Tolerability of Diquas LX on tear film and meibomian glands findings in a real clinical scenario.

Long-acting diquafosol ophthalmic solution (DQS-LX) has significant advantages regarding patient adherence owing to the reduced frequency of required eye drops; however, some patients prefer conventional diquafosol ophthalmic solution (DQS) over DQS-LX. Herein, to clarify the characteristics of patients according to their preference for ophthalmic solutions, dry eye (DE) and meibomian gland (MG) findings were retrospectively investigated. This study enrolled 341 patients with DE (mean age, 62.1 ± 11.7 years) treated at the Itoh Clinic between November 8, 2022, and July 31, 2023, who switched from DQS to DQS-LX. Patients were divided into two groups: those who continued DQS-LX administration (DQS-LX group) and those who wished to revert to conventional DQS (DQS group). Data regarding subjective symptoms assessed using the Standard Patient Evaluation of Eye Dryness (SPEED) questionnaire, tear film breakup time (BUT), tear meniscus height (TMH), corneal and conjunctival fluorescein staining (CFS), conjunctival hyperemia/papilla, meiboscore, plugging, vascularity, meibum grade, and Schirmer's score at the time of DQS-LX switch were evaluated. Of the 341 patients, 31 (9.1%) wished to revert to conventional DQS. In total, 16 eyes of 16 patients in the DQS group and 32 eyes of 32 patients in the DQS-LX group-for whom complete data were available-were included in the analysis. The DQS-LX group had higher SPEED scores, lower TMHs (P < 0.001, respectively), shorter FBUTs, greater CFS findings, larger meibum grades, lower Schirmer scores, and more pluggings compared with the DQS group (P = 0.005, 0.001, 0.001, 0.046, 0.003, respectively). Meiboscores and vascularity did not differ significantly between the two groups (P = 0.73 and 0.39, respectively). In conclusion, patients with low tear film volume and DE complicated by moderate or severe meibomian gland dysfunction (MGD) preferred DQS-LX, while those with allergic findings preferred conventional DQS.

Diquafosol Improves Corneal Wound Healing by Inducing NGF Expression in an Experimental Dry Eye Model.

Dry eye disease (DED) is caused by inflammation and damage to the corneal surface due to tear film instability and hyperosmolarity. Various eye drops are used to treat this condition. Each eye drop has different properties and mechanisms of action, so the appropriate drug should be used according to clinical phenotypes. This study aims to compare the therapeutic mechanisms of cyclosporine A (CsA) and diquafosol tetrasodium (DQS). An experimental in vivo/in vitro model of DED using hyperosmolarity showed decreased cell viability, inhibited wound healing, and corneal damage compared to controls. Treatment with cyclosporine or diquafosol restored cell viability and wound healing and reduced corneal damage by hyperosmolarity. The expression of the inflammation-related genes il-1ß, il-1α, and il-6 was reduced by cyclosporine and diquafosol, and the expression of Tnf-α, c1q, and il-17a was reduced by cyclosporine. Increased apoptosis in the DED model was confirmed by increased Bax and decreased Bcl-2 and Bcl-xl expression, but treatment with cyclosporine or diquafosol resulted in decreased apoptosis. Diquafosol increased NGF expression and translocation into the extracellular space. DED has different damage patterns depending on the progression of the lesion. Thus, depending on the type of lesion, eye drops should be selected according to the therapeutic target, focusing on repairing cellular damage when cellular repair is needed or reducing inflammation when inflammation is high and cellular damage is severe.

Rosmarinic acid-grafted gelatin nanogels for efficient diquafosol delivery in dry eye disease therapy.

Dry eye disease (DED) is a prevalent ocular disorder characterized by unstable tear film condition with loss of aqueous or mucin, excessive oxidative stress, and inflammation, leading to discomfort and potential damage to the ocular surface. Current DED therapies have shown restricted therapeutic effects such as frequent dosing and temporary relief with potential unwanted side effects, urgently necessitating the development of innovative efficient therapeutic approaches. Herein, we developed rosmarinic acid (RosA) conjugated gelatin nanogels loading diquafosol sodium (DQS), DRGNG, for simultaneous ROS-scavenging and mucin-secreting DED treatment. Mechanically, DRGNG suppressed the ROS production, reduced inflammatory factors, and prompted mucin secretion in vitro and in vivo. The whole transcriptome RNA sequencing in vitro further provided a detailed analysis of the upregulation of anti-oxidant, anti-inflammatory, and mucin-promotion pathways. Therapeutically, both in evaporative DED and aqueous deficient DED models, the dual-functional DRGNG could prolong the retention time at the ocular surface, efficiently suppress the oxidative stress response, reverse ocular surface morphology, and recover tear film homeostasis, thus alleviating the DED when the dosage is halved compared to the commercial Diquas®. Our findings contribute to developing innovative therapies for DED and offer insights into the broader applications of nanogels in ocular drug delivery and oxidative stress-related conditions.

Multifunctional Cerium Oxide Nanozyme for Synergistic Dry Eye Disease Therapy.

Dry eye disease (DED) is a chronic multifactorial ocular surface disease mainly caused by the instability of tear film, characterized by a series of ocular discomforts and even visual disorders. Oxidative stress has been recognized as an upstream factor in DED development. Diquafosol sodium (DQS) is an agonist of the P2Y2 receptor to restore the integrity/stability of the tear film. With the ability to alternate between Ce3+ and Ce4+, cerium oxide nanozymes could scavenge overexpressed reactive oxygen species (ROS). Hence, a DQS-loaded cerium oxide nanozyme was designed to boost the synergistic treatment of DED. Cerium oxide with branched polyethylenimine-graft-poly(ethylene glycol) as nucleating agent and dispersant was fabricated followed with DQS immobilization via a dynamic phenylborate ester bond, obtaining the DQS-loaded cerium oxide nanozyme (defined as Ce@PBD). Because of the ability to mimic the cascade processes of superoxide dismutase and catalase, Ce@PBD could scavenge excessive accumulated ROS, showing strong antioxidant and anti-inflammatory properties. Meanwhile, the P2Y2 receptors in the conjunctival cells could be stimulated by DQS in Ce@PBD, which can relieve the incompleteness and instability of the tear film. The animal experiments demonstrated that Ce@PBD significantly restored the defect of the corneal epithelium and increased the number of goblet cells, with the promotion of tear secretion, which was the best among commercial DQS ophthalmic solutions.

Comparing two mucin secretagogues for the treatment of dry eye disease: a prospective randomized crossover trial.

This study aimed to compare the clinical efficacy and investigate patients' preferences for two mucin secretagogues in the treatment of dry eye disease (DED). Thirty patients with DED were randomly treated with either 3% diquafosol or 2% rebamipide ophthalmic solution for 4 weeks, followed by an additional 4-week treatment using the other eye drop after a 2-week washout period. Objective and subjective assessments, including the corneal and conjunctival staining score, tear breakup time (TBUT), Schirmer 1 test, tear osmolarity, tear matrix metalloproteinase-9 (MMP-9), lipid layer thickness (LLT) and ocular surface disease index (OSDI), were performed at baseline, 4 weeks, 6 weeks, and 10 weeks. Patient preferences were assessed based on four categories (comfort, efficacy, convenience, willingness to continue) using a questionnaire and the overall subjective satisfaction score for each drug was obtained at the end of the trial. In total, 28 eyes from 28 patients were included in the analysis. Both diquafosol and rebamipide significantly improved the OSDI (p = 0.033 and 0.034, respectively), TBUT (p < 0.001 and 0.026, respectively), and corneal (p < 0.001 and 0.001, respectively) and conjunctival (p = 0.017 and 0.042, respectively) staining after 4 weeks of treatment. An increase in Schirmer test scores was observed only after rebamipide treatment (p = 0.007). No significant changes were detected in tear osmolarity, MMP-9, and LLT following both treatments. The patients' preference was slightly greater for diquafosol (46.4%) than rebamipide (36.7%), presumably due to rebamipide's bitter taste. The self-efficacy of both drugs and overall satisfaction scores were comparable. These findings indicate that two mucin secretagogues showed comparable effects in ameliorating symptoms and improving signs (TBUT, corneal and conjunctival staining) in patients with DED.

Efficacy of 3% diquafosol long-acting eye drops in dry eye patients treated for three months.

PURPOSE: To investigate changes in the ocular surface and subjective symptoms during a three months administration of 3% diquafosol long-acting (DQL) eye drops. STUDY DESIGN: Prospective observational study. METHODS: DQL eye drops were administered as the sole treatment for all patients, including those in the group where DQL eye drops were newly prescribed (New DQL) and the group who switched from 3% diquafosol (DQS) eye drops (Switched DQL) in this prospective study. Each group underwent assessment of tear meniscus height (TMH), ocular surface disease index (OSDI), fluorescein break-up time (FBUT), fluorescein score, and Schirmer 1 test before DQL administration, at one month, and at three months. Changes in ocular surface scores and subjective symptoms at each time point were analyzed. RESULTS: The study included a total of 63 eyes of 63 patients, with a mean age of 60.3 ±14.6 (SD). Among them, 29 patients (20 women) were in the New DQL group, and 34 patients (24 women) were in the Switched DQL group. Both the New DQL and Switched DQL groups showed significant improvements in TMH, OSDI, FBUT, Fluorescein Score, and Schirmer 1 test after three months of DQL eye drop administration. CONCLUSION: DQL eye drops have the potential to improve ocular scores and subjective symptoms in patients with DE over a three months period, regardless of whether it is newly initiated or as a switch from DQS eye drops.

Effects of a Long-Acting Diquafosol Ophthalmic Solution on the Ocular Surface, Tolerability, and Usability in Dry Eye Disease.

INTRODUCTION: This study aimed to investigate the tolerability of high-viscosity diquafosol tetrasodium (DQS) ophthalmic solution (DIQUAS LX; DQSLX) and examine its usability and effect on clinical findings in patients with dry eye disease (DED). METHODS: This interventional retrospective study included 66 eyes of 66 patients with DED who switched from conventional DQS to DQSLX ophthalmic solution. Tear function assessments (tear film breakup time [BUT], keratoconjunctival vital staining [VS] score), evaluations of DED symptom relief, and a four-item usability questionnaire ("comfort upon instillation," "irritation upon instillation," "eye mucus discharge," "convenience of instillation frequency") assessed using a visual analog scale from 0 (worst) to 10 (best) were administered 4 weeks after switching to DQSLX. Factors associated with drug tolerability were assessed using multiple regression analysis. RESULTS: The symptoms improved by 64.2% after switching to DQSLX. The BUT value, VS score, and the questionnaire items "comfort upon instillation" and "convenience of instillation frequency" were significantly improved after switching to DQSLX. DQSLX tolerability was reported as acceptable in 56 (84.8%) and unacceptable in 10 (15.2%) patients. Overall, DQSLX tolerability was significantly associated with "comfort upon instillation" and "convenience of instillation frequency" and tended to be associated with a VS score ≥ 1. DQSLX tolerability depended on symptom and VS score improvements and absence of excessive "eye mucus discharge" in patients with a VS score ≥ 1 (39 patients), but on "comfort upon instillation" and absence of excessive "eye mucus discharge" in patients with a VS score = 0 (27 patients). CONCLUSION: The high-viscosity DQSLX ophthalmic solution was generally considered acceptable in the study population. However, drug tolerability seemingly differed between patients with DED with and without epithelial damage. The former were affected by improvements in symptoms and clinical findings, whereas the latter were affected by comfort upon instillation. TRIAL REGISTRATION: University Hospital Medical Information Network identifier, UMIN000051390.

Development and evaluation of hot-melt-extruded diquafosol tetrasodium formulations for ophthalmic inserts: A design of experiments approach.

This study aimed to develop, optimize, and evaluate hot-melt-extruded ophthalmic inserts capable of sustained release of diquafosol tetrasodium (DQS) via a design of experiments approach. DQS, a tear stimulant for dry eye management, faces challenges of frequent administration and low bioavailability. The developed insert uses biodegradable polymers in varied proportions to achieve sustained release. Optimized through mixture design, the insert completely dissolved within 24 h and maintained a stable drug content, thickness, and surface pH over three months at room temperature. In vitro corneal permeation studies on excised rabbit corneas demonstrated increased bioavailability, suggesting a reduced dosing frequency compared with conventional eye drops. Therefore, this insert has potential to enhance treatment outcomes by improving patient compliance and providing sustained drug effects.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

Effects of Diquafosol Sodium Ophthalmic Solution on Tear Film Matrix Metallopeptidase-9 and Corneal Nerve Density in Patients with Type 2 Diabetic Dry Eye.

Purpose: Diabetes mellitus has been associated with increased dry eye disease (DED) and exacerbates DED's pathology. This preliminary short-term study aimed to evaluate the effects of 3% Diquafosol Sodium ophthalmic solution (DQS) on ocular surface inflammation and corneal nerve density in diabetic dry eye (DDE) patients. Methods: In this perspective, participants used 1 drop of 3% DQS (Diquas; Santen Pharmaceutical Co., Ltd., Osaka, Japan) 6 times daily for 8 weeks. Non-invasive tear breakup time (NITBUT), tear film lipid layer (TFLL), conjunctival hyperemia [redness score (RS)], corneoconjunctival staining (CFS), corneal sensitivity (CS), Meibomian gland quality (MGQ) and Meibomian gland expressibility (MGEx), corneal nerve fiber density (CNFD), and Standard Patient Evaluation Eye Dryness (SPEED) questionnaire were assessed at baseline, at weeks 4, and up to 8 weeks. Matrix metalloproteinase-9 (MMP-9) of tear samples was measured at baseline and weeks 8. Results: The mean age was 61.27 ± 11.68 years. At baseline NITBUT = 5.89 ± 2.81 s, tear meniscus height = 0.17 ± 0.05 mm, TFLL = 2.74 ± 0.51, CFS = 4.35 ± 0.68, CS = 53.83 ± 9.63 mm, MMP-9 = 49.10 ± 10.42 ng/mL, RS = 1.65 ± 0.44, MGEx = 1.85 ± 0.72, MGQ = 2.65 ± 0.50, CNFD = 20.36 ± 8.20 no./mm2, and SPEED = 12.62 ± 3.91. At week 4, significant improvements were found in all parameters except RS (1.59 ± 0.46, P = 0.172) and CNFD (21.46 ± 8.41, P = 0.163). Finally, at week 8, all parameters had significant improvements. Conclusion: Preliminary short-term findings suggest that treatment of DDE patients with DQS was found to be safe and efficacious in improving dry eye parameters. In addition, inflammatory marker and corneal nerve density were significantly improved. This study was registered with ClinicalTrials.gov (NCT05193331).

RNA structural probing of guanine and uracil nucleotides in yeast.

RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.

Comparison of matrix metallopeptidase-9 expression following cyclosporine and diquafosol treatment in dry eye.

PURPOSE: We sought to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in dry eyes treated with 0.05% cyclosporin A and 3.0% diquafosol tetrasodium. METHODS: One-hundred ninety-five eyes of 195 patients with dry eye were divided into three groups as follows: group 1, cyclosporin group (n = 69); group 2, diquafosol group (n = 59); and group 3, artificial tears eyes (n = 67). All eyes were treated and followed up for three months. Schirmer I Test, corneal staining, tear-film break-up time (TBUT), and tear-film MMP-9 content were measured at three months and compared between groups. The expression of MMP-9 was confirmed using a point-of-care test device (InflammaDry®; RPS Diagnostics, Sarasota, FL, USA) and graded as zero to four points. RESULTS: At the third month, MMP-9 expression was lower in group 1 as compared with in groups 2 and 3 (p = 0.020 and 0.006, respectively). The mean MMP-9 grade according to point-of-care testing was also lower in group 1 than in groups 2 or 3 (p = 0.002 and 0.038, respectively). MMP-9 showed a correlation with corneal staining in both groups 1 and 2 (all p < 0.001) and with Schirmer I Test and TBUT in group 1 (p = 0.018 and 0.015, respectively). CONCLUSIONS: MMP-9 expression and grade were lower after treatment with cyclosporin than after treatment with diquafosol in the dry eye disease. Anti-inflammatory treatment can decrease ocular MMP-9 levels in dry eye disease.

MMP-9 expression and grade were lower after treatment with cyclosporin than after treatment with diquafosol in the dry eye disease. Anti-inflammatory treatment can decrease ocular MMP-9 levels in dry eye disease.

Safety and efficacy of topical diquafosol for the treatment of dry eye disease: An updated meta-analysis of randomized controlled trials.

Dry eye disease (DED) is a common multi-factorial disease that is characterized by tear film instability. Diquafosol tetrasodium (DQS), an ophthalmic solution, has been shown to be beneficial in the treatment of DED. The goal of this study was to provide an update on the safety and efficacy of topical 3% DQS in treating DED patients. A thorough search for all the published randomized controlled trials (RCTs) up to March 31, 2022 in CENTRAL, PubMed, Scopus, and Google Scholar databases was performed. Data were reported as standardized mean difference (SMD) with 95% confidence interval (CI). Modified Jadad scale was used for sensitivity analysis. Funnel plot and Egger's regression test assessed the publication bias. Fourteen RCTs evaluating the safety and efficacy of topical 3% DQS treatment in DED patients were included. Eight included RCTs reported data on the DED after cataract surgery. Overall findings suggest that 3% DQS treatment in DED patients was associated with significantly better improvement at 4 weeks in tear breakup time, Schirmer test, fluorescein staining scores, and Rose Bengal staining score as compared to patients treated with others eye drops including artificial tears or 01% sodium hyaluronate. However, no significant difference in ocular surface disease index was observed. Our findings suggest that 3% DQS treatment is safer and had a superior efficacy compared to artificial tears or sodium hyaluronate for treating DED in general and DED after cataract surgery.

Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors.

BACKGROUND: Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. METHODS: MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. CONCLUSIONS: Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

Diquafosol sodium reduces neuronal activity in trigeminal subnucleus caudalis in a rat model of chronic dry eye disease.

Patients with persistent and severe dry eye disease (DED) have corneal hypersensitivity, resulting in ocular pain, and diquafosol sodium, a potent P2Y2 receptor agonist, is commonly used to improve the resultant tear film stability. This study determined the effects of diquafosol instillation on the suppression of trigeminal subnucleus caudalis (Vc) neuronal activity and ocular pain by enhancing tear film stability in the model for chronic DED. The effects of diquafosol on the ocular surface were assessed by the topical application for 28 days, starting from the 14th day since unilateral exorbital gland removal (chronic DED). Loss of tear volume secretion in chronic DED rats was significantly reversed by diquafosol instillation after 28 days, compared with saline treatment. The number of eyeblinks and pERK-IR neurons in the superficial laminae of Vc following hypertonic saline administration to the ocular surface was lower in diquafosol-treated chronic DED rats than in saline-treated rats. The neuronal activity evoked by hypertonic saline and mechanical stimulation along with the spontaneous neuronal activity in the superficial laminae of the Vc were suppressed in diquafosol-treated chronic DED rats. These findings suggest that ocular surface instillation of diquafosol for 28 days attenuates the neuronal hyperactivity in the Vc and the ocular pain that often occurs in chronic DED.

Diquafosol ophthalmic solution enhances mucin expression via ERK activation in human conjunctival epithelial cells with hyperosmotic stress.

Purpose: To evaluate the effect of diquafosol tetrasodium on the expression of secretory and membrane-associated mucins in multi-layered cultures of primary human conjunctival epithelial cells (HCEC) using intracellular extracellular signal regulated kinase (ERK) signaling. Methods: HCECs were treated with hyperosmotic stress (400 mOsm/l) for 24 h after air-liquid interface cell culture followed by treatment with diquafosol. HCECs were stimulated for 1 h with or without PD98059, an ERK inhibitor, then treated with diquafosol for 6 h and 24 h. Mucin 1 (MUC1), mucin 16 (MUC16), and MUC5AC mRNA and protein expression levels were analyzed, and cell viability was detected using an MTT assay. Western blot analysis was used to examine p44/42 MAPK (Erk1/2) and phosphorylated p44/42 MAPK (Erk1/2) expression. Results: Hyperosmotic stressed HCECs demonstrated increased MUC5AC secretion and gene expression when treated with diquafosol. MUC1 mRNA levels increased significantly at 24 h (p<0.01), and expression of MUC16 mRNA levels increased at 6 h and were maintained until 24 h (p<0.05).There was no significant difference in cell viability compared to the control group. Immunostaining results for MUC1, MUC16, and MUC5AC in diquafosol tetrasodium-treated HCECs at 24 h showed more positive cells than in the control group. Phosphorylation of p44/42 MAPK (Erk1/2) signaling molecules significantly increased from 5 min to 60 min (p<0.05). The effects of diquafosol on mucin expressions in hyperosmotic stressed HCECs were significantly inhibited by PD98059, an ERK inhibitor, at 6 h and 24 h. Conclusions: ERK signaling may regulate the expression levels of MUC1, MUC16, and MUC5AC induced by diquafosol in hyperosmotic stressed HCECs.