DIBI,a polymeric hydroxypyridinone iron chelator,reduces ocular inflammation in local and systemic endotoxin-induced uveitis

Abstract

BACKGROUND/OBJECTIVE:

Non-infectious uveitis is an inflammatory disease of the eye commonly treated by corticosteroids, though important side effects may result. A main mediator of inflammation are oxygen free radicals generated in iron-dependent pathways. As such, we investigated the efficacy of a novel iron chelator, DIBI, as an anti-inflammatory agent in local and systemic models of endotoxin induced uveitis (EIU).

METHODS:

Firstly, the effects of DIBI in systemic EIU in Lewis rats were established. 2 hours post intravenous LPS or LPS/DIBI injections, leukocyte activation and functional capillary density (FCD) were examined using intravital microscopy (IVM) of the iridial microcirculation. Secondly, the toxicity of DIBI was evaluated in BALB/C mice for both acute and chronic dosages through gross ocular examination, intraocular pressure measurements and hematoxylin-eosin staining of ocular tissue. Lastly, three groups of BALB/C mice, control, LPS or DIBI + LPS, were studied to evaluate the effectiveness of DIBI in treating local EIU. Five hours post-local intravitreal (i.v) injection, leukocyte activation and capillary density were examined via IVM.

RESULTS:

Treatment of systemic EIU with DIBI resulted in a reduction of leukocyte activation and FCD improvement within the iridial microcirculation. Toxicity studies suggested that acute and chronic DIBI administration had no adverse effects in the eye. In the local EIU model, DIBI was shown to reduce leukocyte activation and restored the FCD/DCD ratio, providing evidence for its anti-inflammatory properties.

CONCLUSIONS:

Our study has provided evidence that DIBI has anti-inflammatory effects in experimental uveitis. Additionally, no local ocular toxicity was observed.

Keywords

Iron chelation ocular inflammation endotoxin intravital microscopy microcirculation

1 Introduction

Uveitis accounts for approximately 10% of all cases of visual deficits and blindness, with the disease largely affecting individuals of working age [1]. The Standardization of Uveitis Nomenclature (SUN) Working Group classifies uveitis according to its anatomical site as being either anterior, intermediate, posterior or panuveitic [2]. Uveitis can further be categorized as infectious or non-infectious according to its etiology. Current treatments for non-infectious uveitis focus on immunosuppressive therapies to control inflammation [3]. They include non-steroidal anti-inflammatory drugs and topical or systemic administration of corticosteroids. However, prolonged use of topical steroids may lead to cataracts, corneal-epithelium toxicity and steroid-induced glaucoma [4]. Additionally, the use of adjunct immunomodulatory therapy has been shown to be responsible for a wide array of serious side effects [5]. These side effects exemplify the need for novel anti-inflammatory agents in the treatment of non-infectious uveitis.

Iron, through its catalysis of the Fenton and Haber-Weiss pathways that produce reactive oxygen species (ROS), is an important player in inflammation [6]. Iron levels are highly regulated with iron being an essential cofactor in biologic processes such as oxygen transport by hemoglobin, metabolism and redox regulation [7]. During an inflammatory response, ROS such as superoxide and hydrogen peroxide radicals are primarily produced by neutrophils to degrade internalized particles and bacteria [8, 9]. However, this defense mechanism can cause cellular damage such as lipid peroxidation or DNA damage in the case of dysregulated or excessive ROS production [6 , 11]. The sequestration of iron by an iron chelator, rendering iron less bioavailable for ROS production, could therefore be a potentially useful anti-inflammatory strategy in the treatment of non-infectious uveitis.

DIBI is a novel iron chelator comprising of nine pyridinone chelating monomers copolymerized to vinyl pyrrolidone [10, 12]. Given the role that iron plays in inflammation, we have investigated the effects of DIBI on ocular inflammation by measuring its effects on microcirculatory parameters in experimental models of both systemic and local endotoxin-induced uveitis. Specifically, our study utilized non-invasive intravital fluorescence imaging to assess changes in the iridial microcirculation, and measure leukocyte-endothelial interactions as well as capillary blood flow. The evaluation of these parameters has shown to be a reliable indicator of the degree of inflammation in inflammatory diseases [13 –16]. We hypothesized that iron chelation by the novel iron chelator, DIBI, significantly reduces leukocyte adhesion and improves capillary blood flow in the iridial microcirculation in both models of EIU.

2 Material and methods

2.1 Animals

Lewis rats (250–300 g) and BalB/C mice (25–28 g) were obtained from Charles River, Wilmington, MA, USA. All animals were housed in the Carleton Animal Care Facility at Dalhousie University, Halifax, Nova Scotia, Canada and given a one-week acclimation period prior to use in the study. The study was conducted in accordance with protocols approved by the Dalhousie University Committee on Laboratory Animals in keeping with the guidelines established by the Canadian Council on Animal care (protocol #14-040). In addition, all animal experiments were also in compliance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research.

2.2 Experimental groups

i. Systemic endotoxin-induced uveitis

Three groups of rats were used (n = 3 per group). The first group (Control) received normal saline (0.9% sodium chloride, Hospira, Montreal, QC, Canada) administered intravenously (i.v.) at an equal volume of LPS as described below. The second group (LPS) received an i.v. dose of 5 mg/kg LPS (Escherichia coli, 026:B6, Sigma-Aldrich, Oakville, ON, Canada). The third group (LPS + DIBI) received an i.v. dose of 5 mg/kg LPS immediately followed by an i.v. dose of 10 mg/kg DIBI (Chelation Partners Inc., Halifax, NS, Canada). Two hours post i.v. injection, the iridial microcirculation of all animals’ left eye was examined using IVM.

ii. Corneal toxicity and tolerability

DIBI was dissolved in phosphate buffered saline (PBS, pH 7.4, Sigma) and filter sterilized (0.2μm filters) before use.

DIBI was applied acutely to the eye of interest with single or multiple dosing, involving either one and five repeat applications of the drug over a 2-hour time period, respectively. Chronic administration of DIBI was also assessed with a dosing regimen of twice daily (BID) for 7 days. Animals served as internal controls as DIBI was applied topically (5μL) to a randomly assigned eye, while the other eye of the animal received only the PBS vehicle. DIBI concentrations of 0.1, 1, 10, 100 and 1000μg/mL for both chronic or acute dosing were assessed.

Measures of corneal toxicity and tolerability were observed by gross ocular examination. This examination included slit lamp examination (SLE) of the anterior eye for any pathology of the cornea, photomicrographs of the cornea with and without fluorescein addition and direct measurement of intraocular pressure (IOP). Anterior eye examination was performed prior to IOP measurements to ensure that any disruptions found on the cornea were not due to short term artifacts from the tonometry probe. SLE was performed using a single lens handheld slit lamp and photomicrographs were taken with an iPhone 4 fitted with a 60x microscope attachment. Corneal haze and superficial punctate keratitis (SPK) were evaluated using established grading criteria for clinical scoring (Table 1).

Table 1
Grading criteria for clinical scoring: Superficial punctate keratitis and corneal haze. Bron et al. with modifications (Bron et al. 2003)

Grade SPK Haze

0 None Clear cornea Clear cornea

0.5 Trace Minimal epithelium disruption in any quadrant of the cornea Any size haze in any quadrant of the cornea not involving central cornea

1 Mild Involving 2 quadrants of the cornea and central cornea remaining mostly clear Corneal haze with partial opacity not involving the central cornea

2 Moderate Involving 2-3 quadrants of the cornea with central cornea affected minimally Corneal haze with partial opacity of cornea with peripheral and/or central cornea affected

3 Severe Involving 2-3 quadrants of the cornea with central cornea fully affected Severe corneal haze with complete opacity of cornea with peripheral and/or central cornea affected

Grade		SPK	Haze
0	None	Clear cornea	Clear cornea
0.5	Trace	Minimal epithelium disruption in any quadrant of the cornea	Any size haze in any quadrant of the cornea not involving central cornea
1	Mild	Involving 2 quadrants of the cornea and central cornea remaining mostly clear	Corneal haze with partial opacity not involving the central cornea
2	Moderate	Involving 2-3 quadrants of the cornea with central cornea affected minimally	Corneal haze with partial opacity of cornea with peripheral and/or central cornea affected
3	Severe	Involving 2-3 quadrants of the cornea with central cornea fully affected	Severe corneal haze with complete opacity of cornea with peripheral and/or central cornea affected

IOP measurements were measured using rebound tonometry (Tono-lab tonometer; Colonial Medical Supply Company, Windham, NH, USA). Ten IOP measurements were taken from each eye and the average was taken as the absolute pressure.

At the end of the in vivo data collection phase, each animal was sacrificed and the eyes were enucleated. Tissue sections were examined microscopically following haematoxylin-eosin staining.

iii. Local endotoxin-induced uveitis

Three groups of mice were used (n = 5 per group; Control, LPS, and LPS + DIBI). The first group (Control) received an i.v injection of normal saline (5μL), followed by an i.v injection of PBS (5μL), The second group (LPS) received an i.v injection of LPS (250 ng in 5μL of saline), followed by an i.v. injection of PBS (5μL). The third group (LPS + DIBI) received an i.v. injection of LPS (250 ng in 5μL of saline), followed by an injection of DIBI (10μM, 450 μg/μL).

All compounds were administered using the following standard intravitreal injection procedure: Prior to all intravitreal injections, a 30-gauge needle was used to puncture the superior nasal sclera. The intravitreal injections were given using a Hamilton syringe (Hamilton Company, Reno, NV, USA) with a 17-gauge needle. 5μL of either saline or LPS was injected into the left eye of all animals in each group, and 15 minutes later the PBS or DIBI was administered into the initially punctured hole. The punctured hole was then sealed with 3M Vetbond Tissue Adhesive (3M Animal Products, St. Paul, MN, USA). Five hours post-injection, the iridial microcirculation of each animal’s left eye was examined using IVM.

2.3 Anesthesia and preparation

i. Endotoxin-induced uveitis

Animals (rats and mice) were anesthetized with by intraperitoneal (i.p.) injection of sodium pentobarbital (54 mg/kg, Ceva Santé Animale, Montreal, QC, Canada), and placed on top of a heating pad to maintain a body temperature of 37°C. Following sedation, the animal’s eyes were lubricated with Tear-Gel (Novartis Pharmaceuticals Canada Inc., Dorval, QC, Canada) to prevent dehydration. 15 minutes prior to the initiation of IVM and in the absence of a toe-pinch reflex, a tail vein injection of a mixture of two fluorochrome dyes was performed. The fluorochrome dye mixture consisted of rhodamine 6G (1.5 mL/kg) (Sigma-Aldrich, ON, Canada) and fluorescein isothiocyanate (FITC)-albumin (1 mL/kg) (Sigma-Aldrich, ON, Canada). The anesthetized animals were then placed on a stereotactic frame (Kopf Instruments, Tujumga, CA, USA), and a cover slip (circular for mice and rectangular for rats) was placed on top of the cornea of the eye. At the end of the experiment, the animals were sacrificed under anesthesia by an i.v. injection of KCL (0.142 mL/kg).

ii. Corneal Toxicity and tolerability

All animals were observed under sodium pentobarbital anesthesia (54 mg/kg) i.p.

2.4 Intravital microscopy

IVM was used in both models of EIU (systemic and local) for in vivo imaging of leukocyte-endothelial interactions and capillary blood flow [13]. For both rats and mice, IVM of the left eye was performed by randomly examining one region of interest in each of the four quadrants of the eye (30 seconds recording/visual field).

Assessment of capillary blood flow and leukocyte-endothelial interactions was performed off-line by analysis of videotaped images using ImageJ software (National Institute of Health, USA). Rolling leukocytes were defined as cells that interacted only temporarily with the endothelium. Adhering leukocytes were defined as cells that were attached to the endothelial surface for the complete observation period of 30 seconds. The number of adherent leukocytes in an arbitrary vessel was reported as the number of cells per square millimeter of endothelial surface area. The rolling leukocytes were reported as the number of rolling leukocytes per minute.

When analyzing capillary blood flow, for each video, a single rectangular field covering the maximum area possible was drawn, and capillaries containing FITC-albumin marked plasma found in the rectangular field were chosen. Capillaries with absent flow and intermittent flow were characterized as dysfunctional capillaries (dysfunctional capillary density, DCD), whereas the capillaries with continuous flow, regardless of the speed of the flowing cells, were characterized as functional capillaries (functional capillary density, FCD). Summing the length of all corresponding capillaries, and dividing the sum of the lengths with the measured area of the rectangular field calculated the FCD and DCD.

2.5 Statistics

Following confirmation of Gaussian distribution of data by Kolmogorov-Smirnov test, statistical significance was determined via a One-way-ANOVA. Corneal haze, SPK and IOP values were presented for the treated and control eyes of each individual animal within a group assigned a specific DIBI dosing regimen. Statistical significance between the different dosing regimen groups was established via T-test analysis. For all data, a P value of p < 0.05 was used as the threshold for statistical significance.

3 Results

3.1 Systemic endotoxin-induced uveitis

Figure 1A shows the number of firmly adhering leukocytes in the iris microvasculature two hours post an i.p. injection of either saline, LPS, or LPS + DIBI. There is a significant increase in the number of adherent leukocytes in untreated LPS animals in comparison to healthy control animals. Conversely, there is a significant reduction in leukocyte adhesion in the DIBI treated animals in comparison to the untreated LPS animals, and no significant difference between leukocyte adhesion in DIBI treated and healthy control animals.

Fig.1

(A) Leukocyte adherence in the iridial microcirculation of rats. Groups: CON – healthy control group, LPS – 5 mg/kg lipopolysaccharide i.v., LPS+DIBI – 5 mg/kg lipopolysaccharide i.v. +10 mg/kg DIBI i.v., n = 3 per group. Mean±SEM. *p < 0.05 vs. CON, ^#p < 0.05 vs. LPS. (B) Functional capillary density in the iridial microcirculation of rats. Groups: CON – healthy control group, LPS – 5 mg/kg lipopolysaccharide i.v., LPS+DIBI – 5 mg/kg. lipopolysaccharide i.v. +10 mg/kg DIBI i.v., n = 3 per group. Mean±SEM. *p < 0.05 vs. CON.

Figure 1B shows the FCD in the iridial microcirculation of rats two hours post i.p. injection of either saline, LPS, or LPS + DIBI. There was a significant reduction in FCD following systemic EIU in comparison to healthy control and DIBI treated animals. Conversely, no significant change in FCD was observed in DIBI treated rats as compared to healthy control animals.

3.2 Corneal toxicity and tolerability

3.2.1 Gross ocular examination

Slit lamp examination using photomicroscopy provided sensitive and reliable detection of corneal haze and SPK when occurring, as shown in Fig. 2. No statistically significant incidences or dose-dependent occurrences of SPK were seen across all DIBI doses for any of the treatment groups, i.e., for acute single, acute multiple or chronic dosed eyes (Fig. 3). Furthermore, there were no significant incidences or dose-dependent occurrences of corneal haze observed across all tested dosing regimens, acute single, acute multiple and chronic, or dosing concentrations (0.1, 1.0, 10, 100, and 1000μg /ml; Fig. 4).

Fig.2

Photomicrographs of murine corneas treated with DIBI. (A), Example of a clear normal cornea which was treated acutely with a single dose of 1μg/mL DIBI; (B), fluorescein dye of that same eye highlighting intact corneal epithelial tissue; (C), Example of a cornea treated acutely with 5 doses of 0.1μg/mL DIBI showing grade 1.0 SPK; (D), fluorescein dye of that same eye highlighting corneal epithelial erosions; (E), Example of a cornea treated chronically with 100μg/mL DIBI with 14 doses over 7 days showing grade 2.5 corneal haze; (F), fluorescein dye of that same eye highlighting intact corneal epithelial tissue.

Fig.3

Incidences of superficial punctate keratitis across different DIBI concentrations observed after different dosing regimens. (A), acute, single dose of DIBI AT 0.1, 1, 10, 100 and 1000μg/μl (n = 10); (B), acute, 5 doses at the 5 stated concentrations over 2 hours (n = 10–12); (C), chronic dosing with 14 doses at the 5 stated concentrations over 7 days (n = 5–12).

Fig.4

Incidences of corneal haze across different DIBI concentrations observed after different dosing regimens. (A), acute, single dose (p > 0.05, n = 10); (B), acute, 5 doses over 2 hours (p > 0.05, n = 10–12); and (C), chronic dosing with 14 doses over 7 days (p > 0.05, n = 5–12).

3.2.2 Intraocular pressure

There were no significant increases or decreases in IOP when comparing PBS vehicle to DIBI treated eyes across all tested dosing regimens or dosing concentrations of 0.1, 1, 10, 100, and 1000μg /ml for DIBI (Fig. 5).

Fig.5

Intraocular pressure across different concentrations and dosing regimens. (A), mean IOP recorded after acute, single dose of DIBI (n = 10); (B), mean IOP recorded after acute, multiple doses with 5 doses over 2 hours (n = 10–12); (C), mean IOP recorded after chronic dosing with 14 doses over 7 days (n = 5–12).

3.2.3 Histology

Hematoxylin-eosin staining of ocular tissue revealed similar normal histological observations for DIBI treated eyes as well as for untreated controls, regardless of the dose evaluated (Fig. 6).

Fig.6

Histology. DIBI was applied as single or multiple doses (1000μg/mL). Histological examination revealed no significant change in the ocular tissue. At the histological level, the administration of DIBI was well-tolerated with no histological indications of tissue toxicity. Representative images of Haematoxylin-Eosin stained corneal tissue of control and DIBI-administered ocular tissue.

3.3 Local endotoxin-induced uveitis

Figure 7A shows the number of firmly adhering leukocytes in the microvasculature of the mouse iridial microcirculation five hours post double intravitreal injection of either saline/PBS, LPS/PBS, or LPS + DIBI. There was a significant increase in the number of adherent leukocytes in untreated LPS mice compared to healthy control mice. Additionally, leukocyte adhesion in DIBI treated mice was significantly lower in comparison to untreated LPS mice, but was significantly higher than healthy control group levels.

Fig.7

(A) Leukocyte adherence in the iridial microcirculation of mice. Groups: CON – healthy control group, LPS – local intravitreal endotoxin, LPS+DIBI – local intravitreal LPS/DIBI. n = 5 per group. Mean±SEM. *p < 0.05 vs. CON, ^#p < 0.05 vs. LPS. (B) Functional capillary density in the iridial microcirculation of mice. Groups: CON – healthy control group, LPS – local intravitreal endotoxin, LPS+DIBI – local intravitreal LPS/DIBI. n = 5 per group. Mean±SEM. (C) Dysfunctional capillary density in the iridial microcirculation of mice. Groups: CON – healthy control group, LPS – local intravitreal endotoxin, LPS+DIBI – local intravitreal LPS/DIBI. n = 5 per group. Mean±SEM. *p < 0.05 vs. CON (D) Sum of the functional and dysfunctional capillary densities in the iridial microcirculation of mice. CON – healthy control group, LPS – local intravitreal endotoxin, LPS+DIBI – local intravitreal LPS/DIBI. n = 5 per group. Mean±SEM.

Figure 7B shows the number of functional capillaries in the iridial microcirculation of the mice – five hours post a double intravitreal injection of either saline/PBS, LPS/PBS, or LPS/DIBI. There was no significant change in FCD following local EIU in comparison to healthy control and DIBI treated LPS animals.

Figure 7C shows the number of dysfunctional capillaries in the iridial microcirculation of mice – five hours post a double intravitreal injection of either saline/PBS, LPS/PBS, or LPS/DIBI. There was a significant increase in the number of dysfunctional capillaries in untreated LPS mice compared to healthy control mice.

The graph in Fig. 7D was created by combining FCD and DCD data collected from the local EIU experiments. The bars of the graphs represent the sum of the capillaries measured within the defined areas of the iridial microcirculation and their respective proportions of DCD and FCD. It is evident by the larger bar for the untreated LPS group that additional capillaries were opened in this group. We observed no changes in the sum of capillaries measured per mm² in both the healthy control and DIBI treated mice.

4 Discussion

In our present study, we observed a significant decrease in leukocyte adhesion and a significant improvement in FCD in systemic EIU by i.v. DIBI administration. The corneal toxicity studies showed no adverse effects of local DIBI administration. In local EIU we confirmed the anti-inflammatory effects of iron chelation by DIBI.

The imbalance between the generation of ROS and the ability to counteract the pathophysiology resulting from the generation of ROS is referred to as a state of oxidative stress. Oxidative stress has shown to play a causative role in the pathophysiology of uveitis [17]. The presence of free iron has been shown to be responsible for catalyzing the redox reactions involved in the generation of ROS due to its ability to accept and donate electrons [18]. Given the role of iron in inflammation, we sought to study the effects of depleting free iron with the novel iron chelator, DIBI, on leukocyte-endothelial interactions and capillary blood flow in the iridial microcirculation.

Studies have previously demonstrated the efficacy of iron chelators in inflammatory conditions. For over 40 years, Desferrioxamine (DFO) has been the standard chelator to treat conditions of iron-overload and more recently, inflammatory diseases [19]. DFO is a natural microbial product, also known as a siderophore, produced by Streptomyces spp. [20], and it can be described as an ideal iron chelator as it possesses the following properties: iron specificity, stability and biocompatibility [21]. Vulcano et al. reported anti-inflammatory DFO activity in endotoxin-induced septic shock in mice [22]. Another experimental sepsis study employing DFO in adjunct with N-acetylcysteine showed a reduction in oxidative stress and neutrophil infiltration, indicating DFO’s anti-inflammatory abilities [23]. A similar study conducted by our group observed a significant reduction in leukocyte recruitment in rats challenged with Colon Ascendens Stent Peritonitis-induced sepsis [15]. Specific to ocular pathology, it was found that the chelators diethylnetriaminepentacetic acid (DTPA) and DFO reduce hydrogen radical production and resulting tissue damage in Eales disease, an inflammatory vasculopathy of the retina [24]. Similarly, Rao et al. found that DFO reduced ocular inflammation in experimental uveitis [25]. Our results are in line with the abovementioned studies that have investigated the use of iron chelators for iron sequestration during ocular inflammation.

Both local (intraocular or topical) and systemic (oral, intravenous or intraperitoneal) routes of administration have been used in the literature, and both routes seem to be equally effective [26 –29]. A study demonstrating the efficacy of an intraperitoneal injection of the iron chelator alpha-lipoic acid in a model of light induced retinal degeneration confirmed the effectiveness of iron chelation in protecting the retina against light damage [30]. Song et al. also investigated iron chelation using deferiprone in light induced retinal damage. However, this study administered a similar iron chelator systemically, which proved to be protective of light induced retinal damage and showed to markedly reduce oxidative stress in the retina [27]. Conversely, Picard et al. recently provided evidence of intravitreal injection of transferrin protecting the rat retina against light-induced retinal degeneration [28].

Following administration of DIBI, reduced leukocyte adhesion and improved FCD were observed. It is interesting to observe that DIBI’s inhibitory effect correlates to the strength of the inflammation. In the local endotoxin model, where leukocyte adhesion is extensive, DIBI inhibits about 40% of the adhesion (Fig. 7). DIBI is capable of completely reversing the increased leukocyte adhesion in the more modest leukocyte adhesion levels in response to systematic endotoxin (Fig. 1). This suggests that DIBI has significant inhibitory power that reverses early/developing systemic inflammation completely and blocks severe local inflammatory reactions to a significant degree. Although the exact role of DIBI in endotoxin-induced infections has yet to be established, we propose that DIBI’s anti-inflammatory effects can be explained by its role in leukocyte adhesion. LPS is considered a pathogen-associated molecular pattern (PAMP) and interacts with Toll-like receptor 4 (TLR4) found on leukocytes. This interaction activates intracellular inflammatory pathways (e.g. NF-κB), resulting in increased expression of adhesion molecules on the surface of the cell [31, 32]. We believe that due to DIBI’s high molecular weight (9.5 kDa) it is unlikely that it has any direct intracellular effects. However, it is possible that DIBI sterically blocks the interaction between leukocyte and endothelial adhesion molecules. Furthermore, TLR4 on leukocytes also detects an array of endogenous molecules called danger-associated molecular patterns (DAMPs), released as a consequence of cell death or necrosis, and cause leukocyte activation [31]. Hence, given DIBI’s role in reducing the generation of ROS and the resulting decrease in tissue damage, it follows that there would be a reduction in the production of DAMPs which would reduce de novo leukocyte activation.

The improvement in FCD following DIBI treatment is in line with our hypothesized mechanisms of action of DIBI as an anti-inflammatory agent [33]. Reduced leukocyte adhesion can increase FCD following DIBI therapy by opening previously blocked capillaries. Furthermore, the increase in the sum of measured capillaries per defined area the in LPS group can be explained by opening of previously closed arterio-venous shunts in the microcirculation [14]. However, there was no increase in the sum of capillaries observed per defined area in the DIBI treated mice. These results are suggestive of DIBI’s proposed role in acting as an anti-inflammatory agent through modulation of leukocyte adhesion rather than recruiting new vessels.

Iron chelation therapy was initially designed to diminish the toxic effects stemming from iron overload in the host. In contrast to most classical iron chelators such as DFO where the bound iron is still accessible for pathogenic microorganisms, DIBI, through its modified hydroxypyridinone activity, has also proved to cease iron-related growth of bacteria and fungi [10]. It is, therefore, possible that DIBI could also play an antibacterial role in infectious uveitis, though further studies are required.

The results of the corneal toxicity studies of DIBI are suggestive of its safety for the treatment for uveitis. In comparison, DFO administered in patients with chronic iron overload has been illustrated to cause acute deterioration in visual acuity, scotomas, night blindness, and color vision [34]. On the contrary, despite only being investigated in an experimental setting, our results suggest no development of adverse effects due to the administration of DIBI. Moreover, the administration of DIBI did not induce any significant changes in SPK or corneal haze, as assessed by SLE or histological examination, or caused any significant changes in IOP as compared to the vehicle control treatment. All three DIBI treatment regimens and doses were well tolerated. Neither serious adverse reactions nor animal deaths were reported during the course of the study and there were no treatment withdrawals due to treatment-induced adverse events.

The results of this study are promising regarding treatment of non-infectious uveitis. However, the extent to which EIU in rats and in mice accurately represents the course of uveitis in humans is unknown. Despite this limitation, we are now planning dose response studies to investigate the pharmacokinetics of DIBI in experimental endotoxin-induced uveitis. Also, investigations comparing the efficacy of DIBI in comparison to other known chelators such as DFO or Deferiprone in treating uveitis have yet to be conducted.

5 Conclusion

Iron chelation by DIBI showed anti-inflammatory properties by decreasing leukocyte adhesion and improving FCD in the iridial microcirculation during experimental endotoxin-induced uveitis without local adverse effects in the eye.

Author disclosure statement

No competing financial interests exist in the writing of this manuscript.

Footnotes

Acknowledgments

We would like to thank Junru Chen for experimental support.

The work is dedicated to Prof. Friedrich Jung on occasion of his 70. Birthday.

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