Preparation of an Ultrafine Rebamipide Ophthalmic Suspension with High Transparency.

A 2% commercially available, milky-white, rebamipide micro-particle suspension is used to treat dry eyes, and it causes short-term blurring of the patient's vision. In the current study, to improve the transparency of a rebamipide suspension, we attempted to obtain a clear rebamipide suspension by transforming the rebamipide particles to an ultrafine state. In the initial few efforts, various rebamipide suspensions were prepared using a neutralizing crystallization method with additives, but the suspensions retained their opaque quality. However, as a consequence of several critical improvements in the neutralizing crystallization methods such as selection of additives for crystallization, process parameters during crystallization, the dispersion method, and dialysis, we obtained an ultrafine rebamipide suspension (2%) that was highly transparent (transmittance at 640 nm: 59%). The particle size and transparency demonstrated the fewest level of changes at 25°C after 3 years, compared to initial levels. During that period, no obvious particle sedimentation was observed. The administration of this ultrafine rebamipide suspension (2%) increased the conjunctival mucin, which was comparable to the commercially available micro-particle suspension (2%). The corneal and conjunctival concentration of rebamipide following ocular administration of the ultrafine suspension was slightly higher than that of the micro-particle suspension. The ultrafine rebamipide suspension (eye-drop formulation) with a highly transparent ophthalmic clearness should improve a patient's QOL by preventing even a shortened period of blurred vision.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PURPOSE: Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. METHODS: Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. RESULTS: Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. CONCLUSIONS: Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

Rebamipide increases mucin-like substance contents and periodic acid Schiff reagent-positive cells density in normal rabbits.

PURPOSE: The effects of rebamipide on the number of periodic acid Schiff reagent (PAS)-positive cells in the conjunctiva, the mucin content in the cornea and conjunctiva of normal rabbits, and desiccation-induced corneal damage in vivo were examined. METHODS: Rebamipide (0.1%-3%) was applied 6 times a day for 14 days, and the PAS-positive cell count in the bulbar conjunctiva was measured by impression cytology. The amount of conjunctival and corneal mucin-like substances was measured by Alcian blue binding. The corneal damage model was created by desiccation from air flow at room temperature. The level of corneal damage was determined by scoring the area stained with rose bengal and fluorescein dye. RESULTS: Rebamipide increased the number of PAS-positive cells in the conjunctiva when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances of the conjunctiva and cornea. Moreover, 1% rebamipide was also found to lower the rose bengal scores of the cornea in the corneal damage model by desiccation. CONCLUSIONS: Rebamipide is a possible candidate drug for treatment of cornea and conjunctival epithelial damage due to its mucin-like substance increasing action, for instance, in the treatment of dry eye disease.

Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.

The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.

OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.

PURPOSE: To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture. METHODS: Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody. RESULTS: As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4. CONCLUSIONS: The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Rebamipide increases the amount of mucin-like substances on the conjunctiva and cornea in the N-acetylcysteine-treated in vivo model.

PURPOSE: Rebamipide increases the amount of mucin-like substances in the stomach. We aimed to determine the effects of rebamipide on the amount of mucin-like substances in the conjunctiva and cornea of N-acetylcysteine-treated eyes. Furthermore, we attempted to evaluate the effects of rebamipide on the wound healing of N-acetylcysteine-treated eyes. METHODS: The model was created by instilling 10% N-acetylcysteine solutions into rabbit eyes. Rebamipide was then applied on the day following the completion of N-acetylcysteine treatment. The amount of mucin-like substances on the conjunctiva and cornea was measured using the Alcian-blue binding method. The degree of damage was evaluated using scores based on the areas and densities of the cornea and conjunctival after staining using a rose Bengal solution under blind conditions. RESULTS: Rebamipide increased the level of mucin-like substances on the conjunctiva of N-acetylcysteine-treated eyes when instilled at concentrations of 0.3% or higher, and 1% rebamipide increased the amount of mucin-like substances covering the cornea. Moreover, 1% rebamipide improved the rose Bengal scores of the cornea and conjunctiva in N-acetylcysteine-treated eyes. CONCLUSIONS: Rebamipide increased mucin-like substances on the cornea and conjunctiva of N-acetylcysteine-treated eyes. In accordance with the mucin-increasing effects, rebamipide improved the rose Bengal scores for the cornea and conjunctiva of N-acetylcysteine-treated eyes. However, the relevance of these findings to dry eyes is unclear because it is not known whether the change in mucus expression in the N-acetylcysteine model is similar to what occurs in aqueous tear deficiency. Consequently, it may be worth trying on an animal model of keratoconjunctivitis sicca.