In this study, a sensitive quantitative method based on high performance liquid chromatography combined with high-resolution mass spectrometry, Q ExactiveTM-Orbitrap® was set up and applied for the determination of the immunosuppressor agents cyclosporine A and tacrolimus in novel ethanol-free ophthalmic formulations for the treatment of Vernal keratoconjunctivitis. Different storage parameters in terms of storage temperatures and practical usage conditions were investigated to assess the stability of all formulations during shelf life simulating the real conditions as well to confirm the feasibility of use of ethanol-free products. The methodology was linear (r2 = 0.995) over the concentration range 0–200 ng/mL, and its selectivity, precision, accuracy and recovery were all within the required limits. Under different conditions (storage period 0–90 days, 5–25 °C, unopened/usage simulated conditions), our results revealed that both active pharmaceutical ingredients (API) show satisfactory stability up to 30 days of storage/usage, with a significant and consistent concentration decline of cyclosporine A after this time point when its hydroalcoholic formulation was kept at 25 °C.

Background Children with vernal keratoconjunctivitis (VKC) present symptoms that are similar to other ocular allergies, but more pronounced, and are controlled using topical steroids. To avoid excessive and prolonged use of topical steroid eye drops, over the past 20 years galenic eye drops of cyclosporine with a concentration of 1% to 2% and tacrolimus with a concentration of 0.1% have been introduced as a treatment for the severe and unresponsive forms. The main symptoms of VKC occur most frequently during the spring and tend to get worse during the summer, meaning that affected children tend to avoid exposure to sunlight. The aim of this study was to assess the most common cell types present in the conjunctiva of children with VKC, how ocular treatment can influence them, and whether affected children express a typical conjunctival pattern, which could be useful as a pathognomonic pattern of VKC, allowing us to study this rare eye disease. Method This was a cohort study of 56 children, of whom 17 were not receiving any treatment at the time of testing, 14 were using steroid eye drops or had taken them in the previous 10 days, and 25 were treated with cyclosporine eye drops or tacrolimus eye drops 0.1%. Result Children in group 1 (no topical therapy) express more epithelial cells, neutrophils, mast cells, eosinophils, and lymphocytes than the other two groups. Conclusion Given the ease of performance, when conducting further longitudinal studies, the conjunctival cytology examination could be used, on the one hand, to diagnose VKC, especially when the clinical diagnosis is uncertain, and, on the other, to follow disease evolution and monitor the response to topical treatment.

The aim of this study was to evaluate whether the control of ocular symptoms with cyclosporine or with tacrolimus in eye drops allows to improve sun exposure and therefore serum level of vitamin D (VD; 25OHD), in the more severe forms of vernal keratoconjunctivitis (VKC). Out of 242 children followed for active VKC, 94 were treated with 1% cyclosporine or 0.1% tacrolimus eye drops, while the other 148 with mild VKC did not need to be treated with immunomodulators. VD serum levels were measured in spring and autumn in 71 children. In total, 60 of them were treated with cyclosporine eye drops (first group) and 11 (not responding to cyclosporine therapy previously) with 0.1% tacrolimus eye drops (second group) between March and November 2016. Pre-treatment median values of VD were 23.7 ng/mL in the first group and 23.8 in the second group, and post-treatment values increased up to 32.8 and 32.9 ng/mL, respectively. Before treatment, 33% presented a deficiency (25OHD < 20 ng/mL), and at the end of summer, only 4% were deficient. The overweight children had lower improvement in VD serum levels than children with a body mass index (BMI) lower than 85th percentile. Children in therapy with cyclosporine, but requiring the administration of local steroid therapy during the summer for control of the symptoms, showed a greater improvement in 25OHD serum levels in ng/mL (23–37 ng/mL) than children who did not require steroid therapy (24–35 ng/mL). Furthermore, there was a significant difference in change of 25OHD in children presenting limbal VKC (21–41 ng/mL) versus tarsal VKC (24–35 ng/mL) ( P = 0.04). Our study suggests that ocular treatment carried out with immunomodulator eye drops could allow for an improvement in 25OHD serum levels. In children with active VKC and at risk of 25OHD deficiency, likely due to avoidance of sun exposure, the role of other risk factors (BMI, phototype and treatment) on 25OHD serum levels should be considered.

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