The effects of diclofenac (Dic), an acetic acid derivative‐type nonsteroidal anti‐inflammatory drug, were examined on the function of transient receptor potential (TRP) melastatin (TRPM) 3 (TRPM3) in human embryonic kidney 293 cell‐line (HEK293) cells with recombinant human TRPM3 isoforms (TRPM31325, TRPM3‐3, TRPM3‐9, and TRPM3‐S) and in a neuroblastoma cell line human neuroblastoma IMR‐32 cells (IMR‐32 cells) derived from human peripheral neurons. TRPM3 responses evoked by pregnenolone sulfate (PregS) were effectively inhibited by Dic in a concentration‐dependent manner in Ca2+ measurement and electrophysiological assays. The apparent IC 50 for PregS‐induced Ca2+ response of TRPM31325, TRPM3‐3, and TRPM3‐9 was calculated to be 18.8, 42.5, and 7.1 μmol/L, respectively. The TRPM3‐dependent Ca2+ responses evoked by nifedipine, another TRPM3 agonist, were also significantly inhibited by Dic. In contrast, aceclofenac, an acetoxymethyl analog of Dic, had no effects on PregS‐induced TRPM3 responses. Constitutive channel activity of TRPM3‐S without TRPM3 agonists was substantially inhibited by Dic, ruling out the possibility of interaction of Dic against TRPM3 agonists to the channel binding sites. Moreover, Dic reversibly inhibited TRPM3 single‐channel activity recorded in excised outside‐out patches without affecting the channel conductance. In differentiated neuronal IMR‐32 cells with endogenous TRPM3, Dic inhibited PregS‐evoked Ca2+ responses with an apparent IC 50 of 17.1 μmol/L. Taken together, our findings demonstrate that Dic inhibits human TRPM3 without interacting with the channel pore.

Glaucoma is a chronic optic neuropathy that leads to visual field loss. Elucidating the mechanisms underlying glaucoma is essential for developing new treatments, such as neuroprotective drugs. Various glaucoma models based on the induction of intraocular pressure (IOP) elevation have been established for use in glaucoma studies. However, the time-dependent pathological changes accompanying IOP elevation have not been fully elucidated. In this study, rat conjunctival fibroblasts were injected into the anterior chamber of rat eyes, and IOP elevation was induced for 28 days. Glaucomatous signs such as optic nerve head cupping, retinal thinning, glial activation and apoptotic signaling in the retina were obvious in the cell-injected eyes on the 14th day after injection. The pattern of retinal ganglion cell (RGC) loss differed by the magnitude of IOP elevation. The number of RGCs decreased by 37.5% in eyes with IOP lower than 50 mmHg (Under-50) and by 88.0% in those with IOP higher than 50 mmHg (Over-50) 28 days after cell injection. The RGC counts were correlated with IOP in the Under-50 group but not in the Over-50 group. Our model may contribute to the investigation of pathogenic mechanisms of glaucoma and the development of new glaucoma treatments.

Background microRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability. Methods miRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3′-UTR of human IGFBP5. Results Microarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity. Conclusions In this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.

Intravitreal injection therapy of anti-VEGF antibody or steroids is the mainstream for patients with age-related macular degeneration (AMD). However, since intravitreal injection is invasive administration, side effects such as endophthalmitis are major problems. In this study, we selected eye drops as a non-invasive treatment method, and aimed to develop eye drops that can deliver TAK-593 (VEGF receptor tyrosine kinase inhibitor) to the posterior segment of the eye.Since TAK-593 is a poorly water-soluble drug, the TAK-593 emulsion was formulated. The solubility of TAK-593 in various oils was measured, and the oil used for the emulsion was selected. Furthermore, viscosity enhancers were added to the emulsion in order to improve the drug delivery into the eye.As viscosity enhancer, xanthan gum was selected based on the properties and the viscosity of the emulsion. The delivery of TAK-593 to the posterior eye was increased by the formulation concentration and the addition of viscosity enhancers.In the laser-induced choroidal neovascularization model, TAK-593 emulsion eye drops showed the same angiogenesis-suppression efficacy as anti-VEGF antibody intravitreal injection. From these results, it was revealed that TAK-593 with an effective drug concentration can be delivered to the posterior eye by non-invasive eye drop administration.