A synthetic metalloporphyrin SOD mimetic protects corneal epithelial cells from oxidative stress-induced damage in vitro and in vivo

Summary: Keratoconjunctivitis sicca (KCS) is a multifactorial disease of the lacrimal glands and ocular surface1. Pathological findings in patients with KCS includes a reduction in goblet cells and the presence of inflammatory cell infiltrates in the lacrimal glands2. KCS usually involves tear hyperosmolarity and ocular surface inflammation. Increased tissue levels of reactive oxygen species (ROS) and the ensuing oxidative stress are common hallmarks KCS and thought to contribute to the associated ocular surface damage. Superoxide dismutases (SODs) are enzymes which are part of antioxidant system that protects against ROS through dismutation of superoxide to produce hydrogen peroxide2,3. The aim of this study was to determine the efficacy of Mn-TM-2-PyP (metalloporphyrin SOD mimetic) against oxidative-stress induced damage in corneal epithelial cells in in vitro, and in an in vivo controlled desiccating environment model.

Introduction: Keratoconjunctivitis sicca (KCS) is a multifactorial disease of the lacrimal glands and ocular surface¹. Pathological findings in patients with KCS includes a reduction in goblet cells and the presence of inflammatory cell infiltrates in the lacrimal glands². KCS usually involves tear hyperosmolarity and ocular surface inflammation. Increased tissue levels of reactive oxygen species (ROS) and the ensuing oxidative stress are common hallmarks KCS and thought to contribute to the associated ocular surface damage. Superoxide dismutases (SODs) are enzymes which are part of antioxidant system that protects against ROS through dismutation of superoxide to produce hydrogen peroxide^2,3. The aim of this study was to determine the efficacy of Mn-TM-2-PyP (metalloporphyrin SOD mimetic) against oxidative-stress induced damage in corneal epithelial cells in in vitro, and in a controlled desiccating environment in vivo.

Materials and Methods

In vitro

Human corneal epithelial cells (HCE-T, RIKEN, Japan)⁴ were seeded in 96-wellplates grown in DMEM/F12 (1:1) supplemented with 5% fetal bovine serum, 100U/ml penicillin-100 μg/ml streptomycin, 5 μg/ml insulin, 10 ng/ml human EGF and 0.5% dimethyl sulfoxide in a humidified atmosphere of 5% CO₂ at 37°C for 72 hr. Oxidative stress was chemically induced by tert-butylhydroperoxide (tBHP) (1 μM-30 mM) for 4 hr. Cells were pre-treated overnight with either Mn-TM-2-PyP (0.0001-0.5% for cytotoxicity analysis and 0.001-0.05% (w/v) for cytoprotection assays) or control (growth media). Hyperosmolar conditions were induced by supplementation of the growth media with NaCl (5-150 mM).

In vivo

C57BL/6J male mice, 8-10 weeks of age were used. KCS was induced via a desiccating stress/scopolamine model. Mn-TM-2-PyP (0.1% w/v in saline) was administered topically in both eyes (10 μl), 3 times a day, for 2 weeks. Preclinical efficacy of Mn-TM-2-PyP was compared against saline and twice daily administration of 0.05% cyclosporine ophthalmic emulsion. After 2 weeks, acute effect on tear volume was determined using a sterile phenol red-soaked cotton thread. Corneal fluorescein staining was scored to quantify the ocular surface damage. Histological examination included grading of immune cell infiltration into the lacrimal gland and quantification of goblet cells in the inferior conjunctiva.

Results in vitro

Figure 1: Mn-TM-2-PyP protects HCE-T cells from oxidative insult in vitro. A Mn-TM-2-PyP exerted potent dose-dependent cytoprotection against tBHP. B Mn-TM-2-PyP (0.05%) did not protect HCE-T cells from hyperosmolar insult.

Apparent permeability coefficient for Mn-TM-PyP was 1.1 ± 0.1 × 10-6 cm/s and the mass balance was 62 ± 1%, suggesting low permeability but significant cellular uptake of Mn-TM-2-PyP.

Results in vivo

Dosing with saline, Mn-TM-2-PyP or cyclosporine did not result in a statistically significant increase in tear volumes at the end of the two-week study (Table 1). However, Mn-TM-2-PyP reduced the median corneal fluorescein score (median score: 2) compared with untreated (median score: 3) and saline-treated (median score: 3) (Fig. 2). Mn-TM-2-PyP reduced lacrimal gland pathology significantly by one score (Fig. 3 A,B). There was no statistically significant difference in the number of goblet cells in saline, Mn-TM-2-PyP or cyclosporine treated eyes (Fig. 3 C,D).

Parameter	Comparison	No. of eyes (n)	Test	p-value
Tear volume	Mn-TM-2-PyP vs. Saline vs. Cyclosporine	9-10	One way ANOVA	0.16
Corneal fluoresceinstaining	Mn-TM-2-PyP vs. Untreated	29 vs. 27	Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test	<0.05
Mn-TM-2-PyP vs. Saline	29 vs. 17	Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test	<0.01
Lacrimal glandpathology	Mn-TM-2-PyP vs. Saline	15 vs. 8	Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test	<0.05
Cyclosporine vs. Saline	8 vs. 8	Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test	0.08
Mn-TM-2-PyP vs. Cyclosporine	15 vs. 8	Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test	0.99
Loss conjunctivalgoblet cells	Mn-TM-2-PyP vs. Cyclosporine	13 vs. 8	One way ANOVA	0.99

Figure 2: Topical administration of Mn-TM-2-PyP reduced corneal fluorescein staining. A Representative images of corneal fluorescein staining for each treatment group. B Corneal Fluorescein Scores. * P < 0.05 compared with untreated; ^## P < 0.01 compared with saline treated.

Figure 3: A, B Mn-TM-2-PyP reduced immune cell infiltration in the lacrimal gland. C, D Mn-TM-2-PyP did not prevent loss of mucin-producing conjunctival goblet cells.

Conclusions

Topical administration of the superoxide dismutase mimetic, Mn-TM-2-PyP, showed efficacy against corneal surface damage, infiltration of immune cells into the lacrimal gland and degeneration of parenchymal tissue in a preclinical model for KCS.