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Topical Tacrolimus 0.03% as Sole Therapy in Vernal KeratoconjunctivitisA Randomized Double-Masked StudyGuilherme Gubert Mller, M.D., Newton Kara Jos, Ph.D., Rosane Silvestre de Castro, Ph.D.Eye Contact Lens.2014;40(2):79-83.Abstract and IntroductionAbstractObjective: This study sought to evaluate the efficacy of the isolated use of tacrolimus compared with the combined use of tacrolimus and olopatadine for the treatment of severe vernal keratoconjunctivitis (VKC).Methods: Twenty-one patients with severe VKC were randomized into two groups: one treated with 0.03% tacrolimus ointment combined with 1% olopatadine ophthalmic solution and the other with 0.03% tacrolimus ointment combined with placebo eye drops. The clinical signs and symptoms were graded from 0 to 3, and the efficacy of treatment was determined by the difference between the score at the beginning of treatment and after 30 days. The clinical impression of improvement as perceived by the evaluator and the self-assessment provided by the patient were scored at day 30 of treatment and compared between the groups.Results: The scores for symptoms decreased between the assessments in both groups (1.73.9 in the experimental group; 0.61.6 in the control group), with no significant difference between groups (P=0.205). The scores for clinical signs decreased between the assessments in the experimental group (1.12.7) and increased in the control group (0.30.9) but with no significant differences (P=0.205). There was no significant difference between the groups regarding the self-assessment (P=0.659) and the clinical impression of the evaluator (P=0.387).Conclusions: The isolated use of tacrolimus and the combined use of tacrolimus and olopatadine seems to have the same efficacy, although controlled studies with larger samples are required to confirm this hypothesis.IntroductionVernal keratoconjunctivitis (VKC) is a chronic condition that affects children and young adults. This condition appears before 10 years of age and lasts for 2 to 10 years, with spontaneous recovery during puberty. The diagnosis is essentially clinical, with intense itching and photophobia, papillary reaction of the upper tarsal or limbal conjunctiva, formation of giant papillae, epithelial keratitis, and shield ulcer.[13] Vernal keratoconjunctivitis differs from seasonal allergic conjunctivitis and perennial allergic conjunctivitis because it is a condition mediated by Th2 lymphocytes. However, the precise roles of mast cells, eosinophils, fibroblasts, and their cytokines in the inflammatory process and the remodeling of conjunctival tissue remain poorly established.[46] The topical use of antihistamines, mast cell stabilizers (MCSs), and, more recently, drugs with both effects, termed dual-action drugs (DADs), represent the first-line treatment for VKC. In the more severe forms, corticosteroids are used for a short period to induce the remission of the allergic crisis.[79] However, there are cases where it is not possible to withdraw the corticosteroid without any clinical worsening, thus leaving patients susceptible to risks caused by the prolonged use of these drugs, such as cataract, glaucoma, and corneal complications.[10,11] For the past two decades, immunomodulators have been used as substitutes for corticosteroids in allergic crisis control and the maintenance of asymptomatic VKC patients because of the need for alternative therapies with potent anti-inflammatory action and fewer side effects.[12] Tacrolimus, a macrolide derived from the bacterium Streptomyces tsukubaensis, is a potent immunomodulator capable of decreasing the production of inflammatory mediators by T lymphocytes through the inhibition of calcineurin, an intracytoplasmic protein essential for interleukin (IL)-2 and IL-4 transcription.[13,14] Tacrolimus was first used as an immunosuppressant in liver transplants but was subsequently introduced for other solid-organ transplants.[15,16] In addition, this drug has been successfully used in the treatment of atopic dermatitis, vitiligo, and other skin disorders for more than 10 years.[17] There are numerous reports of the successful use of tacrolimus for the treatment of autoimmune diseases of the ocular surface, such as dry eye, scleritis, Mooren ulcer, cicatricial conjunctivitis, atopic, and VKC.[1822] Recent clinical trials have also shown that, like corticosteroids, tacrolimus and other immunosuppressive drugs have similar efficacy in allergic crisis control and maintenance therapy for VKC but with a low incidence of side effects.[2325] In addition to inhibiting the activation of CD4 lymphocytes, tacrolimus has also been reported to inhibit the release of histamine and its action. This drug has also been shown to inhibit prostaglandin synthesis in mast cells and basophils,[26,27] similar to the effect of DAD; therefore, the need for the concomitant use of these two drugs should be evaluated.A prospective randomized double-masked trial was developed to evaluate the efficacy of the isolated use of tacrolimus when compared with the combined use of tacrolimus and olopatadine for the treatment of severe VKC.Materials and MethodsThe present study was conducted in a single center after selecting VKC patients refractory to conventional therapy in which topical tacrolimus was introduced to replace corticosteroids. Refractory, in this context, meant that the clinical condition was maintained or worsened during the use of topical corticosteroids or that there was a relapse after withdrawal.Individuals concomitantly using topical corticosteroids, those who underwent subconjunctival corticosteroid injection in the previous 4 months, or who refused to sign the informed consent form were excluded from the study. Patients with a history of ocular herpes were excluded because of reports of reactivation of the infection by the tacrolimus.[23] The use of tacrolimus before recruitment ranged between 1 and 6 months.Individuals with symptoms of recurrent or persistent conjunctivitis, pruritus, mucus hypersecretion, photophobia and watering combined with the presence of a papillary reaction in the upper tarsal conjunctiva with at least one giant papilla, characterizing the tarsal form of VKC, or the presence of limbal papillae with at least one Horner Tantras dots, characterizing the limbal form of VKC, in both eyes were considered VKC patients. All patients enrolled to the study presented the tarsal form of VKC.For the sample size calculation, a symptom reduction rate of 30% for tacrolimus treatment was assumed based on previous studies.[23] Therefore, a sample of 46 patients (23 per group) was required to have an 80% probability of detecting a difference as small as 50% in the experimental group. Twenty-five subjects were selected between August and November 2012. Because additional participants were not included in the subsequent months, it was decided to continue the study with the sample already established.In both groups, 0.03% tacrolimus ointment (Protopic; Astellas Pharma Tech Co, Toyama, Japan) was applied directly to the conjunctival fornix every 12 hours. This dermatological formulation was chosen because of reports in the literature of good tolerance with a low toxic effect on the ocular surface.[2832] In the experimental group, in addition to tacrolimus ointment, lubricant eye drops (Systane; Alcon, Fort Worth, TX) were prescribed for use every 12 hours as placebo. In the control group, 0.1% olopatadine ophthalmic solution (Patanol; Alcon) was prescribed. Both eye drops had similar flasks, with no identification, and were given to the patients together with the ointment. In addition to the medication given, only the use of lubricant eye drops was allowed in both groups. If other topical medications were being used, the patients were asked to provide this information to the team, and, if necessary, these subjects were removed from the study.For double masking of the study, the eye drop flasks were numbered and contained no identification of the drug. The content of the flasks was only revealed after the end of the data collection period.The randomization was performed using a block system. Envelopes were prepared, with each one containing four flasks of eye drops (two experimental and two control). To open a new envelope, all flasks from the previously opened envelope had to be empty.After the initial approach and after the individuals were assigned to the groups, a protocol for the objective assessment of signs and symptoms, which was adapted from clinical trials with similar methods and objectives,[23,3335] was applied. Symptoms of itching, tearing, foreign body sensation, discharge, and photophobia were assessed, in addition to clinical signs of conjunctival hyperemia, tarsal papillary reaction, epithelial keratitis, limbal neovascularization, and conjunctival fibrosis. Each symptom and sign was graded on a scale from 0 to 3 ( and ). The symptoms and signs were assessed before the introduction of therapy and after 30 days, when the follow-up was ended. The variables were analyzed individually and grouped by summing the scores for comparison between the assessments and groups.Table 1. Scores of VKC SymptomsItching0: no desire to scratch

1+: occasional desire to scratch

2+: frequent desire to scratch

3+: continuous desire to scratch

Tearing0: normal production of tears

1+: occasional tearing

2+: Intermittent tearing

3+: constant tearing

Foreign body sensation0: absent

1+: discrete, occasional foreign body sensation

2+: mild, frequent foreign body sensation

3+ severe, continuous foreign body sensation

Photophobia0: no photophobia

1+: mild, squints in bright light

2+: moderate, needs sunglasses

3+: severe, cannot withstand bright light even when wearing sunglasses

Discharge0: no discharge

1+: little, in the fornix

2+: moderate, in the fornix, with crusting on eyelashes

3+: abundant, wakes up with eyes stuck shut or washes them several times a day

Table 2. Scores for Clinical SignsConjunctival hyperemia0: absent

1+: dilation of some blood vessels

2+: dilation of several blood vessels

3+: generalized dilation of blood vessels

Tarsal papillary reaction0: no papillae

1+: papillary reaction without giant papillae

2+: some giant papillae

3+: giant papillae all over the tarsal conjunctiva

Punctate keratopathy0: intact epithelium

1+: punctate in 1/3 of the cornea

2+: punctate in 2/3 of the cornea

3+: diffuse punctate

Limbal neovascularization0: no neovessels

1+: neovessels in 1 quadrant

2+: neovessels in 2 quadrants

3+: neovessels in 3 or 4 quadrants

Conjunctival fibrosis0: no scars

1+: subepithelial fibrosis

2+: fornix shortening

3+: symblepharon

Clinical impression of the progress of each case and the self-assessment provided by the patient were assessed using an objective 0 to 5 scale (0=cure, 1=significant improvement, 2=mild improvement, 3=unchanged condition, 4=mild worsening, 5=significant worsening). For the significance analysis and the comparison between the groups, scores 2 (clinical improvement) and 3 (worsening or maintenance of the condition) were grouped. Because of the bilateral nature of VKC, the whole evaluation was based on the examination of both eyes, always considering the eye with findings of greater intensity.To assess the safety and side effects of the treatment, intraocular pressure, lens opacification, secondary infections, or other possible complications were assessed. The incidence of cataract was evaluated during biomicroscopical evaluation by LOCS II cataract grading system. Infections and others complications were also assessed during biomicroscopy.The Student's t test for independent samples and the nonparametric MannWhitney test were used to compare the variation of the results between the two groups. To compare the results for the two assessment times, within each group, the Student t test for paired samples and the nonparametric Wilcoxon test were used. When comparing the 2 groups regarding the qualitative variables, Fisher exact test and the chi-square test were used. P-values less than 0.05 were considered significant.The present study was conducted in accordance with the Declaration of Helsinki and was initiated after approval by the Research Ethics Committee of the School of Medical Sciences of the University of Campinas (Faculdade de Cincias Mdicas da Universidade de CampinasUnicamp) was granted.ResultsFrom the 25 subjects recruited, two refused to participate after reading the informed consent form. The others were assigned to the study groups, including 12 to the experimental group and 11 to the control group. One patient was excluded from each group during the study because of the introduction of a topical corticosteroid. The remaining 21 subjects completed the treatment and had their data analyzed (Fig. 1). From these 21 patients, 7 were female and 14 male, aged between 5 and 23 years. Regarding gender and age, there was no significant difference between the groups (). Regarding the initial score, there was also no significant difference between the groups (P=0.673).Table 3. Homogeneity of the Treatment GroupsCharacteristicExperimentalControlTotalP

Gender0.537

Male8614

Female347

Age (SD)10.8 (5.2)10 (2.8)10.4 (4.1)0.667

Figure 1.Participant flowchart.There was no significant difference when comparing the variation in the score for symptoms between the groups at the end of 30 days (P=0.205). There was an improvement in both groups, with a mean decrease of 1.7 points in the sum and a standard deviation of 3.9 between assessments in the experimental group and 0.6 points with a standard deviation of 1.6 in the control group. In the individual assessment of each symptom, there were no significant variations between the assessments or the groups (Fig. 2).

Figure 2.Distribution of the scores for ocular symptoms.There was no significant difference when comparing the variation in the score for clinical signs between groups at the end of 30 days (P=0.205). Improvement was observed in the experimental group, with a mean decrease of 1.1 points in the sum and a standard deviation of 2.7 between the assessments, whereas in the control group, there was a worsening of the signs, with an average increase of 0.3 points and standard deviation of 0.9, although both were not significant. In the individual assessment of each clinical sign, there was a significant worsening only in conjunctival hyperemia in the control group when compared with the experimental group (P=0.035) (Fig. 3).

Figure 3.Distribution of the scores for ocular clinical signs.There was no significant difference in the score for the clinical impression between the groups (P=0.387). The condition was worse or unchanged in 7 patients (70%) and improved in 3 patients (30%) from the control group. In the experimental group, the impression was improved in 6 patients (54.6%) and worse or unchanged in 5 patients (45.5%).In the self-assessment, there was no significant difference between the groups (P=0.659). Three patients (30%) from the control group reported worsening or maintenance of the condition, and 7 patients (70%) reported improvement. In the experimental group, 5 patients (45.5%) reported a worse or unchanged clinical condition, whereas 6 patients (54.6%) reported improvement in their condition ().Table 4. Relationship Between the Self-assessment and Clinical ImpressionClinical Impression

Significant WorseningMild WorseningUnchanged ConditionMild ImprovementSignificant ImprovementCureTotal

Self-assessmentSignificant worsening11

Mild worsening112

Unchanged condition55

Mild improvement4318

Significant improvement134

Cure11

Total111045021

The main complaint was associated with burning during the application of tacrolimus, which was reported by 81% of the patients. During the study period, there was no significant change regarding intraocular pressure, lens opacification, secondary infections, or other factors.DiscussionVernal keratoconjunctivitis is known to be one of the most severe forms of ocular allergy, with the potential to cause corneal damage and permanent visual loss. Sacchetti et al.[36] observed that VKC patients with corneal involvement or more than one recurrence per year have an increased risk of permanent visual loss. In another study, Bonini et al.[10] observed that there was a good ocular prognosis with permanent visual loss in only 6% of the patients treated with antihistamines, MCS, and short periods of corticosteroids; however, in 52% of the patients, the symptoms became worse or persisted after treatment.Sacchetti et al.[36] also suggested grading VKC based on larger clinical signs (presence of ocular symptoms, punctate keratitis, shield ulcer, and photophobia) for selecting the therapy. However, these authors did not determine which approach should be used in patients who do not show improvement even when using strong topical corticosteroids for a short period. Moreover, tacrolimus has been shown to be effective when DAD combined with corticosteroids for a short period is not enough to control the disease.Several studies have considered topical tacrolimus an effective and safe alternative for allergic crisis control and the maintenance of VKC symptoms.[24,28,32,37,38] In a randomized clinical trial, Ohashi et al.[23] obtained significant clinical improvement in the group that used tacrolimus in comparison with the placebo group. Labcharoenwongs et al.[25] compared 0.1% tacrolimus with 2% cyclosporine and reported clinical improvement in both groups, with no significant difference between the two drugs. When comparing tacrolimus to corticosteroids, Shoji et al.[39] found similar efficacy in suppressing subconjunctival eosinophilic and lymphocytic infiltration in an animal model.In the same assay, Ohashi et al.[23] delivered a dosage-ranging study with tacrolimus ophthalmic suspension 0.01%, 0.03%, and 0.1%. The 0.1% concentration showed stronger improvement with safety similar to the others. However, Moscovici et al.[18] showed that absorption of tacrolimus 0.03% eye drops were well below that at which adverse effects were reported when administered systemically. As in our experience, the 0.03% ointment showed great clinical improvement with minimum discomfort.The lack of a commercial tacrolimus ocular presentation (drops or ointment) is still a limiting factor. The development of such formulations will be of great value in the treatment of allergy and others inflammatory ocular surface diseases.The use of antihistamines, MCS, and DAD was not controlled in any of the aforementioned studies. Apparently, because tacrolimus is able to control the signs and symptoms of VKC, the need for the concomitant use of these drugs can be questioned. However, the small variation observed in the score for signs and symptoms in both groups from the present study supports the hypothesis that the isolated use of tacrolimus is sufficient. Moreover, the unchanged condition between the assessments in most cases, both in the clinical impression and in the self-assessment, points to the same conclusion. Additionally, because these are drugs with preservative additives (e.g., benzalkonium chloride) that need to be used for a prolonged period, there may be secondary damages to the ocular surface.In addition to burning sensation, there was no other adverse effect observed. Also, no great complications have been described in literature about ocular use of tacrolimus (ointment or drops). Only one case of herpes keratitis and other with throat irritation were described in one study.[23] One individual was excluded from each group because of the need for the introduction of corticosteroids. This result indicates that, even with good efficacy, tacrolimus alone or combined with olopatadine was not sufficient to control the disease in these cases. Moreover, the significant worsening of the conjunctival hyperemia in the control group compared with the experimental group may be because of the use of olopatadine, although the sample size is a limiting factor for this conclusion.In the present study, the isolated use of tacrolimus and the combined use of tacrolimus and olopatadine showed similar efficacy. Controlled studies with larger samples are needed to confirm this hypothesis; we suggest the individualized use of DAD on a case-by-case basis.References1. Bonini S, Coassin M, Aronni S, et al. Vernal keratoconjunctivitis. Eye(Lond) 2004;18:345351.2. Kumar S. Vernal keratoconjunctivitis: A major review. Acta Ophthalmol 2009;87:133147.3. Friedlaender MH. Conjunctivitis of allergic origin: Clinical presentation and differential diagnosis. Surv Ophthalmol 1993;38(Suppl):105114.4. 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Double-masked, randomized, parallel-group study comparing olopatadine 0.1% ophthalmic solution with cromolyn sodium 2% and levocabastine 0.05% ophthalmic preparations in children with seasonal allergic conjunctivitis. Curr Ther Res Clin E 2004;65:186199.34. D'Arienzo PA, Leonardi A, Bensch G. Randomized, double-masked, placebocontrolled comparison of the efficacy of emedastine difumarate 0.05% ophthalmic solution and ketotifen fumarate 0.025% ophthalmic solution in the human conjunctival allergen challenge model. Clin Ther 2002;24:409416.35. Spangler DL, Abelson MB, Ober A, et al. Randomized, double-masked comparison of olopatadine ophthalmic solution, mometasone furoate monohydrate nasal spray, and fexofenadine hydrochloride tablets using the conjunctival and nasal allergen challenge models. Clin Ther 2003;25:22452267.36. Sacchetti M, Lambiase A, Mantelli F, et al. Tailored approach to the treatment of vernal keratoconjunctivitis. Ophthalmology 2010;117:12941299.37. Vichyanond P, Kosrirukvongs P. Use of cyclosporine A and tacrolimus in treatment of vernal keratoconjunctivitis. Curr Allergy Asthma Rep 2013;13:308314.38. Vichyanond P, Tantimongkolsuk C, Dumrongkigchaiporn P, et al. Vernal keratoconjunctivitis: Result of a novel therapy with 0.1% topical ophthalmic FK-506 ointment. J Allergy Clin Immunol 2004;113:355358.39. Shoji J, Sakimoto T, Muromoto K, et al. Comparison of topical dexamethasone and topical FK506 treatment for the experimental allergic conjunctivitis model in BALB/c mice. Jpn J Ophthalmol 2005;49:205210.The authors have no funding or conflicts of interest to disclose.Eye Contact Lens.2014;40(2):79-83.2014Lippincott Williams & Wilkins