Eye drop composition for treating xerophthalmos containing recoflavone

Abstract

The present invention relates to an eye drop preparation containing recoflavone for treating xerophthalmia. Specifically, in order to maximally improve the symptoms of xerophthalmia, the present invention relates to an eye drop composition containing a main component at a specific concentration or more and at least one polyoxyethylene castor oil derivative or poloxamer as a penetration enhancer, so that the active ingredient recoflavone can sufficiently penetrate the cornea in vivo and exhibit effective medical effects.

Description

[Technical Field]

[0001] Technical field The present invention relates to an ophthalmic composition for treating dry eye disease, containing recoflavone, a 3',4',5-trimethoxyflavone derivative compound. Specifically, in order to maximize the improvement of dry eye symptoms, the present invention relates to an ophthalmic composition containing a specific concentration of the active ingredient recoflavone and at least one of a penetration enhancer, polyoxyethylene castor oil derivative or poloxamer, so that the active ingredient recoflavone can sufficiently penetrate the cornea in vivo and exhibit a remarkable therapeutic effect.

[0002] background art Dry eye disease is a well-known condition in which inflammation occurs on the surface of the eyeball and causes discomfort in the eye due to increased osmotic pressure of the tear film and loss of tear homeostasis caused by tear deficiency or excessive evaporation of tears. On the other hand, there was much debate until just a few years ago regarding the proper definition of dry eye, and in 2017 the Tear Film and Ocular Surface Society (TFOS) defined dry eye disease as follows: "Dry eye disease is a multifactorial disease of the ocular surface characterized by loss of homeostasis of the tear film and accompanied by ocular symptoms, in which tear film instability, hyperosmolarity, inflammation and damage of the ocular surface, and neurosensory abnormalities play a pathogenic role (TFOS DEWS II introduction. Ocul. Surf. 2017, 15, 269-275). Diagnosis of dry eye disease is made using methods such as symptom history, tear volume, tear film breakup time, tear osmolality, corneal and conjunctival staining, and analysis of the lipid layer or meibomian glands, and treatment is used depending on the severity of the dry eye disease, including artificial tears, anti-inflammatory drugs, tear secretion stimulants, autologous serum, and ptosis treatment (J. Korean Med. Assoc. 2018, 61(6)). 352-364).

[0003] For the treatment of dry eye disease, the most commonly used approach is tear replenishment with ocular lubricants, such as artificial tears, typical examples of which are eye drops containing sodium hyaluronate or sodium carboxymethylcellulose. For anti-inflammatory therapy, cyclosporine preparations are used to reduce immunoactive markers (e.g., human leukocyte antigen-antigen D-related), apoptosis markers (e.g., Fas), and inflammatory cytokines (e.g., interleukin-6) in the corneal and conjunctival epithelium, treating inflammation on the surface of the eyeball and improving dry eye symptoms. Diquafosol (tetrasodium diquafosol), a recently developed preparation, is a P2Y2 receptor agonist that promotes the secretion of aqueous and mucous components of tears. In Japan, rebamipide suspension preparations have been proven effective in improving goblet cell density in the eyeball and increasing the mucous and aqueous components of tears, and are used as a therapeutic agent for dry eye disease.

[0004] Recoflavone is a 3',4',5-trimethoxyflavone derivative compound C 20 H 18 It is O8, and the molecular weight of 2-((2-(3,4-dimethoxyphenyl)-5-methoxy-4-oxo-4H-chromen-7-yl)oxy)acetic acid is 386.4.

[0005] KR Nos. 10-0447918, 10-0327621, and 10-0644928 disclose the applicability of recoflavone to gastrointestinal diseases, such as gastritis and gastric ulcers, and inflammatory bowel diseases, such as Crohn's disease and colitis, due to its anti-inflammatory effects and gastric mucus secretion-promoting properties. KR No. 10-0930467 discloses the conjunctival mucus secretion-promoting effects of 3',4',5-trimethoxyflavone derivative compounds, including recoflavone, and their applicability as agents for treating and preventing dry eye diseases.

[0006] KR No. 10-0644928 states that while recoflavones can be used directly in their anhydrous form, their hygroscopic properties cause problems in handling and management, and these problems can be improved by using them in hydrate form.

[0007] Lecoflavones are almost insoluble in water. Lecoflavones have low solubility at low pH levels. As the pH increases, lecoflavones ionize, and their solubility increases. However, even when dissolved, lecoflavones have poor cell permeability due to their high degree of ionization, which can lead to problems with absorption into the body. In fact, when a 3% eye drop solution was administered to rabbits and the drug distribution in various eye tissues was evaluated over time, the results showed that lecoflavones were hardly distributed in tissues located inside the eyeball, but were mainly distributed in peripheral tissues outside the eyeball, such as the cornea, conjunctiva, and sclera. In particular, the drug concentration in the cornea was high immediately after administration, but decreased rapidly over time, indicating that the drug was mainly distributed in the conjunctiva and sclera (data not shown).

[0008] Therefore, in order to demonstrate a significant therapeutic effect on the overall symptoms of dry eye disease, it is necessary to be able to penetrate a certain amount or more of the cornea and conjunctiva with recoflavonoid eye drops in vivo.

[0009] Prior art documents Patent Documents (Patent Document 1) KR No. 10-0327621 (Patent Document 2) KR No. 10-0447918 (Patent Document 3) KR No. 10-0644928 (Patent Document 4) KR No. 10-0930467 Non-patent literature (Non-patent document 1) TFOS DEWS II introduction. Ocul. Surf. 2017, 15, 269-275 (Non-patent document 2) J. Korean Med. Assoc. 2018, 61(6), 352-364 (Non-patent document 3) Invest. Ophthalmol. Vis. Sci., 2014, 55(10), 6569~6574

[0010] Disclosure of the invention technical challenges The present invention relates to an ophthalmic composition containing recoflavone for treating dry eye disease, and provides an ophthalmic composition that allows the active ingredient to penetrate the cornea and conjunctiva in vivo in a certain amount or more, so that recoflavone can exhibit a remarkable therapeutic effect on the overall symptoms of dry eye disease.

[0011] KR No. 10-0930467 discloses that recoflavones have shown some evidence for dry eye disease at low drug concentrations, such as promoting mucus secretion, and that they have the potential to be developed as a treatment for dry eye disease.

[0012] Normally, when the cornea and conjunctiva of the eye are injured, the expression of major mucin proteins, such as MUC1, MUC4, MUC16, and MUC5AC, increases. On the other hand, patients with dry eye disease complain of discomfort because, despite having a more fragile cornea, they have insufficient mucus secretion. A study of the pharmacological mucin secretion induced by recoflavone in human corneal and conjunctival epithelial cells confirmed that the thickness of the mucin layer increased significantly when a 100 μM solution of the active ingredient was applied. Meanwhile, real-time polymerase chain reaction (PCR) confirmed that MUC1, MUC4, MUC16, and MUC5AC, mucin proteins associated with dry eye symptoms, showed a significant increase after 4 hours, followed by a rapid decrease. Furthermore, Western blot analysis showed that only MUC5AC showed an increasing trend, while the others did not (Investigative Ophthalmology and Visual Science, 2014, 55(10), 6565~6574). These results suggest that, considering that dry eye disease involves various symptoms caused by various factors, the pharmacological action of recoflavones may only be able to improve some of the symptoms of dry eye, potentially limiting their potential for development as an effective treatment.

[0013] In the process of developing a formulation containing recoflavones as a treatment for dry eye disease, it was confirmed in in vivo studies that low concentrations of the drug in a simply dissolved state were insufficient to effectively improve the overall symptoms of dry eye. This invention has shown that if recoflavones can penetrate tissues and cells in a certain amount or more in vivo, they can effectively improve the overall symptoms of dry eye. Subsequently, this led to the development of a practical treatment for dry eye disease. Therefore, in this invention, there is a need to develop a recoflavones-containing formulation that can efficiently penetrate the cornea and significantly improve the overall symptoms of dry eye in the tissues and cells of the eye.

[0014] Solution to the problem The present invention provides an ophthalmic composition containing recoflavone that efficiently penetrates the cornea and conjunctiva and significantly improves the symptoms of dry eye in the tissues and cells of the eye. Specifically, the present invention provides an ophthalmic composition that, a) The effective concentration of recoflavones in the eye drops is 5% (w / v) or higher. b) The ophthalmic composition comprises at least one penetration enhancer, c) The ophthalmic composition may further include a viscosity agent, an isotonic agent, a buffer, a pH adjuster, and a preservative. To provide ophthalmic compositions.

[0015] Advantageous effects of the present invention The present invention can effectively improve the symptoms of dry eye disease by increasing the in vivo corneal and conjunctival penetration of a recoflavone-containing ophthalmic composition for treating dry eye disease. In particular, the present invention provides a practical and efficient ophthalmic composition for treating dry eye disease by improving the overall signs of dry eye disease, such as tear secretion volume, corneal fluorescence staining score, corneal epithelial cell exfoliation, corneal surface irregularities, conjunctival goblet cell density, and mucin cell density.

[0016] The effects of the present invention are not limited to those described above, and various other effects can be included within the scope that will be obvious to those skilled in the art from the following description. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the cumulative corneal penetration of recoflavone in miniature pig corneas over 6 hours. [Figure 2] Figure 2 shows a comparison of tear secretion volume between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 3]Figure 3 shows the comparison of corneal fluorescence staining scores between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 4] Figure 4 shows the comparison of the unevenness of the corneal surface between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 5] Figure 5 shows the comparison of the number of detached corneal epithelial cells between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 6] Figure 6 shows the comparison of goblet cell density between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 7] Figure 7 shows a photograph comparing the mucin cell density between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse. [Figure 8] Figure 8 shows a graph comparing the mucin cell density between the ophthalmic composition of Comparative Example 1 and the ophthalmic composition of Example 1 in a dry eye experimental model mouse.

[0018] Best Mode for Carrying Out the Invention Hereinafter, the present specification will be described in more detail.

[0019] Each of the descriptions and embodiments disclosed in the present invention can be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this specification are included in the scope of the present invention. In addition, the scope of the present invention should not be considered to be limited by the specific descriptions described below.

[0020] In the process of developing a formulation containing recoflavone as a therapeutic agent for dry eye disease, the inventors found that at low drug concentrations in a simply dissolved state, it was difficult to effectively improve the overall symptoms of dry eye in in vivo studies. In their research to develop recoflavone as an effective therapeutic agent for dry eye disease, the inventors discovered that when the formulation contains 5% (w / v) or more of recoflavone and a penetration enhancer such as polyoxyethylene castor oil derivative or poloxamer, either alone or in combination, in an amount of 0.1-5.0% (w / v), the amount of corneal and conjunctival penetration in vivo increases, effectively improving the symptoms of dry eye. Based on this, the inventors completed the present invention.

[0021] The following provides a more detailed explanation of this specification.

[0022] Herein, as used herein, the presence of 5% (w / v) or more of recoflavone or its pharmaceutically acceptable salt, hydrate, anhydrous or solvate means that it contains 5% (w / v) or more of recoflavone, and for both the content listed as an example and similar content, it refers to the concentration of recoflavone. In addition, the concentration of recoflavone also refers to the concentration of recoflavone as a whole.

[0023] Ophthalmic composition for treating dry eye disease The ophthalmic composition of the present invention comprises 5% (w / v) or more of recoflavones and one or more penetration enhancers. This ophthalmic composition may further contain a viscous agent, an isotonic agent, a buffer, a pH adjuster, and a preservative.

[0024] Specifically, the present invention provides an ophthalmic composition for treating dry eye disease, comprising 5% (w / v) or more of recoflavone or a pharmaceutically acceptable salt thereof, and 0.1 to 5.0% (w / v) of polyoxyethylene castor oil derivative or poloxamer alone or in combination.

[0025] The recoflavones used as the active ingredient in the present invention can be used in the form of anhydrous or solvates including hydrates, or as one of its pharmaceutically acceptable salts.

[0026] In the present invention, recoflavones can be prepared directly by conventionally known manufacturing methods or by commercially available products.

[0027] In the present invention, the term "recoflavone" refers to a 3',4',5-trimethoxyflavone derivative compound having the structure of the following chemical formula 1. [ka]

[0028] KR No. 10-0644928 discloses that recoflavones, as represented by their chemical formula, are hygroscopic in their anhydrous form, but are non-hygroscopic and stable in their hydrate form, and can exist in various solvates. Therefore, for solid dosage forms, such as tablets and capsules, it is preferable to use solvates containing hydrates from the viewpoint of stability, but for liquid dosage forms, such as eye drops, various forms of recoflavones, such as anhydrous, solvates containing hydrates, and pharmaceutically acceptable salts thereof, can be used.

[0029] Regarding the concentration of recoflavone, KR No. 10-0930467 and prior art use a concentration of 1-3% (w / v) as the active ingredient. On the other hand, in the present invention, in order to significantly improve the overall symptoms of dry eye, it is necessary for a certain amount or more of recoflavone to penetrate the tissue in vivo, and in this case, it has been found that the concentration of recoflavone in the formulation should be 5% (w / v) or more.

[0030] Preferably, the concentration of recoflavone is 5% (w / v) or higher and 10% (w / v) or lower. Recoflavone concentrations exceeding 10% (w / v) are difficult to apply to eye drop formulations due to osmotic pressure.

[0031] In the present invention, "penetration enhancer" refers to a substance that, when an ophthalmic composition containing recoflavone is administered to the eye, plays a role in allowing a certain amount or more of recoflavone to penetrate the cornea in vivo in order to significantly improve the overall symptoms of dry eye, and can be a certain type of surfactant that enhances the cell membrane permeability of recoflavone.

[0032] Specifically, the penetration enhancer includes a polyoxyethylene castor oil derivative or a poloxamer, more specifically, the polyoxyethylene castor oil derivative may be polyoxyl 35 castor oil, and the poloxamer may be poloxamer 407.

[0033] In the present invention, polyoxyethylene castor oil derivatives or poloxamers can be used alone or in combination, and when included alone or in combination, they can be included in the ophthalmic composition in an amount of 0.1 to 5.0% (w / v).

[0034] In the present invention, a viscosity agent commonly used in eye drops may be included as needed. For example, nonionic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxyethylcellulose, and methylcellulose; ionic polymers such as sodium carboxymethylcellulose, polycarbophil, and carbomer; sugars such as glucan, chitosan, lehalose, and gums such as xanthan gum may be used and may be included in the ophthalmic composition in an amount of 0 to 3.0% (w / v).

[0035] In the present invention, isotonic agents commonly used to achieve isotonicity with tears can be used. For example, nonionic compounds such as mannitol, glycerin, propylene glycol, polyethylene glycol, maltose, sucrose, sorbitol, trehalose, and glucose, or ionic compounds such as sodium chloride, sodium nitrate, and potassium nitrate can be used. These isotonic agents can be used alone or in any combination of two or more to adjust the osmotic pressure to be close to that of tears, and can be included in the ophthalmic composition in an amount ranging from 0.01 to 1.0% (w / v).

[0036] In the present invention, buffers or pH adjusters commonly used in the field of ophthalmic formulations can be used to adjust and maintain the pH within a range that does not irritate or damage the cornea or conjunctiva. For example, phosphate, citrate, acetate, bicarbonate, carbonate, gluconate, lactate, propionate, TRIS, or borate can be used as buffers, and conventional acids, such as hydrochloric acid, lactic acid, acetic acid, phosphoric acid, or conventional bases, such as sodium hydroxide and potassium hydroxide, can be used individually or in any combination of two or more of them as pH adjusters. In this case, these can be included in the ophthalmic composition in a range of 0.01 to 3.0% (w / v).

[0037] In the present invention, for the purpose of preventing the eye drops from being contaminated with bacteria or fungi, preservatives commonly used in eye drops may be used as needed, depending on whether a multi-use container or a disposable container is used as packaging for the eye drop formulation. For example, tertiary ammonium salts, such as benzalkonium chloride and benzethonium chloride; parahydroxybenzoic acid esters, such as methylparaben and propylparaben; alcohols, such as chlorobutanol and benzyl alcohol; chlorhexidine acetate and sodium edetate may be used individually or in any combination of two or more of these, and may be included in the ophthalmic composition in an amount of 0 to 0.1% (w / v).

[0038] In this invention, for an ophthalmic composition containing recoflavones for treating dry eye, the cumulative penetration amount of recoflavones in the cornea of ​​a miniature pig over 6 hours is 130 μg / cm³. 2 This is greater than or equal to three times greater than that of an ophthalmic composition that does not contain a penetration enhancer. In this case, the cumulative penetration amount of recoflavones refers to the total amount of recoflavones that penetrated the miniature pig cornea in 6 hours when tested according to Experimental Example 1.

[0039] The cornea has a protective function for the eye and acts as a barrier to drug absorption; therefore, ensuring an appropriate amount of drug penetration into the cornea is necessary for the successful development of eye drops.

[0040] Recoflavone has the advantage of allowing the preparation of eye drops as a completely clear solution, even at high concentrations of 3% (w / v) or 5% (w / v). On the other hand, eye drops containing recoflavone dissolved at high concentrations did not show significant improvement in the overall symptoms of dry eye disease in in vivo studies, and corneal penetration tests confirmed that even with high concentrations of recoflavone dissolved, a sufficient amount of recoflavone could not be penetrated. In the present invention, it was confirmed that when a penetration enhancer was added to the recoflavone-containing ophthalmic composition, the amount of recoflavone that penetrated the cornea increased significantly, and it was confirmed that the overall symptoms of dry eye disease were improved by adding the penetration enhancer.

[0041] In the present invention, considering the characteristics of eye drops administered directly to the eye and the physicochemical properties of recoflavones, an ophthalmic composition for treating dry eye disease can have a pH range of 4.0 to 8.0, and preferably, this range can be set to a pH of 5.0 to 7.0.

[0042] Dosage and administration of ophthalmic compositions With respect to the ophthalmic composition of the present invention, sufficient exposure of the eye to the active ingredient allows for the delivery of a certain amount or more of the drug to the inside and outside of the corneal and conjunctival cells of the eye, which is advantageous for significantly improving the overall symptoms of dry eye. Therefore, the preferred method for administering the ophthalmic composition of the present invention is to instill 1 to 2 drops at a time, three or more times a day.

[0043] Specifically, one drop can be instilled three or more times, four or more times, five or more times, or six or more times at a time; more specifically, one drop can be instilled three to six times a day.

[0044] The ophthalmic composition of the present invention can be administered in combination with other eye drops, in which case the two can be administered at intervals of at least 5 minutes.

[0045] The following preferred embodiments are provided to aid in understanding the present invention. However, these embodiments are provided to make the present invention easier to understand, and the scope of the present invention is not limited by these embodiments.

[0046] Examples 1-4 and Comparative Examples 1-2: Preparation of Ophthalmic Compositions The ophthalmic composition was prepared according to the proportions shown in Table 1 below. First, sodium hydroxide was added to an appropriate amount of purified water while stirring and dissolved, and then recoflavonoid monohydrate was added and dissolved. Hydrochloric acid was added to this solution to adjust the pH to 4-8, and then the osmosis enhancer, isotonic agent, buffer and preservative were added and dissolved. Separately, a viscosity agent was added to an appropriate amount of purified water and dissolved by heating or keeping at room temperature depending on the type, and then added to the above solution and mixed. Additional purified water was added to ensure the correct volume. After confirming that the pH was 4-8, the pH was adjusted as needed using a pH adjuster. This solution was passed through a filter of 0.22 μm or less to prepare a clear ophthalmic composition.

[0047] [Table 1]

[0048] Experimental Example 1: Corneal Penetration Test To evaluate the degree of penetration of ophthalmic compositions into the cornea and conjunctiva, the penetration rate was measured using miniature pig corneas, which are relatively impermeable and suitable for corneal penetration testing. Generally, the conjunctiva is reported to be 2 to 3 times more permeable than the cornea.

[0049] In vitro corneal permeability tests of the ophthalmic compositions of the comparative examples and examples were performed using a vertical Franz diffusion cell apparatus. Corneas and the surrounding sclera were collected from the eyes of miniature pigs weighing 40-50 kg for the tests. To fix the corneas in the Franz diffusion cell, the corneal region was exposed only in the central part of the receptor chamber. Then, the donor chamber was covered and fixed with a joint. Care was taken to remove air from the area where the receptor chamber and the cornea were in contact. 5 mL of pH 7.4 phosphoric acid solution (PBS buffer) and a magnetic stirrer were placed in the receptor chamber. 1 mL of the test solution was placed in the donor chamber, then the donor chamber was closed with a lid and sealed with Parafilm. During the permeability test, the stirrer was kept at approximately 500 rpm, and the Franz diffusion cell was kept at 36.5°C. After 0.5, 1, 2, 4, and 6 hours, 200 μL of each sample was taken and replenished with the same amount of phosphoric acid solution (PBS buffer). The drug concentration in the collected samples was measured using an HPLC analyzer. The concentration measured in each sample was converted to the permeation rate per hour, and the cumulative permeation amount of recoflavones over 6 hours was calculated by correcting for the amount of diluent. Results from an in vitro permeability test using miniature pig corneas confirmed that the permeation enhancer included in the example increased the cumulative permeation amount of recoflavones.

[0050] The cumulative penetration amount per unit area for each individual sample was plotted as the average value over the sampling time, and linear regression analysis was performed. The slope of the equation was calculated as the average penetration rate (flux) of the drug over 6 hours. The relative penetration enhancement rate was calculated as the ratio of the average penetration rates over 6 hours for each example compared to Comparative Example 1 (see Equation 1).

[0051]

number

[0052] Using the method described above, the average cumulative penetration amount per unit area of ​​the cornea, the average penetration rate over time, and the relative penetration enhancement rate of the ophthalmic compositions of Examples 1-3 and Comparative Examples 1 and 2 were calculated and are shown in Table 2.

[0053] Referring to Table 2, Examples 1 to 3 confirmed that, compared to Comparative Example 1, which contains only the active ingredient as in conventional eye drop compositions, the permeability of the drug differs depending on the presence or absence of a penetration enhancer at the same drug concentration. In addition, Example 1 confirmed that, compared to Comparative Example 2, the permeability improves depending on the concentration of recoflavones at the same composition.

[0054] In addition, it was confirmed that Comparative Example 2, which contained a penetration enhancer despite having a lower concentration of recoflavones, exhibited higher penetration than Comparative Example 1.

[0055] The results showed that in Example 1, the average penetration rate after 6 hours was 27 μg / cm³. 2 Since the rate was / hr, it was shown that the relative penetration enhancement rate increased 3.4 times compared to Comparative Example 1, which did not contain a penetration enhancer.

[0056] Furthermore, the improvement in dry eye symptoms in vivo was compared and evaluated in the experimental examples described below. The results showed that the overall improvement by Example 1 was significantly better than the improvement by Comparative Example 1, which did not contain a penetration enhancer.

[0057] Therefore, rather than simply improving laboratory data, we pursued a comprehensive improvement in in vivo dry eye symptoms. As a result, compared to Comparative Example 1, which does not contain a penetration enhancer, the relative penetration enhancement rate in the cornea of ​​miniature pigs at 6 hours should preferably be 3.0 or higher, or the cumulative penetration amount should preferably be 130 μg / cm³. 2 It was confirmed that this should be the case.

[0058] [Table 2]

[0059] Experiment Example 2: Tear secretion In a mouse model of dry eye, the effect of ophthalmic compositions on improving corneal permeability on tear secretion volume was evaluated.

[0060] NOD.B10.H2 in 12-14 week olds b Mice (Jackson Laboratory, Bar Harbor, USA) were acclimatized for 7 days and then subcutaneously injected with 0.5 mg / 0.2 mL of scopolamine hydrobromide into the hind limbs four times a day (8:00 AM, 11:00 AM, 2:00 PM, and 5:00 PM) for 10 days under dry stress with an average ambient humidity of less than 40%. After confirming the development of dry eye, placebo (a composition without the active ingredient in Example 1), Comparative Example 1, and Example 1 were each instilled into both eyes four times a day at a dose of 5 μL for 21 days.

[0061] To measure tear volume, a phenol red-impregnated cotton thread was placed at the outer corner of the eye for 20 seconds. The length of the thread from which the tears were absorbed was converted to microliters (μL).

[0062] Figure 2 shows the results of tear secretion in a dry eye experimental model mouse using the method described above. In Example 1, where corneal permeability was improved, tear volume was improved, but in Comparative Example 1, which contained 5% (w / v) recoflavones similar to Example 1, tear volume was almost the same as that of the placebo.

[0063] Experimental Example 3: Corneal Fluorescein Staining (CFS) The effect of ophthalmic compositions on improving corneal permeability in corneal fluorescence staining (CFS) in a mouse model of dry eye was evaluated.

[0064] Under the same conditions as in Experimental Example 2, placebo (a composition not containing the active ingredient of Example 1), Comparative Example 1, and Example 1 were instilled into the eyes for 10 days. 1 μL of 1% fluorescein reagent was applied to the eyes to stain the cornea, and the eyes were washed with physiological saline. Images were captured using a digital slit lamp, scored, and the degree of epithelial damage (degree of fluorescence staining) was evaluated. A score of 1 to 3 points was assigned to each of the five regions of the cornea, and the scores were totaled.

[0065] Figure 3 shows the results of corneal fluorescence staining (CFS) as a direct indicator of improvement in dry eye disease using the method described above. In Example 1, a significant decrease in corneal fluorescence staining (CFS) was observed. This means that the symptoms of dry eye were significantly improved. On the other hand, in Comparative Example 1, no improvement was observed compared to placebo.

[0066] Experimental Example 4: Evaluation of Corneal Surface Irregularities The effect of ophthalmic compositions on improving corneal permeability on corneal surface irregularities in a mouse model of dry eye was evaluated.

[0067] Under the same conditions as in Experimental Example 2, a placebo (composition without the active ingredient of Example 1), Comparative Example 1, and Example 1 were instilled into the eye for 10 days. The white ring image from the corneal surface was reflected and acquired using a fiber optic ring illuminator on a stereomicroscope (SZX7, Olympus). The corneal surface unevenness score was assigned to the reflected ring: 0 points: no distortion; 1 point: distortion in 1 / 4 of the ring; 2 points: distortion in 2 / 4; 3 points: distortion in 3 / 4; 4 points: distortion in all 4 / 4; 5 points: severe distortion.

[0068] To confirm the effect on corneal healing, the surface irregularities of the corneal surface were evaluated using the method described above (Figure 4). In Example 1, the surface irregularities of the corneal surface were reduced by approximately 30%. This indicated a significant improvement in the symptoms of dry eye. On the other hand, no difference was observed compared to the placebo in Comparative Example 1.

[0069] Experimental Example 5: Detachment of corneal epithelial cells The effect of improving corneal permeability of ophthalmic compositions on corneal epithelial cell exfoliation in a mouse model of dry eye was evaluated.

[0070] Under the same conditions as in Experimental Example 2, a placebo (a composition not containing the active ingredient of Example 1), Comparative Example 1, and Example 1 were instilled into the eyes for 21 days, and the animals were euthanized.

[0071] The periorbital region and lacrimal gland excised during surgery were fixed in 10% formalin for 3 days and embedded in paraffin. 5 μm tissue was microtome-cut and dried on a slide for 1 hour. This tissue was prepared by hematoxylin-eosin (H&E) staining, and the detached epithelial cells were examined using a virtual microscope (Nanozoomer 2.0RS, Hamamatsu, Japan) at a depth of 1 mm. 2 The corneal region was evaluated. To accurately compare the degree of improvement in dry eye symptoms, eye tissue immediately before drug administration after dry eye had been fully induced (initial stage) was compared with eye tissue from normal mice that had not been exposed to drug or dry environment stress for 10 days (normal eye).

[0072] Figure 5 shows the results of evaluating detached corneal epithelial cells. In normal eyes, only a small amount of detached epithelial cells were present, but exposure to a dry eye-inducing environment for 10 days (initial stage) significantly increased detachment. Comparative Example 1 showed a clear improvement compared to placebo. Example 1 showed an even better result, restoring the eye to a normal state. This indicates an effective reduction in detached epithelial cells.

[0073] Experimental Example 6: Conjunctival Goblet Cell Density The effect of improving corneal permeability of ophthalmic compositions on goblet cell density for mucus secretion in a mouse model of dry eye was evaluated.

[0074] Under the same conditions as in Experimental Example 5, paraffin-embedded tissue was sectioned and placed on slides. Staining was performed according to the instructions for the PAS (periodate Schiff reaction) kit, and goblet cell density was measured at 1 mm using a virtual microscope (Nanozoomer 2.0RS, Hamamatsu, Japan).2 It was evaluated in the conjunctival region.

[0075] Figure 6 shows the results of evaluating the conjunctival goblet cell density by the method described above. The initial conjunctival goblet cells showed a significant decrease compared to normal eyes after being exposed to the dry eye-inducing environment for 10 days. No recovery of conjunctival goblet cells was shown in the placebo. In Comparative Example 1, a clear improvement effect was shown compared to the placebo. On the other hand, in Example 1, a further improvement from Comparative Example 1 was shown. This showed a better result of recovering until approaching the level of normal eyes.

[0076] Experimental Example 7: Distribution of mucin The effect of improving the corneal permeability of an ophthalmic composition on the distribution of mucin, which is the main component of mucus in a dry eye experimental model mouse, was evaluated.

[0077] Under the same conditions as in Experimental Example 5, paraffin-embedded tissues were sectioned and placed on slides. Staining was performed according to the instructions of an Alcian blue staining kit, and in a virtual microscope (Nanozoomer 2.0RS, Hamamatsu, Japan), in the corneal and conjunctival regions of 1 mm 2 It was evaluated.

[0078] Figures 7 and 8 show the distribution of mucin by the method described above. The initial mucin showed a significant decrease compared to normal eyes after being exposed to the dry eye-inducing environment for 10 days. No recovery of mucin, which is the main component of mucus, was shown in the placebo. In Comparative Example 1, a clear improvement effect was shown compared to the placebo. On the other hand, in Example 1, a further improvement from Comparative Example 1 was shown. This showed a better result of recovering until approaching the level of normal eyes. These were consistent with the results of goblet cell density for mucin secretion in Experimental Example 6.

[0079] As described above, specific parts of the present invention have been explained in detail, but to those skilled in the art, these specific parts are merely preferred embodiments and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents.

Claims

1. An ophthalmic composition for treating dry eye disease, 5% (w / v) or more of recoflavones or a pharmaceutically acceptable salt thereof, Alone or in combination, containing 0.1-5.0% (w / v) of a polyoxyethylene castor oil derivative or poloxamer, Ophthalmic composition.

2. The ophthalmic composition for treating dry eye disease according to claim 1, wherein the polyoxyethylene castor oil derivative is polyoxyl 35 castor oil.

3. An ophthalmic composition for treating dry eye disease according to claim 1, wherein the poloxamer is poloxamer 407.

4. The ophthalmic composition for treating dry eye disease according to claim 1, wherein the ophthalmic composition for treating dry eye disease comprises 5% (w / v) to 10% (w / v) of recoflavones or a pharmaceutically acceptable salt thereof.

5. The ophthalmic composition for treating dry eye disease according to claim 1, wherein the ophthalmic composition for treating dry eye disease has a pH in the range of 5.0 to 7.0.

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