (IBUnano-gel formulation). In addition, we demonstrated their anti-inflammatory effect by using arthritis model rat (adjuvant-induced arthritis rat, AA rat). The gel formulations were prepared using additives (Carbopol 934, 2-hydroxypropyl-ß-cyclodextrin and methylcellulose) and bead mill-method. The IBU particle size in the IBUnano-gel formulation was 208 nm. The increase in inflammation of the hind feet of AA rats was attenuated by the treatment with the IBUnano-gel formulation, and preventive effect was higher than that of a gel formulation containing IBUmicroparticles (IBUmicro-gel formulation, mean particle size 85.4 µm); the accumulation and permeability through the skin of IBU from the IBUnano-gel formulation were significantly larger in comparison with the IBUmicro-gel formulation. Further, no gastrointestinal lesions were observed in AA rats following the repetitive administration of the 5% IBUnano-gel formulation (0.30 g) for 42 days (once a day). These results suggest that the dermal application of IBU-nanoparticles provide effective and efficient therapy that spares patients from unwanted side effects.

An Ophthalmic Formulation of Disulfiram Nanoparticles Prolongs Drug Residence Time in Lens.

Disulfiram (DSF) is a dimer of diethyldithiocarbamate (DDC) that we previously added to a solution of 2-hydroxypropyl-ß-cyclodextrin (DSF solution). We found that the instillation of this DSF solution delayed lens opacification in a hereditary cataractous ICR/f rat. In this study, we attempted to design an ophthalmic formulation containing DSF nanoparticles for use as a lens targeted drug delivery system (nano-DSF suspension), and investigated the changes in drug content in the lens after the instillation of DSF solution or nano-DSF suspension. The nano-DSF suspension was prepared by a bead mill method to yield a mean particle size of nano-DSF of 181 nm. Following the instillation of 1.4% DSF solution or the nano-DSF suspension, DDC was detected only in the aqueous humor and lens; in both, the area under the curve (AUC) and mean residence time (MRT) for the nano-DSF suspension were higher than for the DSF solution. In addition, we found that the DDC residence time in the cortex and nucleus of the lens was higher than in the capsule-epithelium. Although DDC was not detected in the cortex and nucleus of lenses following the instillation of the 1.4% DSF solution, the instillation of a 1.4% nano-DSF suspension led to the accumulation of DDC in both areas. In conclusion, it is possible that the instillation of a nano-DSF suspension can supply more DDC into the aqueous humor and lens than a conventional formulation, and these findings provide information significant for the prevention of cataracts and the design of a lens targeted drug delivery system.

Size effect of rebamipide ophthalmic nanodispersions on its therapeutic efficacy for corneal wound healing.

In a variety of tissues including gastrointestinal mucosa, rebamipide (REB) provides cytoprotection, prevents inflammation, and promotes wound healing. Clinically, REB ophthalmic dispersions are used to treat diabetic keratopathy. In this study, we investigated the optimal particle size of REB to promote corneal wound healing using a model of diabetic keratopathy, the debrided corneal epithelium from Otsuka Long-Evans Tokushima Fatty (OLETF) rats. First, we prepared three dispersions with different REB particle sizes (REB735, REB150, REB45) by treatment with zirconia beads and Bead Smash 12 (a bead mill). The mean particle sizes of the REB735, REB150, REB45 dispersions were approximately 735 nm, 150 nm and 45 nm, respectively. Next, we measured the amounts of REB in the corneal and conjunctival tissues of rats following the instillation of the REB dispersions. The amounts of REB in the corneal and conjunctival tissues following the instillation of REB dispersions was increased by using the mill method, and the amount of REB in rats instilled with the REB150 dispersion was significantly higher than in rats instilled with the REB45 dispersion. Moreover, the corneal wound healing rate for rats instilled with the REB150 dispersion was significantly higher than for rats instilled with the REB735 or REB45 dispersions. In addition, these REB dispersions enhanced corneal epithelial cell growth, resulting an enhancement of corneal wound healing rate. Thus, we found that the ocular drug accumulation and therapeutic effect on corneal wound healing of REB dispersions is enhanced by preparing particles with a size of ca. 150 nm. These findings provide significant information that can be used to design further studies aimed at developing ophthalmic dispersions.

Hyperglycemia Enhances the Production of Amyloid ß1-42 in the Lenses of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Type 2 Diabetes.

PURPOSE: It has been reported that the accumulation of amyloid ß1-42 (Aß1-42) in human lenses can cause some forms of lens opacification. However, the factors leading to changes in the accumulation of Aß in the lens remain obscure. In this study, we investigate the effect of hyperglycemia on Aß1-42 accumulation in lenses. METHODS: Otsuka Long-Evans Tokushima Fatty (OLETF) rats and the human lens epithelial cell line SRA 01/04 (HLE cells) were used. The expression of mRNA was determined using a quantitative real-time RT-PCR method; Aß1-42 levels were analyzed by an ELISA method. RESULTS: Otsuka Long-Evans Tokushima Fatty rats at more than 20 weeks of age develop diabetes mellitus with hyperglycemia. Additionally, the levels of the mRNAs for Aß1-42, amyloid precursor proteins (APP), ß-(BACE1), and·Î³-secretase (PS) rise in the lenses of OLETF rats with age; high Aß1-42 levels are observed in the lens capsule-epithelium and cortex. The enhanced expression of the genes for APP, BACE1, and PS in the lenses of OLETF rats is prevented by food restriction (25 g/d/rat). When the effect of glucose levels on the production of Aß1-42 was investigated in the human lens epithelial cell line SRA 01/04 (HLE cells), the mRNA levels for APP, BACE1, and PS, as well as Aß1-42 protein levels, were significantly higher under high glucose conditions (20 mM) than under normal glucose conditions (5.6 mM). CONCLUSIONS: High glucose leads to the increased expression of genes related to Aß production, resulting in the accumulation of Aß in the lens.

Increased Expression of Interleukin-18 in Lenses of Ovariectomized Rats.

Previous studies showed an increased prevalence of cataracts in postmenopausal women. In this study, we investigated changes in the levels of calcium ion (Ca(2+)) and interleukin (IL)-18, which are factors in cataract development, in the lenses of ovariectomized (OVX) rats, a model of postmenopausal woman. Although the Ca(2+) content in the blood of OVX rats increased 1 month after ovariectomy and subsequently decreased, the Ca(2+) content in the lenses was unchanged in OVX rats 1-3 months after ovariectomy. The Ca(2+)-ATPase activity in the lenses of OVX rats peaked 1 month after ovariectomy, and the behavior of Ca(2+)-ATPase activity in lenses of OVX rats was similar to that of the Ca(2+) concentration in the blood. It is possible that hypercalcemia increases the Ca(2+) inflow into the lens; however, the enhanced Ca(2+)-ATPase activity prevents the Ca(2+) level from rising. On the other hand, we found that the levels of both IL-18 and interferon (IFN)-γ in the lenses of OVX rats were significantly increased as compared with the lenses of sham (control) rats during the period 1-3 months after surgery. These results suggest that the expression of IFN-γ via IL-18 in the lenses of OVX rats is induced by ovariectomy, and that excessive IL-18 and IFN-γ production in the lenses may be related to cataract development in postmenopausal women. These findings support those of previous studies that assessed lens opacification in postmenopausal women.