Methods for treating ocular surface diseases

Abstract

The present invention provides a method for treating ocular surface damage in a subject due to ocular disease or injury, such as dry eye disease, chemical or physical injury, infection, neurosensory abnormalities, and unspecified etiology, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a microRNA-328 antagonist.

Description

[Technical Field]

[0001] The present invention relates to a method for treating ocular surface damage in a subject due to ocular disease or injury, such as dry eye disease, chemical or physical injury, infection, neurosensory abnormalities, and unspecified etiology, comprising administering to the subject an miRNA-328 antagonist. [Background technology]

[0002] The ocular surface consists of the cornea and conjunctiva, and damage to the ocular surface can cause pain, redness, decreased vision, and ultimately permanent corneal or conjunctival damage.

[0003] Many eye diseases or injuries can cause ocular surface damage. Dry eye disease, the most common ocular surface disease, is a multifactorial disorder involving tears and the ocular surface, causing symptoms of discomfort, visual disturbances, and tear film instability.

[0004] The World Dry Eye Workshop (DEWS), organized by the Tear Film and Ocular Surface Society (TFOS), reported "TFOS DEWS II Definition and Classification" (Ocul Surf. 2017 Jul; 15(3): 276-283), providing a definition of dry eye disease as referred to in this invention. Environmental factors, including exposure to pollutants, ultraviolet (UV) radiation, and ozone, as well as the long-term use of preservative-containing eye drops, such as those used to treat glaucoma, are also often involved in dry eye disease. These factors increase oxidative stress and ocular surface inflammation, further contributing to the progression of dry eye disease.

[0005] Recently, meibomian gland dysfunction has been recognized as another major risk factor for dry eye disease. Typical symptoms of dry eye disease are dryness, burning, and gritty eye irritation, which worsen over time. Both eyes are usually affected. If dry eye persists for some time, micro-abrasions of the ocular surface, corneal erosions, and punctate keratopathy occur. As the disease progresses to the end stage, the epithelium undergoes pathological changes, namely squamous metaplasia and goblet cell loss.

[0006] Chemical injuries, including alkali and acid injuries, to the eye result in extensive damage to the ocular surface. Such injuries can cause permanent vision loss if the cornea is unable to repair itself completely. Physical injuries ranging from foreign bodies, trauma, to metal fragments can result in varying degrees of damage to the ocular surface. Eye infections can cause corneal erosions. If the corneal epithelium does not regrow, corneal erosions can develop into corneal ulcers. If corneal ulcers are not treated, severe vision loss can occur.

[0007] Neurotrophic keratopathy, also known as neuropathic keratopathy (NK), is characterized by reduced or lost corneal sensation due to impaired corneal innervation. Loss of corneal sensation can lead to epithelial keratopathy, epithelial defects, stromal ulcers, and ultimately corneal perforation. It is a rare disease with an estimated prevalence of less than 5 / 10,000 people.

[0008] The etiologies of NK include, but are not limited to, herpes keratitis (shingles and herpes simplex), prolonged use of topical medications containing benzalkonium chloride (BAC), chemical and physical burns, contact lens overuse, and corneal surgery such as laser in situ keratomileusis (LASIK). Human nerve growth factor (NGF) has been shown to effectively treat NK patients. Cenegermin (Oxervate) containing recombinant human nerve growth factor (rhNGF) has been shown to be effective in treating NK patients. TM ) was the first topical NK approved for the treatment of neurotrophic keratitis in the United States on August 22, 2018.

[0009] Goblet cells are restricted to the conjunctival epithelium. Their primary function is to produce and secrete mucins to hydrate and lubricate the ocular surface. Mucins are highly glycosylated glycoproteins. Mucins are classified into two distinct types: transmembrane mucins and secretory mucins. MUC5AC is one of the most studied mucins and is secreted to form the large gels found in the conjunctiva. A decrease in the number of goblet cells in the conjunctiva has been demonstrated in patients with dry eye disease. Furthermore, the use of contact lenses alters goblet cell density.

[0010] Microribonucleic acids (miRNAs) are non-coding, single-stranded RNA molecules approximately 21–23 nucleotides in length. In animals, mature miRNAs are complementary to the 3'-end untranslated region (UTR) of one or more message ribonucleic acids (mRNAs). Annealing of miRNAs with their target mRNAs inhibits protein translation and / or mRNA cleavage. It has previously been reported that microRNA-328 (miR-328) is a risk factor for myopia (Chen et al., Invest. Ophthalmol. Vis. Sci., 53:2732–2739, 2012), and that anti-miR-328 oligonucleotides are used to treat myopia (Juo et al., US10179913B2, 2019). Summary of the Invention

[0011] The present invention relates to a method for treating ocular surface damage in a subject due to ocular disease or injury, such as dry eye disease, chemical or physical injury, infection, neurosensory abnormalities, and unspecified etiology, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a miRNA-328 antagonist. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 shows that miR-328 expression levels increase in a dose-dependent manner with treatment with different concentrations of BAC in the rabbit corneal cell line SIRC. Data are shown as mean ± SEM from three independent experiments. [Figure 2] Figure 1 shows that anti-miR-328 dose-dependently increases NGF in the rabbit corneal cell line SIRC after 10 min exposure to BAC. Data are means ± SEM from three independent experiments. [Figure 3] Representative images of rabbit corneal fluorescent staining after treatment with phosphate-buffered saline (PBS) or anti-miR-328 are shown. PBS was used as a negative control in this study. Dry eye disease was induced by BAC from day 0 to day 21. Treatment with PBS (upper panel) or anti-miR-328 (lower panel) eye drops was initiated from day 8 to day 21. Green fluorescence is corneal staining, indicating corneal damage due to dry eye disease. On day 21, rabbits treated with anti-miR-328 had no corneal staining, while corneal staining was still present in the PBS group. [Figure 4] Representative images of H&E staining of rabbit corneal sections are shown. Rabbits with dry eye disease were treated with PBS or anti-miR-328, while normal rabbits were not treated with eye drops. (A) Representative H&E staining of corneas in the normal, PBS, and anti-miR-328 groups. The corneal epithelium in PBS-treated eyes was thinner and more disrupted, while the epithelial layer in anti-miR-328-treated eyes was thicker and more intact. There was no statistically significant difference in the stroma between normal, PBS-treated, and anti-miR-328-treated eyes. Scale bar = 50 μm. (B) Scatter plot showing the difference in epithelial and stromal thickness among the three groups. [Figure 5]Representative images of rabbit corneal cell apoptosis detected by TUNEL assay are shown. Rabbits with dry eye disease were treated with PBS or anti-miR-328, while normal rabbits were not treated with eye drops. (A) Brown apoptotic cells in the TUNEL assay. Eyes treated with anti-miR-328 had fewer apoptotic cells in the corneal epithelium and stromal layer compared with PBS-treated eyes, while normal eyes had almost no apoptotic cells. Scale bar = 100 μm. (B) Scatter plot showing the difference in apoptotic cells among the three groups. [Figure 6] Representative images of the orifices of meibomian glands in rabbits are shown. The PBS-treated rabbit eye showed hyperkeratosis (arrows) at the orifices of the meibomian glands, but the anti-miR-328-treated rabbit eye did not. Scale bar = 100 μm. [Figure 7] A representative graph shows the dose-dependent effect of anti-miR-328 on mice with dry eye disease. Dry eye disease was induced in the eyes of mice with BAC and treated with anti-miR-328 twice daily (10 minutes apart) for 14 days. Representative photographs were taken on the 14th day. The data showed that the 160 μM dose provided the best therapeutic effect. [Figure 8] Anti-miR-328 repairs corneal abrasions in mouse eyes. Corneal abrasions were inflicted on both eyes using an Alger brush. The left eye was then treated with PBS, and the right eye was treated with anti-miR-328 (160 μM). Eye drops were administered twice daily starting on the day of corneal abrasion. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a method for treating ocular surface damage in a subject due to ocular disease or ocular damage, such as dry eye disease, chemical or physical injury, infection, neurosensory abnormalities, and unspecified etiology, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a microRNA-238 antagonist.

[0014] As used herein, the term "subject" refers to an animal, particularly a mammal. In preferred embodiments, the term "subject" refers generally to a human being, and is not limited to a specific individual or individuals.

[0015] The term "therapeutically effective" is intended to limit the dose of each agent to achieve the goal of reducing disease severity while avoiding adverse side effects, such as those commonly associated with alternative therapies.

[0016] In one embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier, adjuvant or excipient.

[0017] In another embodiment of the present invention, the microRNA-328 antagonist is an anti-miR-328 oligonucleotide comprising a nucleotide sequence complementary to miR-328 or its precursor.

[0018] In one embodiment, antisense miR-328 oligonucleotides (15-22 nucleotides in length) are designed based on the sequence of mature human miR-328 (SEQ ID NO: 1, CUGGCCCUCUGCCCUCCGU). Anti-miR-328 oligonucleotide sequences of 15-22 nucleotides in length are shown in Table 1. The anti-miR-328 oligonucleotides mentioned in this invention are disclosed in the prior patent US10179913B2, the entire contents of which are incorporated herein by reference.

[0019] [Table 1]

[0020] In one embodiment, the length of the anti-miR-328 oligonucleotide ranges from 15 to 22 nucleotides. In another embodiment, the length of the anti-miR-328 oligonucleotide is 16 or 17 nucleotides. In a preferred embodiment, the anti-miR-328 oligonucleotide consists of SEQ ID NO: 3 or SEQ ID NO: 4. In a more preferred embodiment, the anti-miR-328 oligonucleotide consists of SEQ ID NO: 3.

[0021] The present invention provides a pharmaceutical composition comprising an miRNA-328 antisense oligonucleotide and a pharmaceutically acceptable carrier. The pharmaceutical composition is administered topically to the eye. In one embodiment, the pharmaceutical composition is administered topically to the eye. In another embodiment, the pharmaceutical composition is administered in the form of eye drops.

[0022] Example The following examples are non-limiting and merely representative of various aspects and features of the present invention.

[0023] Example 1 In vitro studies of anti-miR-328 oligonucleotides Rabbit cornea (SIRC) cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin at 37°C and 5% CO2. Cells were exposed to benzalkonium chloride (BAC) for 10 minutes, after which the original medium was replaced with fresh medium. To measure miR-328 levels, cells were incubated for an additional 24 hours before harvesting for RNA extraction. To measure NGF performance, cells were exposed to BAC for 10 minutes and then treated with anti-miR-328 for 12 hours.

[0024] Example 2 In Vivo Study in a Mouse Model to Evaluate the Efficacy of Treating Dry Eye Disease with Anti-miR-328 Oligonucleotides

[0025] Dry eye disease mouse model Benzalkonium chloride (BAC) is a commonly used eye drop preservative. However, BAC has toxic effects on the eye, including conjunctival inflammation and fibrosis, tear film instability, corneal cytotoxicity, and anterior chamber inflammation. Due to its toxic effects, high concentrations of BAC are widely used to induce dry eye disease in animal models.

[0026] First, C57BL6 mice were randomly assigned to either an anti-miR-328 group or a PBS-saline group. PBS is the solvent used to prepare anti-miR-328 eye drops. To induce dry eye disease, 5 μL of 0.2% BAC was instilled into both eyes of C57BL6 mice daily for 7 days. On day 8, 5 μL of PBS-saline or anti-miR-328 (10 μM) eye drops were administered to each eye daily for 2 weeks, during which time BAC instillation continued in both eyes.

[0027] The two-week treatment sequence consisted of first instilling PBS or anti-miR-328 into the eye, followed approximately 10 minutes later by BAC, simulating the inability to eliminate the cause of dry eye when patients receive anti-dry eye treatment.

[0028] Evaluating the results The therapeutic effect of anti-miR-328 was evaluated at the end of 2 weeks of PBS / anti-miR-328 treatment. Clinical observations were performed daily, and fluorescent staining was performed weekly. For corneal fluorescent staining, a drop of 1% sodium fluorescein was prepared on a fluorescein paper strip (Madhu Instruments Pvt. Ltd., Okhla Industrial Area, India), and then 5 μL of the solution was instilled into the conjunctival sac. Eyes were examined and graded under a slit lamp SL-15 (Kowa, Tokyo, Japan) with a cobalt blue filter.

[0029] The corneal fluorescein staining grading criteria from a phase 3 clinical trial of Lifitegrast, an FDA-approved drug for dry eye disease, were used with some modifications as follows: The corneal surface was divided into nine equal areas. Each area was graded in 0.5-point increments within a score range of 0 to 4 (maximum score 36), with lower scores indicating better results. Scores of "0" were defined as no staining, "1" as few or rare punctate lesions, "2" as discrete and countable lesions, "3" as too numerous to count, and "4" as confluent lesions.

[0030] If the animals in the two groups had similar corneal staining scores on day 7, a Student's t-test was used to evaluate the treatment effect; otherwise, a paired t-test was used to evaluate the treatment effect.

[0031] result A total of 19 male mice were used in this study, including 9 mice in the placebo group and 10 mice in the anti-miR-328 group. Therefore, the results are based on 18 placebo-treated eyes and 20 anti-miR-328-treated eyes.

[0032] Both the PBS group and the anti-miR-328 group significantly improved corneal fluorescent staining in mice with dry eye disease, but anti-miR-328 treatment appeared to achieve better results (Table 2). The improved staining scores on days 7 and 21 were 31.15 and 17.94 for the PBS group (p=0.0003, paired t-test, Table 2), and 31.70 and 8.2 for the anti-miR-328 group (p=1.67 x 10 -9 , paired t-test, Table 2). It is demonstrated that anti-miR-328 administration has a greater therapeutic effect than PBS administration (P=0.005).

[0033] [Table 2]

[0034] Example 3 In vivo study in a rabbit model to evaluate the therapeutic effect of anti-miR-328 oligonucleotides on BAC-induced dry eye disease

[0035] Dry eye disease rabbit model

[0036] Materials and Methods The study used pigmented Rex rabbits. Before inducing dry eye disease with BAC, the rabbits were randomly assigned to either a PBS treatment group or an anti-miR-328 treatment group. Dry eye disease was induced by instilling 20 μL of 0.15% BAC (Sigma-Aldrich, St. Louis, MO, USA) twice daily (9:00 AM and 5:00 PM) for one week. On day 8, the rabbits began treatment with PBS or anti-miR-328 twice daily for two weeks, during which BAC was still administered twice daily. The treatment sequence for these two weeks was as follows: PBS or anti-miR-328 was administered first, followed by BAC at 10-minute intervals.

[0037] Evaluating the results As described above, the therapeutic effect of anti-miR-328 / PBS was evaluated after 2 weeks of treatment. A second grading system, called the "ocular sum score," was also used to evaluate the therapeutic effect on the rabbits' eyes.

[0038] The "ocular total score" was adapted from the Cornea and Contact Lens Research Unit (CCLRU) grading scale and published guidelines (see Takamura E. et al., Japanese Guidelines for Allergic Conjunctival Disease 2017. Allergol Int. 2017;66:220-229, and Su G., Wei Z., Wang L., et al., Evaluation of Toluidine Blue-Mediated Photodynamic Therapy for Experimental Bacterial Keratitis in Rabbits. Transl Vis Sci Technol. 2020;9:13). The scale is based on four domains: limbal hyperemia, bulbar conjunctival hyperemia, tarsal conjunctival hyperemia, and keratitis. The top three domains have four severity levels (0-3), and keratitis has five severity levels (0-4). Rabbit eyes were also used for histological examination, including the cornea and meibomian glands.

[0039] Histological analysis On day 21, the rabbits were humanely euthanized, after which the right eye was enucleated and immersed in Davidson's fixative (20 ml 37% formalin, 100 ml glacial acetic acid, 350 ml 95% alcohol, and 530 ml water). The tissue was fixed for 48 hours, then washed with tap water and transferred to 10% neutral buffered formalin for storage before being trimmed and processed.

[0040] After removal of the ocular adnexa, they were immediately fixed in 10% buffered formaldehyde solution for 24 hours. The collected samples (cornea, conjunctiva, and meibomian glands) were then dehydrated in a gradient series of ethanol and embedded in paraffin. After microtome sectioning, the embedded tissue blocks were stained with hematoxylin and eosin (H&E) for histological examination. Histological images of all specimens were viewed under an automated digital slide scanner (Pannoramic mini II, 3dhitech Ltd., Budapest, Hungary) and visualized and measured using CaseViewer software (https: / / www.3dhistech.com / caseviewer).

[0041] A TUNEL assay was performed to assess cell apoptosis in the corneal epithelium and stroma. The TUNEL assay was performed using an in situ cell death detection kit (Product ID: 11684 817910) according to the manufacturer's instructions (Roche, Indianapolis, IN). Apoptotic cells were counted in three randomly selected fields under a 40x microscope.

[0042] Hyperkeratosis of meibomian gland orifices in the upper eyelid was assessed. The percentage of hyperkeratosis-induced obstruction of each orifice was calculated on 1200 μm histology slides. The mean value of all orifice obstruction within a slide represents the treatment effect for that particular eye.

[0043] Conjunctival impression cytology Conjunctival impression cytology specimens were collected on days 0, 7, 14, and 21. After instilling 0.5% alcaine and wiping excess intraocular fluid, a 5.5 mm diameter semicircular nitrocellulose filter paper (Toyo Roshi Kaisha, Ltd., Japan) was placed on the superior bulbar conjunctiva. The filter paper was held in place with gentle pressure for 1 minute, then removed from the eye and immediately fixed in 10% neutral buffered formalin. The paper was then stained with a PAS kit according to the manufacturer's instructions (PAS-2-IFU, ScyTek Laboratories, Inc., Logan, USA). The tissue was stained using PAS reagent, and the number of goblet cells was counted under a microscope at 400x magnification. After staining, goblet cell density was quantified.

[0044] Example 4: Evaluation of Corneal Abrasion Caused by Physical Injury and the Effect of Treatment with Anti-miR-328 Oligonucleotides

[0045] Corneal abrasion mouse model

[0046] Materials and Methods C57BL / 6 mice were first anesthetized, and alcaine was instilled into both eyes to further reduce subsequent discomfort during physical corneal injury. A cleaned ophthalmic Algerbrush was used to create corneal abrasions. The eyelids were held separately with the fingers, and one eye was opened at a time. The Algerbrush was then firmly placed on the cornea and moved back and forth across the ocular surface to induce corneal abrasions. Corneal fluorescent staining was performed postoperatively and daily thereafter. The left eye was treated with PBS, and the right eye was treated with anti-miR-328 twice daily (9:00 AM and 5:00 PM).

[0047] The magnitude of the fluorescent staining on the cornea is assessed as the outcome.

[0048] result

[0049] Effect of BAC on miR-328 Rabbit corneal cell line (SIRC) was treated with different concentrations of BAC. miR-328 expression increased in a dose-dependent manner (Figure 1). Data are mean ± SEM from three independent experiments. Treatment of SIRC exposed to BAC with anti-miR-328 resulted in increased NGF expression (Figure 2).

[0050] Corneal staining A total of 40 eyes were treated with PBS and 42 eyes were treated with anti-miR-328. Anti-miR-328 demonstrated therapeutic efficacy in reducing corneal staining in rabbit eyes (Figure 3). After 2 weeks of treatment, anti-miR-328 eye drops significantly reduced the improvement staining score (p = 0.038, paired t-test, Table 3), whereas PBS had no effect on corneal staining (p = 0.699, paired t-test, Table 3).

[0051] Similar results were observed when analyzing the data by ocular total score, where the mean score was significantly worse in the PBS group (p=7.4x10 -8 , paired t-test, Table 3), whereas the anti-miR-328 group showed a slight improvement (p=0.053, paired t-test, Table 3).

[0052] [Table 3]

[0053] Corneal thickness Rabbits with dry eye disease treated with anti-miR-328 or PBS eye drops had a significant difference in the mean thickness of the corneal epithelium (36.4 ± 1.2 μm vs 25.6 ± 1.7 μm, p = 9.4 × 10 -5 ), the average thickness of the corneal epithelium in normal rabbits is 45.4 ± 1.2 μm (Figure 4). The difference in stromal thickness between PBS-treated and anti-miR-328-treated eyes was not statistically significant (p = 0.34) (578.8 ± 25.0 μm vs. 539.5 ± 31.8 μm, Figure 4).

[0054] The stromal thickness of normal rabbit eyes is 521.2 ± 20.4 μm. TUNEL assay showed that the PBS group had more apoptotic cells than the anti-miR-328 group in the corneal epithelium (53 ± 3 cells vs. 39 ± 3 cells, P = 0.002) and stroma (84 ± 7 cells vs. 65 ± 5 cells, P = 0.029) (Figure 5).

[0055] Meibomian gland histology According to histology, the anti-miR-328 group had a lower mean occlusion percentage compared to the PBS group (56.4% ± 7.59% vs. 78.5% ± 8.17%, p = 0.059) although the difference did not reach a significant level of 0.05 (Figure 6).

[0056] conjunctival goblet cells The density of conjunctival goblet cells in the anti-miR-328 group was significantly higher than that in the PBS group (26 vs. 19 cells / mm 2 , p=0.005). This finding is consistent with previous reports of low goblet cell density in dry eye disease.

[0057] Dose-dependent effects To find the dose that achieves the greatest therapeutic effect on dry eye disease, we tested anti-miR-328 doses of 10, 30, 60, 90, 120, and 160 μM in mice with dry eye disease. The data showed that the reduction in corneal staining was dose-dependent, and eyes treated with anti-miR-328 eye drops at a dose of 160 μM had almost no fluorescent staining on day 14 (FIG. 7). Therefore, 160 μM anti-miR-328 may be the optimal dose for treating dry eye disease in mice.

[0058] Dry eye disease was induced in the eyes of mice using BAC, and the mice were treated with anti-miR-328 twice a day (10 minutes apart) for 14 days. Photographs were taken on the 14th day. The data showed that the therapeutic effect was best at a dose of 160 μM. Therefore, 160 μM of anti-miR-328 may be the optimal dose for treating dry eye disease in mice.

[0059] Corneal abrasion caused by physical injury and evaluation of the effect of anti-miR-328 oligonucleotides To demonstrate the effect of anti-miR-328 on corneal repair, mouse corneas were injured using an Algerbrush. After corneal abrasion, corneal staining revealed fluorescein throughout the cornea on day 0, indicating complete abrasion (Figure 8). However, by day 3, eyes treated with anti-miR-328 showed faster and better recovery than eyes treated with PBS (Figure 8).

[0060] The present invention, and the method and process for making and using it, have been described in such full, clear, concise and exact terms as to enable any person skilled in the art to make and use the subject matter of the present invention. The foregoing are preferred embodiments of the present invention, and it should be understood that the scope of the present invention may be construed and modified without departing from the scope of protection of the application. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

1. 1. Use of a pharmaceutical composition for the manufacture of a medicament for treating ocular surface damage in a subject due to an ocular disease or ocular injury, said pharmaceutical composition comprising a therapeutically effective amount of a microRNA-328 antagonist; the eye disease or eye injury is any of dry eye disease and chemical or physical injury; the microRNA-328 antagonist is an anti-miR-328 oligonucleotide comprising an oligonucleotide sequence complementary to the sequence of miR-328 or its precursor; The anti-miR-328 oligonucleotide sequence consists of SEQ ID NO: 3 The above use.

2. The concentration of the anti-miR-328 oligonucleotide is 1 to 500 μM or 10 to 160 μM.

2. The use according to claim 1.

3. Ocular surface damage caused by dry eye disease 2. The use according to claim 1.

4. Ocular surface damage caused by neurotrophic keratitis 2. The use according to claim 1.

5. Ocular surface injury is a corneal abrasion caused by mechanical trauma 2. The use according to claim 1.

6. Ocular surface damage caused by chemical injury 2. The use according to claim 1.

7. Ocular surface damage caused by meibomian gland dysfunction 2. The use according to claim 1.

8. The pharmaceutical composition is administered topically to the eye.

2. The use according to claim 1.

9. The pharmaceutical composition is administered in the form of eye drops.

2. The use according to claim 1.

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