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Created by Simon Kaja on  March 27, 2020  in category: Scientific publications

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 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 the SiccaSystemTM in vivo model.

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

in vitro, cytotoprotection, hyperosmolar insults

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).

Table 1: Statistical findings of in vivo parameters shows efficacy of Mn-TM-2-PyP against KCS.



No. of eyes (n)



Tear volume

Mn-TM-2-PyP vs. Saline vs.



One way ANOVA


Corneal fluorescein


Mn-TM-2-PyP vs. Untreated

29 vs. 27

Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test


Mn-TM-2-PyP vs. Saline

29 vs. 17

Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test


Lacrimal gland


Mn-TM-2-PyP vs. Saline

15 vs. 8

Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test


Cyclosporine vs. Saline

8 vs. 8

Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test


Mn-TM-2-PyP vs. Cyclosporine

15 vs. 8

Kruskal-Wallis ANOVA; Dunn’s multiple comparisons test


Loss conjunctival

goblet cells

Mn-TM-2-PyP vs. Cyclosporine

13 vs. 8

One way ANOVA


corneal fluorescein staining, Mn-TM-2-PyP

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.

histology, immune cell infiltration, lacrimal glands, conjunctival goblet cells

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.


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.


1. Craig JP, et al. TFOS DEWS II Definition and Classification Report. Ocul Surf 2017; 15:276-283.

2. Wakamatsu T H, Dogru M, Kazuo M. Tearful relations: oxidative stress, inflammation and eye diseases. Arq Bras Oftalmol. 2008; 71 (6 Supl): 72-79.

3. Nita M, Grzybowski A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxidative Medicine Cellular Longevity. 2016, 3164734. Doi 10.1155/2016/3164734.

4. Araki-Sasaki K, Ohashi Y, Sasabe T et al. An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 1995; 36:614-621.