Efficacy and Safety of Diquafosol Ophthalmic Solution in Patients with Dry Eye Syndrome: A Japanese Phase 2 Clinical Trial

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1 Efficacy and Safety of Ophthalmic Solution in Patients with Dry Eye Syndrome: A Japanese Phase 2 Clinical Trial Yukihiro Matsumoto, MD, 1 Yuichi Ohashi, MD, 2 Hitoshi Watanabe, MD, 3 Kazuo Tsubota, MD, 1 for the Ophthalmic Solution Phase 2 Study Group* Objective: To investigate the dose-dependent efficacy and safety of diquafosol ophthalmic solution for the treatment of dry eye syndrome. Design: Randomized, double-masked, multicenter, parallel-group, placebo-controlled trial. Participants: A total of 286 Japanese patients with dry eye who were prescribed topical diquafosol (1%, n 96; 3%, n 96) or placebo ophthalmic solution (n 94). Methods: After a washout period of 2 weeks, qualified subjects were randomized to receive a single drop of 1% or 3% diquafosol or placebo ophthalmic solutions 6 times per day for 6 weeks. Main Outcome Measures: The primary outcome measure was fluorescein corneal staining score assessment. The secondary outcome measures were Rose Bengal corneal and conjunctival staining scores, tear break-up time (BUT), and subjective symptom assessment. Safety measures were clinical blood and urine examination and recording of adverse events. Results: Fluorescein corneal staining scores significantly improved with both 1% and 3% topical diquafosol compared with placebo at 4 weeks, respectively (P 0.037, P 0.002). There was a dose-dependent effect among the groups. Rose Bengal corneal and conjunctival staining scores also improved significantly with both 1% and 3% diquafosol compared with placebo (P and P 0.004, respectively). Subjective dry eye symptom scores significantly improved with both diquafosol ophthalmic solutions (P 0.033), although there were no significant differences in BUT compared with placebo. No significant differences between the treatment groups were observed in relation to the occurrence of adverse events. Conclusions: Both 1% and 3% diquafosol ophthalmic solutions are considered effective and safe for the treatment of dry eye syndrome. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. Ophthalmology 2012;119: by the American Academy of Ophthalmology. *Group members listed online in Appendix 1 (available at Dry eye has been defined as a multifactorial disease of the tears and the ocular surface that results in symptoms such as ocular discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. 1 Dry eye may ensue as a result of failure of lacrimal or meibomian glands on the ocular surface. 2,3 The prevalence of dry eye disease is increasing because of the changes in lifestyles due to increased visual display terminal work, dry room environments resulting from air conditioning, contact lens use, and increased practice of refractive surgery. 4 8 In the United States, the prevalence of dry eye syndrome has been reported to increase with age. The prevalence was 5.7% among women aged 50 years, 7.8% among women aged 50 years, and 9.8% among women aged 75 years in the Women s Health Study. 9 In Japan, the prevalence of dry eye syndrome has been reported to be 10.1% in men and 21.5% in women among visual display terminal users aged between 22 and 60 years in a recent epidemiologic study performed using the same methodology as the Women s Health Study. 10,11 The tear film has been traditionally reported to consist of 3 important components: a mucin layer that coats the ocular surface epithelium, an aqueous layer that is present between the mucin and a lipid layer, and a lipid layer that overlays the surface of the tear film. 12 However, the current belief is that the secreted mucin exists within the aqueous layer of the tear film and that mucins play an integral role in the interactions between the tear film and epithelial cells on the ocular surface. 13 Decreased tear production and alteration of tear components in several ocular surface diseases may result in dryness of the ocular surface epithelium, induce apoptosis of epithelial cells, and cause decreased production of mucin from the goblet cells. The lack of mucins may reduce the stability of the tear film and lead to or aggravate dry eye disease. 14 The 2007 Report of the International Dry Eye Workshop stated that there have been tremendous advances in the treatment of dry eye and ocular surface disease in the last 2 decades, with a commensurate increase in knowledge re by the American Academy of Ophthalmology ISSN /12/$ see front matter Published by Elsevier Inc.

2 Matsumoto et al Treatment in Dry Eyes garding the pathophysiology of dry eye. This has led to a paradigm shift in dry eye management from simply lubricating and hydrating the ocular surface with artificial tears to strategies that stimulate natural production of tear constituents. 15 The workshop report also suggested the possibility that future therapies will focus on replacing specific tear factors that play an essential role in maintaining ocular surface homeostasis such as mucins. is a uridine triphosphate related compound. 16 has been reported to be an agonist of the purinergic P2Y 2 receptor that is expressed in several ocular (including conjunctival epithelium and goblet cells) and pulmonary tissues and is related to the mechanisms of G protein mediated activation of phospholipase C and inositol. At a cellular level, the P2Y 2 receptor is known to contribute to water transfer and mucin secretion In animal studies involving rabbits, diquafosol has been shown to stimulate both water secretion from conjunctival epithelial cells and mucin secretion from conjunctival goblet cells via the P2Y 2 receptors. 18,20 has also been shown to prevent corneal epithelial damage in a rabbit dry eye model. 21 In a rat model of dry eye disease, diquafosol was demonstrated to improve tear secretion and restore the corneal epithelial barrier function. 22 A previous double-masked, placebo-controlled safety and efficacy trial of diquafosol stated that 2% diquafosol was superior to placebo in reducing corneal staining and relieving foreign body sensation. 23 In this prospective, double-masked, randomized, placebo-controlled phase 2 study in Japan, we investigated the efficacy and safety of 2 different concentrations of diquafosol ophthalmic solution (1% and 3%), applied 6 times per day for 6 weeks in the treatment of dry eye disease. Materials and Methods Subjects A total of 286 eligible eyes of 286 Japanese patients with dry eye, aged 20 years, were enrolled in this phase 2 study. Inclusion criteria for the study were as follows: (1) meet the diagnostic criteria of the Japanese Dry Eye Research group, 24 (2) a Schirmer s test I measurement value (without topical anesthesia) of 5 mm, and (3) a fluorescein (FL) corneal staining score of 1 point at the beginning of and after the washout period. Patients were excluded from the study if they had a history of allogeneic hematopoietic stem cell transplantation, refractive corneal surgery, contact lens use, Stevens Johnson syndrome, ocular cicatricial pemphigoid, or chemical or thermal burns. Patients were also excluded if they had any other ocular diseases, had any systemic disease or medication use that would cause dry eyes, or were pregnant at the time of this study. Protocol Design This study was a randomized, multicenter, doubled-masked, placebocontrolled, parallel-group phase 2 clinical study to evaluate the efficacy and safety of 1% and 3% diquafosol ophthalmic solutions in patients with dry eye. The study protocol was designed with a 2-week washout period, followed by a 6-week treatment period (Fig 1). A placebo ophthalmic solution was administered during the washout period. The vehicle of diquafosol sodium ophthalmic Washout period (2 weeks) solution as placebo contains dibasic sodium phosphate hydrate. After the washout period, the patients were randomly divided into 3 groups: 1% diquafosol ophthalmic solution group (1% diquafosol group), 3% diquafosol ophthalmic solution group (3% diquafosol group), and placebo ophthalmic solution group (placebo group). The patients were instructed to apply 1 drop of their ophthalmic solution 6 times daily. Fluorescein corneal staining scores, Rose Bengal (RB) corneal and conjunctival staining scores, and tear break-up time (BUT) examinations were performed in both eyes of each patient, and subjective dry eye symptoms were recorded at each institution every 2 weeks during the study period. The primary end point was set as the change in FL staining score at week 4 (last observation carried forward), and the secondary end points were set as the changes in tear stability, vital staining, and symptom scores at 2, 4, and 6 weeks. The incidence of adverse events was also recorded at the same time. The trial was carried out at 46 investigative sites in Japan, conformed to the Tenets of the Declaration of Helsinki, and was approved by the ethical review board of each institution. All patients who agreed to participate in this study provided written informed consent. This study was also registered at gov under the identifier NCT (Web site registration date, August 24, 2010). Ocular Surface Evaluation Treatment period (6 weeks) (weeks) Double-masked 1% diquafosol 3% diquafosol Figure 1. The study protocol was a randomized, multicenter, doubledmasked, placebo-controlled, parallel-group phase 2 clinical study to evaluate the efficacy and safety of 1% and 3% diquafosol ophthalmic solutions in patients with dry eye with a 2-week washout period followed by a 6-week treatment period. The ocular surface was initially stained with 2 l of 1% preservativefree FL solution instilled into the conjunctival sac using a micropipette. The patients were instructed to blink several times for a few seconds to ensure adequate mixing of the dye. The BUT was determined to be the interval between the last complete blink and the appearance of the first corneal black spot in the stained tear film. The BUT was measured 3 times with a stopwatch, and the mean value of the measurements was calculated. Fluorescein and RB staining were scored according to the protocol described by Shimmura et al. 25 Two microliters of 1% FL or 1% RB preservative-free solution was instilled into the conjunctival sac using a micropipette. In FL staining, the cornea was divided into 3 equal zones: upper, middle, and lower. Each zone had a staining score ranging between 0 and 3 points, with minimum and maximum total staining scores ranging between 0 and 9 points. 25 In RB staining, the ocular surface was divided into 5 zones: nasal and temporal conjunctival, and upper, middle, and lower corneal areas. A staining score between 0 and 3 points was used in each zone, with the minimum and maximum total staining scores ranging between 0 and 15 points. The degree of staining with both FL and RB dyes was scored as follows: 0 no staining, 1 staining of less than half of the corneal/conjunctival area, 2 staining of more than half of the corneal/conjunctival area, and 3 staining in the whole corneal/conjunctival area. 1955

3 Ophthalmology Volume 119, Number 10, October 2012 Table 1. Characteristics of the Study Population 1% 3% No. of randomized subjects No. of subjects completing the study (%) 89 (94.7) 89 (92.7) 90 (93.8) No. of subjects discontinuing the study (%) 5 (5.3) 7 (7.3) 6 (6.3) Reason for discontinuation, n (%) Adverse events 1 (1.1) 3 (3.1) 4 (4.2) Lack of efficacy 2 (2.2) 3 (3.1) 0 Withdrawal of consent by the patient 1 (1.1) 0 1 (1.0) Improper entry 1 (1.1) 1 (1.0) 1 (1.0) Evaluation of Ocular Symptoms The presence of ocular symptoms was evaluated by conducting interviews including symptoms of foreign body sensation, photophobia, itching, eye pain, dry eye sensation, heaviness, blurred vision, asthenopia, ocular discomfort, eye discharge, and tearing. The severity of ocular symptoms was assessed on a 4-point scale from 0 to 3 as follows: 0 no symptoms, 1 mild, 2 moderate, and 3 severe. Safety Evaluations The safety of diquafosol was assessed by recording adverse events, associated symptoms, clinical blood and urine examinations, fundoscopy, visual acuity, intraocular pressure measurements, and slit-lamp examinations. Slit-lamp examination was performed every 2 weeks during the follow-up visits. Visual acuity tests, intraocular pressure measurements, fundoscopy examinations, and clinical laboratory examinations were performed at the first and last visits. Clinical laboratory examinations included complete blood counts (including red blood cell count, leukocyte count, hemoglobin level, hematocrit value, platelet count, and leukocyte fraction), liver function tests (including aspartate transaminase, alanine amino transferase, -glutamyl transferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, total protein, total albumin, albumin/globulin ratio, and total cholesterol levels), blood biochemistry profile (including blood urea nitrogen, serum creatinine, uric acid, sodium, potassium, and chlorine levels), and urine analyses (including urinary glucose, protein, and urobilinogen levels). Statistical Methods Data are shown as mean standard error. Monocular data analyses of the eligible eyes were performed for statistical comparisons. Primary efficacy analyses consisted of assessment of the statistical significance of the change in the FL staining score at the week 4 end point (primary end point). The maximum contrast method (MCM) was used to evaluate dose-response relationships. 26 Secondary efficacy analyses consisted of the assessment of changes in tear stability, FL, RB, and symptom scores at 2, 4, and 6 weeks (secondary end points). Statistical differences between the treatment and placebo groups were evaluated using the unpaired t test. A P value less than 0.05 was considered statistically significant. SAS software version 9.1 (SAS Inc., Cary, NC) was used for statistical analysis. Results A total of 320 patients were enrolled in this study. Thirty-four patients discontinued the study during the washout period before initiation of treatment. Five patients (1.5%) dropped out during the washout period of the study because of aggravation. There was no statistically significant difference in the baseline of FL staining scores between dropouts and nondropouts ( points and points, respectively). A total of 286 patients were randomly assigned to the treatment groups as follows: 96 to the 1% diquafosol group, 96 to the 3% diquafosol group, and 94 to the placebo group. Some 93.7% of the subjects completed the study (Table 1). There were no statistically significant differences between the background characteristics of patients in the 3 treatment groups with respect to gender, age, distribution of Sjögren s syndrome in each group, and severity of dry eye disease in relation to the FL staining scores, RB staining scores, BUT, and Schirmer s test values (Table 2). Ocular Surface Findings and Tear Functions Changes in Fluorescein Corneal Staining Scores. Figure 2 shows the changes in FL corneal staining scores from baseline in each treatment group at all follow-up points. The FL corneal staining scores revealed a statistically significant improvement in both diquafosol groups compared with the placebo group at week 4 (1% diquafosol group, P 0.037; 3% diquafosol group, P 0.002). In addition, the FL staining score in the 3% diquafosol group was significantly better than the placebo group at week 6 (P 0.005). Dose-response analysis using the MCM demonstrated that the linear dose response and dose response saturated with 1% diquafosol were both significant. However, a more marked significance was observed in the linear dose response (linear, P 0.004; Table 2. Baseline Demographics and Background Characteristics of the Patients (n 94) 1% 3% Gender, female 76 (80.9%) 85 (88.5%) 77 (80.2%) Sjögren s syndrome 23 (24.5%) 20 (20.8%) 16 (16.7%) Age (yrs) FL corneal staining score (points) RB corneal and conjunctival staining score (points) BUT (sec) Schirmer s test I (mm) BUT break-up time; FL fluorescein; RB Rose Bengal. 1956

4 Matsumoto et al Treatment in Dry Eyes * * * * *P < 0.05; **P < Figure 2. The change in fluorescein (FL) corneal staining scores from baseline. The FL corneal staining scores revealed a statistically significant improvement in both diquafosol groups compared with the placebo group at week 4. In addition, the FL staining score in the 3% diquafosol group was significantly better than in the placebo group at week 6. saturated with 1% diquafosol group, P 0.006) at the week 4 end point. The topical 3% diquafosol treatment group showed the greatest improvement in FL staining scores, which were and points at weeks 4 and 6, respectively. Rose Bengal Corneal and Conjunctival Staining Score. Figure 3 shows the change in RB corneal and conjunctival staining scores from baseline in each treatment group at all time points throughout the study period. The RB corneal and conjunctival staining scores significantly improved in both 1% and 3% diquafosol treatment groups compared with the placebo group at week 4 (1% diquafosol group, P 0.007; 3% diquafosol group, P 0.004) and week 6 (1% diquafosol group, P 0.003; 3% diquafosol group, P 0.003). Topical 3% diquafosol treatment resulted in the most marked improvement in RB staining scores ( and points at weeks 4 and 6, respectively). Tear Break-up Time. Topical 3% diquafosol resulted in the most marked improvement in BUTs, which were and Figure 4. The change in tear break-up times (BUT) from baseline. Tear BUT value showed the most marked improvement in the 3% diquafosol treatment group at weeks 4 and seconds at weeks 4 and 6, respectively. However, no significant difference in BUT was observed among the 3 groups at any time point throughout the study (Fig 4). Subjective Symptoms A significant improvement in dry eye sensation symptom score was obtained in both diquafosol groups compared with the placebo group at week 4 (1% diquafosol group, P 0.003; 3% diquafosol group, P 0.033) and in the 1% diquafosol group at week 6 (P 0.004) (Fig 5). There were no statistically significant differences in the other subjective symptom scores compared with the placebo group. Safety Assessments The most common adverse reactions encountered during the study are listed in Table 3. No significant differences were found between the treatment groups with regard to the rate of occurrence of adverse reactions. The most frequently reported adverse reaction was eye irritation. No serious treatment-related adverse events occurred in any patient during the study. * ** ** ** *P < 0.05; **P < Figure 3. The change in the Rose Bengal (RB) corneal and conjunctival staining scores from baseline. The RB corneal and conjunctival staining scores significantly improved in both the 1% and 3% diquafosol treatment groups compared with the placebo group at weeks 4 and 6. *P < 0.05; **P < Figure 5. Change from baseline in dry eye sensation. A significant improvement in dry eye sensation symptom score was shown in both diquafosol groups compared with the placebo group at week 4 and in the 1% diquafosol group at week

5 Ophthalmology Volume 119, Number 10, October 2012 There were no systemic adverse effects or significant changes in the clinical laboratory examination values according to the blood and urine examinations (data not shown). Moreover, there were no changes in the slit-lamp findings, intraocular pressure measurements, fundoscopy observations, or visual acuity before and after treatment in the diquafosol or placebo groups. Discussion Table 3. Most Common Adverse Reactions (n 94) 1% 3% No. of subjects with 13 (13.8%) 12 (12.5%) 15 (15.6%) adverse reactions Eye irritation 3 (3.2%) 7 (7.3%) 12 (12.5%) Eye pain 3 (3.2%) 1 (1.0%) 4 (4.2%) Conjunctival hyperemia 3 (3.2%) 3 (3.1%) 1 (1.0%) Foreign body sensation 3 (3.2%) 1 (1.0%) Abnormal sensation 2 (2.1%) 1 (1.0%) Eye discharge 3 (3.2%) 3 (3.1%) Several potential topical pharmacologic agents may stimulate secretion of aqueous tears, mucins, or both. Among such agents, diquafosol, a P2Y 2 receptor agonist, is a novel dinucleotide agent that has been reported to promote secretion of aqueous tears and mucins on the ocular surface eye drops have been favorably evaluated in terms of efficacy and safety in clinical trials in the United States. 23 This investigation is the first prospective, randomized, double-masked, placebo-controlled phase 2 trial in Japanese patients with dry eye, which looked into the efficacy and safety of 2 concentrations of diquafosol after ocular surface washout. The strengths of this study are the lack of statistically significant differences between the background characteristics of the 3 treatment groups with respect to age, gender, distribution of Sjögren s syndrome, and severity of dry eye disease in relation to the vital staining scores, BUT, and Schirmer s test values, as well as presence of a washout, which we believe allowed reliable comparisons among the 3 groups. This trial demonstrated subjective and objective improvements in tear function and ocular surface findings with both concentrations of diquafosol. Initial placebo-controlled trials from the United States confirmed the beneficial effects of diquafosol on ocular surface health in terms of reduction of keratoconjunctival vital staining scores. 23 Our study also shows significant diminution of FL and RB staining scores with both concentrations of diquafosol. Dose-response analysis using the MCM demonstrated marked significance in the linear dose response, with the 3% topical eye drops showing the greatest improvement in vital staining scores at 4 and 6 weeks. Keratoconjunctival staining is the hallmark of dry eye disease that may lead to ocular surface inflammation, infection, scarring, visual disturbance, and various other ocular symptoms. 27,28 The improvements in vital staining scores are consistent with the mechanism of action of diquafosol, which has been shown to stimulate P2Y 2 receptors in rabbit and human conjunctiva, regulating conjunctival aqueous and conjunctival and goblet cell mucin secretion. Of great interest was the improvement in RB staining scores because RB staining of the ocular surface has been linked to the absence of ocular surface mucins, leading to perturbation of the epithelial barrier and dye staining. The improvements observed in RB staining scores in this study provide indirect evidence of possibly increased mucin secretion and improvements in secreted mucin quality with diquafosol treatment. These possible mechanisms need to be investigated in future clinical trials through enzyme-linked immunosorbent assay for tear mucins or real-time polymerase chain reaction of conjunctival epithelial cells for MUC5AC mrna levels to confirm whether mucin secretion is actually increased. Although 3% diquafosol treatment resulted in the most marked improvement in BUTs compared with baseline and the change in BUTs values in the 3% diquafosol treatment group tended to be higher than the other 2 groups at 4 and 6 weeks, no statistically significant differences among all groups were observed at any follow-up time points in our study. Improvements in vital staining scores might have resulted from increased water secretion from the lacrimal glands or, more likely, from the conjunctival epithelial cells. Schirmer s tests, however, were conducted for inclusion eligibility examinations and were not performed thereafter throughout the study period. Phase 3 trials in Japan are also under way, looking into tear quantity alterations with diquafosol treatment. Dry eye sensation symptom scores also showed concurrent improvements with the vital staining scores after diquafosol treatment. The improvement in symptoms is an important finding because symptoms provide a more integrated view of the clinical condition over time and are responsible for the public health burden and the careseeking behavior of patients with dry eye and their desire for therapy. Clinical and extensive laboratory investigations showed both concentrations of diquafosol to be safe for the treatment of dry eye disease. Eye irritation was observed in 7.3% and 12.5% of the 1% and 3% diquafosol treatment groups, respectively. An effect on patient compliance was observed with 3.1% and 4.2% of the patients in the 1% and 3% diquafosol treatment groups, respectively, discontinuing the study because of adverse events. Eye irritation appeared immediately after administration, but the severity was mild and disappeared soon after. sodium itself is a substance that can cause mild eye irritation in patients with dry eye. All adverse reactions disappeared or diminished to a nonproblematic degree during the study period or after the completion of this study. Future clinical trials should test the efficacy of diquafosol in different types of dry eye disease, including short BUT-type dry eye or severe mucin deficiency dry eye states, such as Stevens Johnson syndrome, toxic epidermal necrosis, ocular cicatricial pemphigoid, or dry eye associated with graft-versus-host disease. Future studies should also test the efficiency of diquafosol in superiority inferiority protocols to clarify whether single or combined treatments with artificial tears, hyaluronic acid eye drops, 24,29,30 or other tear 1958

6 Matsumoto et al Treatment in Dry Eyes substitutes provide further improvements in ocular surface health, that is, in terms of improvements in ocular surface epithelial damage, tear film stability, or tear quantity scores. In conclusion, this study confirmed the efficacy and safety of diquafosol ophthalmic solution in a Japanese population. A 3% diquafosol solution was tested for the first time and proved to be at least equally and more effective in the treatment of dry eye in terms of reducing staining scores and symptomatology. A further goal of the ongoing phase 3 diquafosol dry eye treatment clinical trial in Japan will be to evaluate efficacy over a longer treatment span. References 1. Definition and Classification Subcommittee. The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf 2007;5: Mathers WD. 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Prevalence of dry eye disease among Japanese visual display terminal users. Ophthalmology 2008;115: Uchino M, Dogru M, Yagi Y, et al. The features of dry eye disease in a Japanese elderly population. Optom Vis Sci 2006;83: Lemp MA. Tear film: new concepts and implications for the management of the dry eye. Trans New Orleans Acad Ophthalmol 1987;35: Dilly PN. Structure and function of the tear film. Adv Exp Med Biol 1994;350: Danjo Y, Watanabe H, Tisdale AS, et al. Alteration of mucin in human conjunctival epithelia in dry eye. Invest Ophthalmol Vis Sci 1998;39: Management and Therapy Subcommittee. Management and therapy of dry eye disease: report of the Management and Therapy Subcommittee of the International Dry Eye Work- Shop (2007). Ocul Surf 2007;5: Vallejo CG, Lobaton CD, Quintanilla M. Dinucleosidasetetraphosphatase in rat liver and Artemia salina. Biochim Biophys Acta 1976;438: Hosoya KI, Ueda H, Kim KJ, Lee VH. Nucleotide stimulation of Cl(-) secretion in the pigmented rabbit conjunctiva. J Pharmacol Exp Ther 1999;291: Li Y, Kuang K, Yerxa B, et al. Rabbit conjunctival epithelium transports fluid, and P2Y2(2) receptor agonists stimulate Cl(-) and fluid secretion. Am J Physiol Cell Physiol 2001;281: C Jumblatt JE, Jumblatt MM. Regulation of ocular mucin secretion by P2Y2 nucleotide receptors in rabbit and human conjunctiva. Exp Eye Res 1998;67: Murakami T, Fujihara T, Horibe Y, Nakamura M. elicits increases in net Cl- transport through P2Y2 receptor stimulation in rabbit conjunctiva. Ophthalmic Res 2004;36: Fujihara T, Murakami T, Nagano T, et al. INS365 suppresses loss of corneal epithelial integrity by secretion of mucin-like glycoprotein in a rabbit short-term dry eye model. J Ocul Pharmacol Ther 2002;18: Fujihara T, Murakami T, Fujita H, et al. Improvement of corneal barrier function by the P2Y(2) agonist INS365 in a rat dry eye model. 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7 Footnotes and Financial Disclosures Ophthalmology Volume 119, Number 10, October 2012 Originally received: November 17, Final revision: March 28, Accepted: March 29, Available online: June 26, Manuscript no Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. 2 Department of Ophthalmology, Ehime University School of Medicine, Ehime, Japan. 3 Department of Ophthalmology, Kansai Rosai Hospital, Hyogo, Japan. Financial Disclosure(s): The author(s) have made the following disclosure(s): Yuichi Ohashi: consultant for Santen Pharmaceutical Co., Ltd. Hitoshi Watanabe: consultant for Santen Pharmaceutical Co., Ltd. Kazuo Tsubota: consultant for Santen Pharmaceutical Co., Ltd. Sponsored by Santen Pharmaceutical Co., Ltd., Osaka, Japan. Examination procedures were approved by the investigational review board. This work was presented at the 6th International Conference on the Tear Film and Ocular Surface, September 22 25, 2010, Florence, Italy. Correspondence: Yukihiro Matsumoto, MD, Department of Ophthalmology, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku, Tokyo , Japan. ymatsumoto8@aol.com. 1960