The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

Dry eye disease (DED) is a complex, multifactorial pathology characterized by corneal epithelium lesions and inflammation. The aim of the present study was to evaluate the efficacy of a cationic emulsion of cyclosporine A (CsA) in a mouse model that mimics severe dry eye. Eight to 12-week-old female C57BL/6N mice with tail patches of scopolamine were housed in controlled environment chambers to induce dry eye. At day three, following dry eye confirmation by corneal fluorescein staining (CFS, score 0-15) and phenol red thread (PRT) lacrimation test, the mice (n = 10/gp) were either treated 3 times a day in both eyes with drug-free cationic emulsion, a 0.1% CsA cationic emulsion, or 1% methylprednisolone (positive control), or non-treated. Aqueous tear production and CFS scores were evaluated at baseline and throughout the treatment period. The lacrimation test confirmed the scopolamine-induced decrease in aqueous production by the lacrimal gland. A reduction of 59% in induced-CFS was observed following topical treatment with 0.1% CsA. The beneficial effect of the cationic emulsion vehicle itself on keratitis was also clearly evidenced by its better performance over 1% methylprednisolone, -36%, vs. -28% on the CFS scores, respectively. This study indicates that the cationic emulsion of CsA (0.1%) was a very effective formulation for the management of corneal epithelium lesions in a severe DED mouse model. In addition, it performed better than a potent glucocorticosteroid (1% methylprednisolone). This cationic emulsion of CsA (0.1%), combining CsA and a tear film oriented therapy (TFOT), i.e. with vehicle properties that mechanically stabilize the tear film, represents a promising new treatment strategy for the management of the signs of dry eye.

Purpose: Artificial tears (ATs) are used routinely to alleviate the symptoms of mild to moderate dry eye. Preservative-free cationic emulsions (eg, Cationorm®) are an innovative approach for the management of signs and symptoms of dry eye. The aim of the present exploratory experiment was to evaluate the efficacy of this cetalkonium chloride (CKC)-containing cationic emulsion on debrided cornea and to characterize its effects on scraping-induced inflammation.Methods: Four ATs were assessed in a rat model of corneal scraping. The upper part of the corneal epithelium was scraped before a 5-day treatment, followed by clinical evaluations and fluorescein staining to evaluate cornea recovery. The anti-inflammatory efficacy of the ATs was assessed in vivo and in vitro.Results: In vivo confocal microscopy (IVCM) revealed a trend toward better corneal clinical signs (lower IVCM scores) for the animals treated with the unpreserved ATs. Benzalkonium chloride treatment decreased goblet cell count by 37.5%. While the soft-preserved Systane Balance® and Optive® and the preservative-free Vismed® had no effect on the goblet cell count, Cationorm increased this count by almost 40%. Interestingly, inflammatory cell infiltration in the stroma was at its lowest following treatment with the preservative-free Cationorm. Cationorm is also the only AT decreasing IL6- and IL8-stimulated secretion by 59% and 74%, respectively.Conclusion: By restoring an adequately hydrated ocular surface environment, the different ATs promote corneal epithelium healing. These data position Cationorm as a promising AT for the management of signs and symptoms of dry eye in patients with mild to moderate dry eye disease presenting chronic subclinical levels of ocular inflammation.

Purpose/Aim: Inflammation is recognized as playing an etiological role in dry eye disease. This study aimed to assess the efficacy of various topical cyclosporine A (CsA) formulations on cornea inflammatory markers in a mouse model of dry eye. Material and Methods: Six-to 7-week-old mice treated with scopolamine were housed in a controlled environment room to induce dry eye. Following dry eye confirmation by corneal fluorescein staining (CFS), the mice were treated three times a day with: 0.05%CsA (Restasis, Allergan), 0.1%CsA (Ikervis, Santen), 1%CsA oil solution, and 0.5% loteprednol etabonate (LE, Lotemax, Baush+Lomb), or left untreated. Aqueous tear production and CFS scores were assessed during the treatment period, and corneas were collected to measure the expression profile of a selection of inflammatory genes. Results: After 7 days of treatment, the CFS scores were reduced by 21%, 31%, and 44% with 0.05%CsA, 0.1%CsA, and 1%CsA eye drops, respectively. By contrast, 0.5% LE did not decrease corneal fluorescein staining at day 10. A statistically significant dose-dependent CFS reduction was observed only between the 0.05% and 1%CsA formulations. The gene expression profiles indicated that 12, 18, 17 genes were downregulated by 0.05%CsA, 0.1%CsA, 1%CsA, respectively. Among them, the genes significantly downregulated were: IL1A, IL1R1, and TLR4 with 0.05%CsA; H2-Eb1, IL1A, IL1B, IL1RN, IL6, TGFB2, TGFB3, TLR2, TLR3, and TLR4 with 0.1%CsA; IL1B, IL6, TGFB3, and TLR4 with 1%CsA. TGFB1 and TGFBR1 were the only genes upregulated in all groups, but only TGFB1 upregulation reached significance. IL6RA was significantly upregulated by 0.05%CsA. Conclusions: This study indicates that the three CsA formulations effectively modulated TLR4, TGFβ1, IL1, and IL6 pathways to reduce corneal epithelium lesions in a mouse model of severe dry eye. The study also suggests that the different anti-inflammatory eye drops modulated inflammatory genes in a slightly different manner. ARTICLE HISTORY

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Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the Novasorb® technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Cationorm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a predictive alternate toxicity test method.Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this article. Two to 3-month-old female New Zealand white rabbits, n = 6 per group, were treated with ATs (21 instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal microscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the ocular tolerability of the overdosed ATs.Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in 82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of the overdosed ATs was confirmed using Draize and McDonald–Shadduck's test scales.Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.

Purpose/Aim: Dry eye (DE) disease is a multifactorial disease in which uncontrolled inflammation can lead to corneal epithelium lesions and symptoms of discomfort. The aim of the present study was to evaluate the efficacy of two cyclosporine emulsions in a mouse model of DE with corneal epithelium lesions. Materials and Methods: Six-to 9-week-old female C57BL/6 N mice were housed in a controlledenvironment room to induce DE. Following DE development, mice were instilled with: QD 0.1%CsA cationic emulsion (CaEm), BID 0.05%CsA anionic emulsion (AEm), or left untreated. Aqueous tear production and corneal epithelium lesions were assessed throughout the experiment. At the end of the treatment period, left eyes were sampled, fixed, and stained for histology, while the cornea, conjunctiva, and lacrimal gland of right eyes were sampled for transcriptomic analysis. Results: Corneal lesion scores were reduced by 10.4%, 18.4%, and 10.9% at day 6, 10, and 14, respectively, with CaEm (QD), and by 2.6%, 3.0%, and 5.5% at day 6, 10, and 14, respectively, with AEm (BID). Histology demonstrated that 7 out of 10 DE mice presented moderate to severe ocular lesions, while only 2 and 5 out of 10 mice presented slight to moderate ocular lesions when treated with the CaEm (QD) and AEm (BID), respectively. The transcriptomic profile analysis suggests that a different set of inflammatory genes are modulated in the cornea, conjunctiva, and lacrimal gland upon DE development. In addition, the two emulsions distinctively modulate the gene expression profile. Conclusions: This study demonstrates that both emulsions were effective at reducing corneal lesions, with the CaEm (QD) being slightly better than the AEm (BID). Interestingly, this study suggests that ocular tissues may not respond similarly to a dry environment and that a different set of genes is modulated by the two formulations in the ocular tissues.

Purpose: Artificial tears (ATs) are used routinely to alleviate the symptoms of mild to moderate dry eye. Preservative-free cationic emulsions (eg, Cationorm Ò ) are an innovative approach for the management of signs and symptoms of dry eye. The aim of the present exploratory experiment was to evaluate the efficacy of this cetalkonium chloride (CKC)-containing cationic emulsion on debrided cornea and to characterize its effects on scraping-induced inflammation. Methods: Four ATs were assessed in a rat model of corneal scraping. The upper part of the corneal epithelium was scraped before a 5-day treatment, followed by clinical evaluations and fluorescein staining to evaluate cornea recovery. The anti-inflammatory efficacy of the ATs was assessed in vivo and in vitro. Results: In vivo confocal microscopy (IVCM) revealed a trend toward better corneal clinical signs (lower IVCM scores) for the animals treated with the unpreserved ATs. Benzalkonium chloride treatment decreased goblet cell count by 37.5%. While the soft-preserved Systane Balance Ò and Optive Ò and the preservative-free Vismed Ò had no effect on the goblet cell count, Cationorm increased this count by almost 40%. Interestingly, inflammatory cell infiltration in the stroma was at its lowest following treatment with the preservative-free Cationorm. Cationorm is also the only AT decreasing IL6-and IL8-stimulated secretion by 59% and 74%, respectively. Conclusion: By restoring an adequately hydrated ocular surface environment, the different ATs promote corneal epithelium healing. These data position Cationorm as a promising AT for the management of signs and symptoms of dry eye in patients with mild to moderate dry eye disease presenting chronic subclinical levels of ocular inflammation.