Manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin, a superoxide dismutase mimetic, reduces disease severity in in vitro and in vivo models for dry-eye disease

Abstract

Purpose:

To determine the efficacy of the superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro in human corneal epithelial (HCE-T) cells and in vivo in a preclinical mouse model for dry-eye disease (DED).

Methods:

In vitro, HCE-T cultures were exposed either to tertbutylhydroperoxide (tBHP) to generate oxidative stress or to hyperosmolar conditions modeling cellular stress during DED. Cells were pre-treated with Mn-TM-2-PyP or vehicle. Mn-TM-2-PyP permeability across stratified HCE-T cells was assayed, In vivo, Mn-TM-2-PyP (0.1% w/v in saline) was delivered topically as eye drops in a desiccating stress / scopolamine model for DED. Preclinical efficacy was compared to untreated, vehicle- and ophthalmic cyclosporine emulsion-treated mice.

Results:

Mn-TM-2-PYP protected HCE-T cells in a dose-dependent manner against tBHP-induced oxidative stress as determined by calculating the IC₅₀ for tBHP in the resazurin, MTT and lactate dehydrogenase release cell viability assays. Mn-TM-2-PyP did not protect HCE-T cells from hyperosmolar insult. Its permeability coefficient across a barrier of HCE-T cells was 1.1 + 0.05 × 10⁶ cm/s and the mass balance was 62 ± 0.6%. In vivo, topical dosing with Mn-TM-2-PyP resulted in a statistically significant reduction of corneal fluorescein staining, similar to ophthalmic cyclosporine emulsion. Furthermore, Mn-TM-2-PyP significantly reduced leukocyte infiltration into lacrimal glands and prevented degeneration of parenchymal tissue. No protective effect against loss of conjunctival goblet cells was observed. Notably, Mn-TM-2-PyP did not produce ocular toxicity when administered topically.

Discussion:

Our data suggest that Mn-TM-2-PyP, a prototypic synthetic metalloporphyrin compound with potent catalytic antioxidant activity, can improve signs of DED in vivo by reducing oxidative stress in corneal epithelial cells.

1. Introduction

Dry-eye disease (DED) represents a common ophthalmic condition worldwide with prevalence estimates ranging from 5 – 50% [1]. DED is considered a multifactorial disease characterized by tear fluid dyshomeostasis and ocular surface inflammation [2], manifesting as discomfort and visual disturbance. DED is a chronic disease that creates a significant socioeconomic burden as a result of health care costs and workplace productivity in addition to lowering quality of life of affected individuals [1].

Inflammation is critically implicated in the pathogenesis of DED and contributes to the progression of signs and symptoms associated with DED [3]. Two prescription drugs have been approved in the United States: 0.05% cyclosporine ophthalmic emulsion (formulated as Restasis®; “indicated to increase tear production in patients whose tear production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca” [4, 5]) and 5% lifitegrast ophthalmic solution (formulated as Xiidra®; indicated “for the treatment of the signs and symptoms of dry eye disease” [6, 7]). Both drugs target the inflammatory component of the disease, but are associated with a significant incidence of adverse effects in up to 25% of patients [8, 9], highlighting the need for novel safe and well-tolerated therapeutic approaches.

Clinical and translational studies have proposed that oxidative stress in ocular tissues of the anterior segment is a major contributor to the exacerbation of DED symptoms. For example, levels of oxidative stress were significantly higher in the conjunctival epithelium of DED patients compared with controls [10], and late lipid peroxidation markers in the tear film correlated with disease severity [11]. Furthermore, inducing mitochondrial oxidative stress in mice resulted in DED-like pathology [12]. Lastly, in vitro studies support the notion that hyperosmolar conditions can generate oxidative stress in corneal epithelial cells, which may exacerbate ocular surface damage [13]. However, despite the apparent role of oxidative stress in DED, only few studies have tested the efficacy of antioxidants in preclinical models for non-autoimmune DED [14–17]. Herein, we tested the efficacy of manganese(III) tetrakis (1-methyl-4-pyridyl)porphyrin (Mn-TM-2-PyP) in the desiccating stress/ scopolamine murine model for DED.

Manganese-porphyrins belong to the metalloporphyrin group and possess broad antioxidant specificity, which includes scavenging O₂·–, H2O2, ONOO-, NO·, and lipid peroxyl radicals. They are well characterized and proposed to offer protection in a variety of oxidative stress injuries such as stroke, diabetes, radiation injury and ischemia [18–23]. Furthermore, a detailed non-clinical safety assessment of the structurally related manganese (III) meso- tetrakis(N-ethylpyridinium-2-yl)porphyrin in mice and monkeys provided a favorable safety profile following intravenous injection that was not associated with any specific target organ toxicity [24]. Given their ready solubility in aqueous buffers [24, 25], manganese porphyrins are well-suited for topical ocular formulations.

2.1. Tissue culture

Human corneal epithelial (HCE-T) cells [26] were obtained under Material Transfer Agreement from RIKEN Research Institute (Tokyo, Japan) and cultured according to the provider’s instructions as described by us previously [27]. Cells were maintained in standard tissue culture flasks (Techno Plastic Products, MidSci, St. Louis, MO) in DMEM:F12 media (Thermo Fisher Scientific, Waltham, MA) supplemented with 5 μg/ml insulin (Millipore Sigma, St. Louis, MO), 100 U/ml penicillin - 100 μg/ml streptomycin, 10 ng/ml human recombinant epidermal growth factor, 10 ng/ml human epithelial growth factor (all from Thermo Fisher Scientific), 5% fetal bovine serum (Gemini Bio Products, West Sacramento, CA) and 0.5% dimethylsulfoxide (Millipore Sigma).

2.2. Induction of oxidative stress and hyperosmolar conditions

HCE-T were seeded in 96-well plates at 50,000 cells/cm² and grown for 72 hr. Oxidative stress was chemically induced by incubation with increasing concentrations (1 μM - 30 mM) of tert-butylhydroperoxide (tBHP) for 4 hr. Cells were pre-treated for 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 vehicle (growth media).

Hyperosmolar conditions were induced by supplementation of the growth media with NaCl (5 – 150 mM), as described previously [13]. Osmolarity of the media was confirmed using a vapor pressure osmometer (Vapro®, Wescor, Logan, UT).

2.3. Cell viability assays

The resazurin cell viability assay was performed as described by us previously for HCE-T cells [27]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and lactate dehydrogenase (LDH) release assays were conducted essentially as described by us for other ocular cells types [28, 29].

2.4. Permeability assay

Permeation studies were conducted by seeding HCE-T cells at a density of 100,000 cells on 12 mm Snapwell inserts with 0.4 μm pore size polycarbonate membrane (Costar®, Corning Inc., Corning, NY). Cells were grown for one week and subsequently stratified in an air-liquid interface for three weeks [30]. Successful stratification was assessed by staining against the tight junction markers, zona occludens 1 and quantification of the apparent permeability coefficient (P_app) for permeability standards 6-carboxyfluorescein and rhodamine B (data not shown). Stratified HCE-T cell layers were washed using buffer solution (BSS Plus®, Alcon, Hänenberg, Switzerland). The permeation experiments were conducted in orbital shaker at 35 °C. The solution containing 0.05 % Mn-TM-2-PyP was placed in the donor chamber and blank buffer was added in the receiver chamber. Samples were taken from the receiver side and the donor side at 10, 20, 30, 45, 60, 75, 90, 105, 120, 150 and 180 min and replaced with an equal volume of fresh buffer. Absorbance of the samples were measured at 460 nm using Cytation 3 multi-mode reader (BioTek Instruments, Winooski, VT, USA). The rate (the apparent permeability coefficient, P_app, cm/s) was calculated according to the following equation:

$$P_{app}\text{ = }\frac{\Delta Qr/\Delta t}{A\times C_d}$$ Equation 1,

where ΔQr / Δt is the slope of the linear region of the cumulative amount of the Mn-TM-2-PyP in the receiver chamber versus time plot, and where C_d is the average concentration of the Mn-TM-2-PyP in the donor chamber during the experiment, while A is the surface area (1.12 cm²). The mass balance (%) was calculated and defined as the sum of the Mn-TM-2-PyP in the receiver chamber (the amounts of the samples withdrawn at different time points) at 180 min, and the Mn-TM-2-PyP remaining in the donor chamber at 180 min, divided by the initial donor amount. P_app values and mass balances are presented as mean ± standard error of mean (SEM).

2.5. Animals

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the EC Directive 86/609/EEC for animal experiments, using protocols approved and monitored by the Animal Experiment Board of Finland. C57BL/6J mice were purchased from Janvier Labs (Le Genest-Saint-lsle, France) and housed at a constant temperature (22 ± 1 °C) and in a light-controlled environment (lights on from 7 am to 7 pm) with ad libitum access to food and water. Male mice (8 – 10 weeks of age) were used for experiments.

2.6. Induction of chronic experimental DED and drug administration

DED was induced using a combination of desiccating environment (5%-15% humidity and 15 L/min airflow; SiccaSystem™; K&P Scientific LLC, Oak Park, IL) and concurrent transdermal scopolamine administration for a period of two weeks. Mn-TM-2-PyP (0.1% dissolved in physiological saline) was administered topically three times daily as eye drops (10 μl into the conjunctival sac using a P20 micropipettor) for the entire two-week induction period. Preclinical efficacy of Mn-TM-2-PyP was compared against saline (administered topically three times daily) and against twice daily topical administration of 0.05% cyclosporine ophthalmic emulsion (Restasis; Allergan Inc., Irvine, CA).

2.7. Quantification of tear volume

Tear volume quantification was performed using a sterile phenol red-soaked cotton thread (ZoneQuick™) that was applied in the lateral canthus for a duration of 30 s, using forceps. The wetting length of the thread was read by an examiner blinded for treatment group under a microscope and estimated using a ruler. Resolution of the measurements was 0.5 mm. Tear volume was measured in all groups, at baseline and at the end of the two-week follow-up time.

2.8. Quantification of ocular surface inflammation

To quantify ocular surface damage, we scored corneal fluorescein staining, essentially as described by us previously [31].

2.9. Tissue collection and histology

Mice were euthanized by inducing a deep plane of general anesthesia by intraperitoneal administration of a cocktail of 75 mg/kg ketamine and 1 mg/kg xylazine. Eyes and intraorbital lacrimal glands were dissected and post-fixed overnight in 4% paraformaldehyde. Paraffin sections (5 μm) of lacrimal glands and frozen sections (10 μm) of eyeballs were processed for histological analysis using hematoxilin-eosin staining for quantification of immune cell infiltration into the lacrimal gland and Periodic Acid Schiff (PAS) staining to quantify the number of goblet cells in the inferior conjunctiva, as described by us in detail previously [32].

2.10. Corneal 8-hydroxy-2’ -deoxyguanosine (8-OHdG) staining

Cryosections of cornea were labeled with an anti-8-OHdG antibody (clone N45.1, 1:200 dilution, Japan Institute for the Control of Aging, NIKKEN SEIL Co., Ltd., Shizuoka, Japan). Briefly, antigen retrieval was performed using a combination of trypsin (0.1%) and denaturing of nuclear DNA by incubating sections in 2N HCl for 30 min at 37 °C followed by neutralization by immersing sections in 0.1 M borate buffer twice for 5 min each. Sections were incubated in primary antibody overnight at 4 °C. The next day, sections were washed three times for 10 min each with 10 mM phosphate-buffered saline (pH 7.4) and 8-OHdG was detected with Histostain-Plus IHC Kit (Thermo Fisher Scientific) and 3,3 diaminobenzidine substrate (Liquid DAB Substrate Kit, Invitrogen, Thermo Fisher Scientific). Images were acquired using a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany). 8-OHdG immunoreactivity was quantified by measuring density of nuclei in the corneal epithelial layer. Density of eight randomly-selected nuclei in three regions of approx. 100 μm² was measured using Fiji ImageJ software [33]. Values were averaged to obtain the mean for that eye.

2.11. Statistics

Data are presented as mean ± standard error of mean (SEM) or as box and whisker plots showing medians, interquartile ranges, mean and min/max values with individual data points presented as symbols. Data were analyzed using either Kruskal-Wallis ANOVA (for non-parametric data) or One-Way ANOVA (for parametric data). Differences between groups were subsequently determined using either Dunn’s, Dunnett’s or Tukey’s multiple comparisons tests as appropriate. Differences were considered statistically significant at the P< 0.05 level.

3. Results

3.1. Mn-TM-2-PyP protects HCE-T cells from oxidative insult in vitro

In order to determine the possible cytotoxicity of Mn-TM-2-PyP to corneal epithelial cells, we tested various concentrations of Mn-TM-2-PyP (0.0001% - 0.5% w/v in media) in the resazurin cell viability assay. Concentrations up to 0.01% did not show any significant loss of cell viability and the estimated LD₅₀ for Mn-TM-2-PyP was 0.19% (Fig. 1A). Next, we tested the ability of Mn-TM-2-PyP to protect HCE-T cells against chemically-induced oxidative stress using the resazurin assay. Fluorescence values were corrected for the fluorescence of the corresponding Mn-TM-2-PyP concentration in media. Mn-TM-2-PyP at concentrations between 0.001% and 0.05% exerted potent dose-dependent cytoprotection against tBHP insult (n = 4 Fig. 1B). The EC₅₀ values for tBHP were 102.6 μM (control), 187.3 μM (0.001%), 1.95 mM (0.005%) and 3.43 mM (0.05%), as determined by 4^th parameter non-linear curve fitting of the log-dose vs. response curve (Fig. 1B). Given the absorbance of fluorescent light by Mn-TM-2-PyP, we validated our results in the MTT cell viability and proliferation assay (Fig. 1C). We observed similar cytoprotection by Mn-TM-2-PyP with EC₅₀ values for tBHP of 72.8 μM (control) and 2.03 mM (0.005%). Similarly, Mn-TM-2-PyP completely prevented LDH release from HCE-T cells in response to tBHP treatment (Fig. 1D).

Figure 1: Mn-TM-2-PyP exerts potent cytoprotective effects against oxidative stress, but not hyperosmolar insult.

A. Cytotoxicity of HCE-T cells was assessed using the resazurin cell viability assay. Non-linear curve fitting using a four parameter (log)inhibitor vs. response curve yielded an estimated LD₅₀ for Mn-TM-2-PyP of 0.19% w/v. Concentrations of up to 0.01% did not show any significant cytotoxicity after exposure for 16 – 20 hr. B. One hour pre-treatment with Mn-TM-2-PyP exerted potent dose-dependent cytoprotection of HCE-T cells against tBHP-induced oxidative stress. Individual data points were fitted using non-linear 4^th parameter algorithm. Individuals data points represent mean ± S.E.M. from 5 different experiments. C. We confirmed the results from the resazurin cell viability assay using an MTT cell viability and proliferation assay. Estimated EC₅₀ values for tBHP were similar to those derived from the resazurin assay shown in B. Data are shown as mean ± S.E.M. from four separate experiments with eight technical replicates each. D. In a matched experiment, media supernatants from the same wells as from the MTT assay were used to quantify LDH release. Mn-TM-2-PyP completely prevented tBHP-induced LDH release from HCE-T cells. Data are shown as mean ± S.E.M. from four separate experiments with eight technical replicates each and fitted using a four-parameter algorithm. E. In contrast, Mn-TM-2-PyP did not exert any cytoprotective effects against hyperosmolarity-induced loss of cell viability (n = 4 separate experiments). Data were normalized against the 0 μM tBHP condition and are presented as individual data points representing the mean ± S.E.M. and were fitted using a sixth order polynomial equation.

In contrast, Mn-TM-2-PyP (0.05%) did not protect HCE-T cells from hyperosmolar insult. NaCl concentrations of 5 – 150 mM (equivalent to 350 – 1,250 mOsm) resulted in a dose-dependent loss of cell viability of up to 25%, which was not affected by pre-treatment with Mn-TM-2-PyP (Fig. 1E).

3.2. Mn-TM-2-PyP shows significant uptake into corneal epithelial cells

Permeation across the stratified human corneal epithelial barrier was assessed using a snapwell system. Mn-TM-2-PyP (0.05%) showed low permeability, with a P_app of 1.1 ± 0.05 × 10⁻⁶ cm/s as determined based on the cumulative amount of drug in the receiver chamber at multiple time points and a penetration of 3.9 ± 0.1% at 180 min (Fig. 2). The calculated mass balance was 62 ± 0.6%, indicative of significant cellular uptake. In addition, a long lag time (28 ± 4 min) prior to steady-state flux, i.e. linear transport phase of Mn-TM-2-PyP across corneal epithelial cells further suggests intracellular accumulation.

Figure 2: Mn-TM-2-PyP shows significant uptake into corneal epithelial cell.

Permeation across the corneal epithelial barrier was assessed using a snapwell system. Samples were taken at indicated time points for up to three hours and the P_app for Mn-TM-2-PyP was calculated. The dotted line indicates the steady state flux, i.e. the linear transport phase of Mn-TM-2-PyP across corneal epithelial cells, and was determined by linear curve fitting, indicating a lag-time of 28 ± 4 min. The calculated mass balance was 62 ± 1%, indicative of significant cellular uptake.

3.3. Topical application of Mn-TM-2-PyP reduces corneal fluorescein staining

Next we tested the efficacy of Mn-TM-2-PyP in a preclinical mouse model for DED. Tear volume in mice exposed to the desiccating environment/ scopolamine induction paradigm for 14 days showed significantly lower tear volumes compared to naive mice (3.5 ± 0.5 mm vs. 5.8 ± 0.7 mm; n = 9; P < 0.05), suggestive of successful disease induction. Dosing with saline (4.3 ± 0.5 mm), Mn-TM-2-PyP (4.1 ± 0.5 mm) or cyclosporine (4.6 ± 0.9 mm) did not result in a statistically significant increase in tear volumes at the end of the two-week study period (n = 9 – 10; ANOVA P = 0.16; Fig. 3A).

Figure 3: Topical administration of Mn-TM-2-PyP does not increase tear volume but reduces corneal fluorescein staining.

A. Tear volumes were significantly reduced in untreated mice exposed to the DED induction paradigm for two weeks compared with naive mice, indicative of successful induction of DED. No statistically significant differences between treatment arms were detected. B. Mn-TM-PyP was administered three times daily in a desiccating environment/ scopolamine mouse model for DED. Mice treated with Mn-TM-2-PyP showed significantly lower corneal fluorescein staining compared with untreated and saline-treated animals. The effect of Mn-TM-2-PyP on corneal fluorescein staining was similar or better than that of the FDA-approved reference compound, 0.5% ophthalmic cyclosporine emulsion (Restasis). Data are presented as box and whisker plots, indicating individual data points (circles), median (line), mean (+), interquartile ranges (box) and the min/max values (whiskers). * P < 0.05 compared with untreated; ^## P < 0.01 compared with saline-treated. C. Representative images of corneal fluorescein staining are shown for each treatment group. Scale bar: 2 mm.

However, three times daily topical administration of 0.1% Mn-TM-2-PyP in saline statistically significantly attenuated corneal fluorescein staining (Non-parametric Kruskal Wallis ANOVA P < 0.001; Fig. 3B-C). Mn-TM-2-PyP reduced the median fluorescein score (median score: 2; n = 29 eyes) compared with untreated (median score: 3, n = 27 eyes, P < 0.05) and saline-treated (median score: 3, n = 17 eyes, P < 0.01) eyes. While cyclosporine showed a trend toward lower fluorescein score after 2 weeks of treatment, this effect did not reach statistical significance (n = 21, P = 0.29 vs. untreated and P = 0.09 vs. saline-treated eyes).

3.4. Topical application of Mn-TM-2-PyP reduces oxidative DNA damage

In order to determine whether topical administration of Mn-TM-2-PyP reduces oxidative DNA damage, we quantified immunoreactivity of 8-OHdG in corneal epithelial cells. Untreated and saline-treated eyes showed significant nuclear 8-OHdG immunoreactivity, suggestive of oxidative DNA damage. Mn-TM-2-PyP-treated eyes showed a noticeable reduction in 8-OHdG staining, while immunoreactivity in cyclosporine-treated eyes was similar to untreated eyes (Fig. 4A). Quantification of nuclear immunoreactivity (density), revealed a statistically significant difference in 8-OHdG staining between treatment groups (One-Way ANOVA, P < 0.05). Post-hoc analysis using Tukey’s multiple comparisons test revealed a statistically significant reduction in 8-OHdG immunoreactivity in Mn-TM-2-PyP-treated eyes (n = 3) compared with untreated (n = 4, P < 0.05; Fig. 4B) and saline-treated (n = 4, P < 0.05; Fig. 4B) eyes. 8-OHdG staining in cyclosporine-treated eyes was lower, but that difference did not reach statistical significance (n = 4, P = 0.54 vs. untreated eyes and P = 0.45 vs. saline-treated eyes; Fig. 4B).

Figure 4: Topical application of Mn-TM-2-PyP reduces oxidative DNA damage.

A. Corneal sections were labeled for 8-OHdG as marker for oxidative stress-induced DNA damage. Strong immunoreactivity in untreated eyes is suggestive of significant DNA damage in animals exposed to the desiccating stress/ scopolamine model for DED. Nuclear 8-OHdG immunoreactivity was reduced in Mn-TM-2-PyP-treated eyes compared with both untreated and saline-treated eyes (One-Way ANOVA, P < 0.05; Tukey’s multiple comparisons test, P < 0.05 compared with both untreated and saline-treated eyes). In comparison, topical cyclosporine treatment had no statistically significant effect on 8-OHdG staining (P = 0.59 compared with untreated and P = 0.45 compared with saline-treated). Data are presented as mean ± standard error of the mean, individual data points are depicted as circles. * P < 0.05 compared with untreated, ^# P < 0.05 compared with saline-treated. B. Representative grayscale images are shown. Note the lighter nuclear staining in Mn-TM-2-PyP-treated eyes, suggestive of reduced oxidative stress-induced DNA damage. Scale bar: 10 μm.

3.5. Topical application of Mn-TM-2-PyP attenuates lacrimal gland pathology

DED is characterized by infiltration of immune cells into the lacrimal gland. To determine whether topical administration Mn-TM-2-PyP can attenuate lacrimal gland pathology, we scored immune cell infiltration as described by us previously [32]. Lacrimal glands showed a large number of foci of mononuclear cells, and in some cases, degeneration of parenchymal tissue resulting in a median score of 3 in saline-treated animals (n = 8; Fig. 5A, B). In contrast, Mn-TM-2-PyP reduced lacrimal gland pathology significantly by one score (n = 15; Kruskal-Wallis test with Dunn’s multiple comparisons test, P < 0.05 compared with saline). The improvement by Mn-TM-2-PyP was slightly greater in magnitude as observed in cyclosporine-treated eyes (n = 8; P = 0.08 compared with saline, P = 0.99 compared with Mn-TM-2-PyP).

Figure 5: Mn-TM-2-PyP reduced immune cell infiltration in the lacrimal gland.

A. Induction of DED resulted in significant infiltration of immune cells into the lacrimal gland in saline-treated animals. Mn-TM-2-PyP significantly reduced lacrimal gland pathology (Kruskal-Wallis test with Tukey’s multiple comparisons test, P < 0.05). Cyclosporine-treated eyes showed a reduction in the number of infiltrates, however, the difference did not reach statistical significance (P = 0.08). Data are presented as box and whisker plots, indicating individual data points (circles), median (line), mean (+), interquartile ranges (box) and the min/max values (whiskers). * P < 0.05. B. Representative examples of H&E-stained lacrimal gland showing reduced number of infiltrates in both the Mn-TM-2-PyP- and cyclosporine-treated groups and showed a potent prevention of degeneration of parenchymal tissue. Scale bar: 200 μm. C. Mn-TM-2-PyP did not prevent loss of mucin-producing conjunctival goblet cells. The number of goblet cells was similar between saline-, Mn-TM-2-PyP- and cyclosporine-treated groups (One-Way ANOVA, P = 0.99). Data are presented as box and whisker plots, indicating individual data points (circles), median (line), mean (+), interquartile ranges (box) and the min/max values (whiskers). D. Representative examples of PAS staining on conjunctival cryosections. Scale bar: 50 μm.

3.6. Topical Mn-TM-2-PyP treatment does not protect against loss of conjunctival goblet cells

Mucin-producing conjunctival goblet cells were identified using PAS staining on frozen sections of the inferior conjunctiva. Goblet cells and total conjunctival length were quantified. The number of goblet cells was 30.7 per mm conjunctiva in saline-treated eyes (n = 5), representing an approximate 50% reduction compared to naive animals (data not shown). There was no statistically significant difference in the number of goblet cells in Mn-TM-2-PyP-treated eyes (29.8 per mm conjunctiva, n = 13) and cyclosporine-treated eyes (n = 8; One-Way ANOVA P = 0.99; Fig. 5C, D).

4. Discussion

In the present study, we tested the efficacy of the prototypic manganese porphyrin antioxidant, Mn-TM-2-PyP in corneal epithelial cells in vitro and in a desiccating stress/scopolamine mouse model for DED.

Mn-TM-2-PyP exerted strong protection against oxidative stress in human corneal epithelial cells. The observed low permeability across corneal cells is likely a result of cellular uptake and/or binding to the epithelial barrier. Our data thus support the notion that Mn-TM-2-PyP can act as a potent cy to protectant for the corneal epithelium. Furthermore, our data indicate that three times daily topical administration provides a significant therapeutic benefit against ocular surface alterations and lacrimal gland pathology in vivo in a DED model over untreated or vehicle-treated eyes. Notably, Mn-TM-2-PyP showed similar or mildly superior efficacy as 0.5% ophthalmic cyclosporine emulsion (Restasis), an FDA-approved topical immunomodulator indicated to increase tear production in patients with keratoconjunctivitis sicca.

In the present study, we used HCE-T cells to predict the toxicity to and the permeability of Mn-TM-2-PyP across the corneal epithelium. HCE-T cells have been widely used as in vitro model for the corneal epithelium as they form a stratified epithelium with barrier properties and a characteristic morphology (for review, see [34]). However, it must be noted that an altered genomic content suggestive of heterogenous cellular origin has been described for HCE-T cells [35], which needs to be considered when interpreting our in vitro findings. Our mouse model for DED is based on a well-established paradigm that employs low-humidity air flow and concurrent scopolamine administration to induce DED in wild-type mice [36]. The model and its quantitative readouts used to determine DED severity were published by us previously [32] and are of similar magnitude to those reported by us and others using a desiccating stress/ scopolamine model to induce DED in mice [32, 36].

The central role of oxidative stress in ocular surface disease, which has been demonstrated in preclinical and clinical studies, is the basis for the scientific premise of utilizing antioxidants as therapeutics in DED. In aqueous-deficient DED, lacrimal gland dysfunction alters the composition of the tear film causing hyperosmolarity [3]. Hyperosmolar conditions on the corneal epithelium facilitate the generation of oxidative stress by disturbing the cellular balance of generation and elimination of Reactive Oxygen Species, as previously demonstrated in human corneal epithelial cells [13]. Elevated levels of oxidative stress are considered an important contributor to ocular surface inflammation through activation of nuclear factor-κB (NF-κB) by increased Reactive Oxygen Species [37]. NF-κB controls the expression of components of the antioxidant system and is also a mediator of pro-inflammatory signaling through toll-like receptors, such as TLR4, which by itself has been implicated in corneal surface inflammation [38]. Elevated levels of oxidative stress can thus cause apoptosis and damage to the corneal epithelium and facilitate pro-inflammatory signaling [37]. We show the presence of oxidative stress-induced DNA damage in the desiccating stress/ scopolamine model for DED (Fig. 4A, B), which renders support to studying the preclinical efficacy of topical antioxidants for the treatment of DED in this model. Topical treatment with Mn-TM-2-PyP resulted in a statistically significant reduction of corneal oxidative stress-induced DNA damage (Fig. 4A, B). This finding supports our in vitro data from permeability studies that suggest significant cellular uptake of Mn-TM-2-PyP into corneal epithelial cells (Fig. 2). Notably, the effect of manganese porphyrins in preventing NF-κB up-regulation in response to oxidative stress has been well documented [39]. Furthermore, topically applied manganese porphyrins showed potent anti-inflammatory effects in a mouse model of allergic dermatitis [25].

Topical antioxidant treatment using Mn-TM-2-PyP resulted in an attenuated lacrimal gland pathology with a significantly lower number of foci of infiltrating immune cells (Fig. 5). It is well known that mitochondrial oxidative stress in the lacrimal gland can induce lacrimal dysfunction. Specifically, deletion of the mev-1 gene that encodes the succinate dehydrogenase cytochrome b protein resulting in a defect in complex II of the mitochondrial electron transport chain resulted in increased generation of reactive oxygen species in the lacrimal gland and an ocular phenotype reminiscent of dry-eye disease, including reduced tear volumes, increased corneal fluorescein staining, and infiltration of immune cells into the lacrimal gland [12, 40].

Ongoing studies are investigating the extent by which topical administration of Mn-TM-2-PyP can alter the expression of genes and proteins associated with elevated tissue levels of oxidative stress in DED. Data from these studies will provide additional mechanistic insight needed for the translation of antioxidant treatments of DED to the clinic.

In this study, we also compared the efficacy of Mn-TM-2-PyP against 0.05% ophthalmic cyclosporine emulsion (Restasis), which is FDA-approved and the current clinical standard of care for patients with moderate to severe DED [4, 8]. When comparing three times daily topical administration of Mn-TM-2-PyP to twice daily administration of cyclosporine, Mn-TM-2-PyP showed similar or better efficacy in reducing corneal surface inflammation (Fig. 3B, C) and partially preventing the infiltration of immune cells into the lacrimal gland (Fig. 5A, B). Notably, Mn-TM-2-PyP reduced oxidative DNA damage in corneal epithelial cells, while cyclosporine did not show any effect (Fig. 4A, B). Neither drug was able to increase the number of conjunctival goblet cells (Fig. 5C, D), in agreement with previous reports [32].

Manganese porphyrins have an established preclinical safety profile [24] and are currently used in early stage clinical studies administered subcutaneously as adjunctive therapy concurrent with radiation therapy (Clinical trials #NCT03386500 and # NCT02655601). Together with their solubility and stability in aqueous buffers and formulations, manganese porphyrins, including Mn-TM-2-PyP, have the potential for a rapid translation to the clinic. Precedent for the use of topical antioxidants was recently shown by encouraging results of the mitochondrially-targeted antioxidant, SkQ1, in a Phase 2 Safety and Efficacy Trial [41].

5. Conclusions

Topical administration of the superoxide dismutase mimetic, Mn-TM-2-PyP, showed efficacy against corneal surface inflammation, infiltration of immune cells into the lacrimal gland and degeneration of parenchymal tissue in a preclinical model for DED. Efficacy was similar or better than ophthalmic cyclosporine emulsion, which is the current clinical standard of care. Given their chemical and safety profile, manganese porphyrins show great potential for translation to the clinic.

Abbreviations:

ANOVA

analysis of variance

DED

dry eye disease

HCE-T

human corneal epithelial cells

KCS

Keratoconjunctivitis sicca

LDH

lactate dehydrogenase

Mn-TM-2-PyP

manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide

P_app

apparent permeability coefficient

S.D.

standard deviation

S.E.M.

standard error of the mean

tBHP

tert-butylhydroperoxide