Method for treatment of keratitis in dogs

Abstract

FIELD: veterinary science; ophthalmology.SUBSTANCE: as part of complex therapy, dogs are given 1–2 drops of ophthalmic solution in the conjunctival sac 3 times a day for 10–14 days. The solution contains 0.3% sodium hyaluronate solution and water-soluble blueberry extract with an anthocyanin concentration of 0.05%.EFFECT: method accelerates corneal epithelialization, reduces the severity of the inflammatory reaction and oxidative stress in eye tissues, increases the activity of the endogenous antioxidant system of the cornea, and improves the stability of the tear film and the barrier function of the epithelium, which in turn ensures increased effectiveness of keratitis therapy in dogs.1 cl, 2 tbl, 1 ex

Description

[0001] The invention relates to veterinary ophthalmology, specifically to means for the prevention and treatment of corneal diseases in dogs, accompanied by a disruption of the integrity and function of the epithelial layer. Such conditions include superficial and deep keratitis of various etiologies, keratoconjunctivitis, dry eye syndrome, erosions and degenerative lesions of the cornea, arising as a result of mechanical, chemical or infectious factors, as well as against the background of metabolic disorders and severe oxidative stress. The aim of the present invention is to develop a method for the prevention and treatment of inflammatory corneal diseases in dogs, ensuring accelerated restoration of the epithelial layer, a reduction in the severity of the inflammatory reaction and oxidative stress, and an increase in the resistance of the cornea to damaging factors while maintaining high biocompatibility and good tolerability of the ophthalmological agent.

[0002] The canine cornea is highly sensitive to damaging agents and has limited self-healing potential, as its epithelial layer is constantly exposed to external factors (ultraviolet radiation, dust, pathogens, preservatives contained in ophthalmic drops and ointments). Therefore, a pressing issue in veterinary medicine is the use of ophthalmic agents that provide comprehensive epithelial protection, reduce inflammation, stabilize the tear film, and stimulate reparative processes. One of the most common ophthalmoprotective components is sodium hyaluronate, used in modern solutions as a biocompatible moisturizer and cytoprotector. Hyaluronate (hyaluronic acid) stimulates the migration and proliferation of corneal epithelial cells, accelerating wound healing in vitro and in vivo.

[0003] In animal models of corneal injury and cell cultures, sodium hyaluronate reduces inflammation, decreases cell apoptosis, and promotes the restoration of epithelial barrier function. These effects are mediated through the activation of repair factors, including fibronectin, the hyaluronic acid receptor CD44, and connective tissue growth factor, as well as through the suppression of proinflammatory cytokines such as interleukin-1β and extracellular matrix degradation enzymes, including matrix metalloproteinase-9. Hyaluronate also regulates the expression of regulatory microRNAs, such as microRNA-18a, which further contributes to the reduction of inflammation and acceleration of reparative processes [1].

[0004] The use of hyaluronate is reflected in a number of well-known ophthalmic compositions. For example, international patent application WO 2008157140 (published December 24, 2008) and EP 2171025 (published April 7, 2010) describe hyaluronic acid-based formulations primarily focused on modifying its rheological properties and stabilizing the tear film. However, such solutions are limited to physicochemical effects and do not include biologically active antioxidant components capable of enhancing cellular defense.

[0005] In recent years, interest in combination products based on hyaluronate and natural biologically active substances has grown. For example, application EP 3808353 A1 (published April 21, 2012) proposes a composition of hyaluronate with arabinogalactan demonstrating improved viscoelastic properties. However, the effect of this combination is primarily due to the polymeric nature of the components and does not include significant anti-inflammatory or antioxidant activity, which reduces its effectiveness in treating corneal epithelial damage accompanied by oxidative stress.

[0006] A separate approach is presented in US 2024 / 0197649 A1 (published June 20, 2024), which proposes incorporating the lipophilic antioxidant lutein into an aqueous ophthalmic base. This approach requires the use of micellar or microemulsion systems to ensure solubility and stability, which complicates manufacturing and can reduce tolerability due to the presence of surfactants and co-solubilizers required for the formation of micelles or microemulsions. Such components can alter the lipid layer of the tear film, increase its evaporation, exacerbate conjunctival irritation, and increase the risk of a local inflammatory reaction in dogs with sensitive corneas.In addition, carotenoid antioxidants (e.g., lutein) exert their activity primarily through physical quenching of singlet oxygen and stabilization of lipid membranes, whereas water-soluble anthocyanins act directly and, by reducing free radicals, suppress chain reactions of lipid peroxidation, chelate metal ions, regulate the expression of antioxidant enzymes, and reduce proinflammatory signaling.

[0007] Against the backdrop of these decisions, interest in phytocompositions containing standardized anthocyanin extracts is growing. Bilberry extract (Vaccinium myrtillus), rich in phenolic compounds, exhibits pronounced antioxidant, cytoprotective, and anti-inflammatory effects. Anthocyanins have been shown to effectively reduce the formation of reactive oxygen species in cells subjected to oxidative stress, including retinal and vascular endothelial cells, normalize the activity of antioxidant enzymes in the cornea, and induce the expression of heme oxygenase-1 (HO-1) through activation of the Nrf2 signaling pathway [2].

[0008] Furthermore, it has been proven that combining hyaluronate with biologically active substances can enhance the reparative effect. For example, studies have shown that a combination of sodium hyaluronate with B vitamins, taurine, and dexpanthenol enhances epithelialization, reduces the level of inflammatory cytokines, and increases cellular resistance to oxidative damage [3].

[0009] These data confirm that hyaluronate can exhibit synergy with natural antioxidants and metabolic stimulants. The anthocyanin components of the standardized blueberry extract (cyanidin, delphinidin, petunidin, peonidin, and malvidin) exhibit pronounced cytoprotective properties, making them promising substances for combination with sodium hyaluronate.

[0010] Cyanidin is characterized by its ability to reduce apoptosis and strengthen the epithelial barrier function. Delphinidin exhibits strong antioxidant and anti-inflammatory effects, inhibiting pathological angiogenesis. Petunidin exhibits an antioxidant effect and promotes the protection of vascular structures and retinal cells. Peonidin improves photoreceptor stability and supports visual signal transmission. Malvidin reduces endoplasmic reticulum stress and protects retinal cells from damage. Collectively, anthocyanins provide multi-level antioxidant and anti-inflammatory protection due to their ability to modulate the cellular response to oxidative and inflammatory stress. This makes them functionally compatible with sodium hyaluronate and demonstrates the potential for creating combination formulations combining a hydrophilic polymer and a standardized plant extract.

[0011] Thus, the state of the art demonstrates significant interest in sodium hyaluronate as a basic ophthalmic protector. However, existing solutions are either limited to physicochemical modification of the tear film, use plant components without standardization and proven bioavailability, or require complex technological delivery systems. All of this underscores the relevance of developing a new composition combining a biocompatible sodium hyaluronate polymer and a standardized water-soluble blueberry extract with pronounced antioxidant and cytoprotective properties, taking into account the specific nature of ophthalmological diseases in dogs.

[0012] The objective of the invention is to develop the most effective method for the prevention and treatment of corneal diseases in dogs, accompanied by a violation of the integrity and functions of the epithelial layer.

[0013] The technical result of the claimed invention is the acceleration of corneal epithelialization, a reduction in the severity of the inflammatory reaction and oxidative stress of the eye tissues, an increase in the activity of the endogenous antioxidant system of the cornea, as well as an improvement in the stability of the tear film and the barrier function of the epithelium, which ensures an increase in the effectiveness of keratitis therapy in dogs compared to the use of sodium hyaluronate as monotherapy.

[0014] The achievement of the specified technical result is due to the synergistic action of sodium hyaluronate and anthocyanin extract, which consists in a combination of mechanical protection of the corneal surface and biological activation of reparative and cytoprotective processes, leading to accelerated restoration of the epithelial layer and a reduction in inflammatory changes.

[0015] The treatment method is as follows. An ophthalmic solution containing 0.3% sodium hyaluronate and a standardized water-soluble blueberry extract (Vaccinium myrtillus) with anthocyanin content of 0.05% is injected into the conjunctival sac of the affected eye. The solution is administered by instillation, 1-2 drops 3 times daily for 10-14 days, depending on the severity of corneal damage and the dynamics of clinical signs. This method moisturizes the ocular surface, reduces irritation and inflammation, and activates corneal epithelial reparation processes. If necessary, this method can be used as part of a comprehensive treatment for inflammatory ocular diseases in dogs.

[0016] To evaluate the effectiveness of the proposed composition, an experimental model of superficial corneal injury was used. The model is based on the controlled creation of a small epithelial defect (6 mm), allowing for objective assessment of the rate of reparative processes and tear film restoration parameters.

[0017] The injury was reproduced by gentle mechanical removal of the epithelium using a sterile 6 mm diameter corneal trephine to mark the target area and an atraumatic corneal scarifier to carefully remove epithelial cells within the marked area. The instruments used are disposable ophthalmic instruments approved for experimental veterinary research. This technique avoided stromal trauma and ensured that the defect was strictly superficial. This approach ensures model homogeneity and minimizes animal discomfort, allowing for objective comparison of the effects of the test drugs.

[0018] To confirm the efficacy of the proposed ophthalmic composition, a study was conducted on 14 pug dogs (n = 4-5 years old). Prior to the study, all animals were examined using standard ophthalmoscopy and fluorescein staining to confirm comparable corneal epithelial defect area and depth. The animals were divided into two groups of 7 animals, based on the analogous pairing principle:

[0019] The control group received a 0.3% sodium hyaluronate solution. The experimental group received a composition of 0.3% sodium hyaluronate with the addition of 0.05% standardized water-soluble extract of Vaccinium myrtillus.

[0020] The assessment of the dynamics of reparation was carried out on days 0, 3, 7 and 14 (Tables 1, 2).

[0021] Table 1

[0022] Average corneal defect area (mm 2 )

[0023] Day Control group Experimental group 0 6,0 6,0 3 4,5 3,5 7 3,0 1,7 14 1,5 0,5

[0024] The obtained data demonstrate that the inclusion of Vaccinium myrtillus extract in a sodium hyaluronate solution leads to significantly faster restoration of the corneal epithelial layer. By day 3, the defect area in the experimental group was 22–25% smaller than in the control group. By day 7, the difference reached 43–45%, and by day 14, the defect area in the experimental group was three times smaller.

[0025] To evaluate the effect of the proposed composition of sodium hyaluronate 0.3% + standardized water-soluble extract of Vaccinium myrtillus 0.05% on the antioxidant system of the cornea, a modeling of the dynamics of the key components of the antioxidant response was performed: the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the expression level of heme oxygenase-1 (HO-1), which is demonstrated in Table 2.

[0026] Table 2

[0027] Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1) activity

[0028] SOD Day Control group Experimental group 0 1,00 1,00* 3 1,12 ± 0,08 1,35 ± 0,10 7 1,20 ± 0,10 1,62 ± 0,12 14 1,18 ± 0,09 1,55 ± 0,11 CAT 0 1,00 1,00 3 1,08 ± 0,07 1,28 ± 0,09 7 1,18 ± 0,09 1,50 ± 0,11 14 1,15 ± 0,08 1,44 ± 0,10 GPx 0 1,00 1,00 3 1,10 ± 0,08 1,30 ± 0,10 7 1,22 ± 0,10 1,58 ± 0,12 14 1,20 ± 0,09 1,50 ± 0,11 HO-1 0 1,00 1,00 3 1,05 ± 0,06 1,40 ± 0,10 7 1,12 ± 0,08 1,95 ± 0,15 14 1,10 ± 0,07 1,70 ± 0,13

[0029] *p < 0.05

[0030] The control group showed moderate fluctuations in enzymatic activity, characteristic of the natural reparation process. The experimental group showed a marked and stable increase in all studied parameters. Superoxide dismutase activity in the experimental group increased by 20–35% on days 3–7 and remained higher than the control throughout the entire observation period. This indicates enhanced superoxide detoxification. Catalase activity increased by 18–28% relative to the control, indicating an increased ability to neutralize hydrogen peroxide, a key substrate for oxidative damage. Glutathione peroxidase activity increased by 25–30%, reflecting enhanced glutathione-dependent antiradical protection and stabilization of cell membranes. The most significant differences were recorded in HO-1 levels. The expression of the enzyme in the experimental group increased almost 2-fold on the 7th day, indicating the activation of the Nrf2-dependent cytoprotective cascade.

[0031] The data in Tables 1 and 2 taken together confirm that the addition of standardized Vaccinium myrtillus extract to sodium hyaluronate solution provides a bidirectional therapeutic effect, such as acceleration of corneal epithelial repair and pronounced activation of antioxidant protection.

[0032] Literature:

[0033] 1. Guo, Y., Wang, H. Sodium hyaluronate promotes proliferation, autophagy, and migration of corneal epithelial cells by downregulating miR-18a in the course of corneal epithelial injury / / European Journal of Histochemistry, 2023. – Vol. 67. – DOI: 10.4081 / ejh.2023.3663.

[0034] 2. Park, C. Anthocyanins inhibits oxidative injury in human retinal pigment epithelial ARPE-19 cells via activating heme oxygenase-1 / C. Park, H. Hwangbo, S. Kim, J. Noh, S. Park, S. Hong, S. Hong, G. Kim, Y. Choi / / Journal of Microbiology and Biotechnology, 2023. – Vol. 34. – P. 596–605. – DOI: 10.4014 / jmb.2310.10011.

[0035] 3. Lazzara, F. Corneal protective effects of a new ophthalmic formulation based on vitamin B12 and sodium hyaluronate / F. Lazzara, F. Conti, G. Maugeri, V. D’Agata, L. Sotera, C. Bucolo / / Frontiers in Pharmacology. 2025. – Vol. 16. – DOI: 10.3389 / fphar.2025.1548213.