EYE DROP COMPOSITION FOR PREVENTING OR TREATING EYE DISEASE

Abstract

The present invention relates to an ophthalmic preparation containing an active ingredient selected from 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound or a pharmaceutically acceptable salt thereof. The eye drop according to the present invention is excellent in stability and safety and shows an excellent effect on prevention or treatment of eye diseases in such a way that an active ingredient thereof reaches a posterior segment of an eyeball simply through instillation rather than a direct injection into an eyeball.

Claims

1. A method for preventing or treating eye diseases, the method comprising: administering an eye drop comprising 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol represented by a following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient; a solubilizing agent; and a buffer agent and also having a pH range is 3.5 to 8.5 into a subject in need of treatment: ##STR00002##

2. A method for preventing or treating eye diseases, the method comprising: administering an eye drop comprising 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol represented by a following formula 1 or a pharmaceutically acceptable salt thereof by 0.1 w/v % to 2.0 w/v % as an active ingredient; a solubilizing agent; and a buffer agent and also having a pH range is 3.5 to 8.5 into a subject in need of treatment: ##STR00003##

3. A method for preventing or treating eye diseases, the method comprising: administering an eye drop comprising 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol represented by a following formula 1 or a pharmaceutically acceptable salt thereof by 0.1 w/v % to 2.0 w/v % as an active ingredient; a solubilizing agent; and a buffer agent and also having a pH range is 4.5 to 8.5 into a subject in need of treatment: ##STR00004##

4. The method according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride.

5. The method according to claim 2, wherein the pharmaceutically acceptable salt is hydrochloride.

6. The method according to claim 3, wherein the pharmaceutically acceptable salt is hydrochloride.

7. The method according to claim 1, wherein a concentration of the solubilizing agent is 0.1 w/v % to 25.0 w/v %.

8. The method according to claim 2, wherein a concentration of the solubilizing agent is 0.1 w/v % to 25.0 w/v %.

9. The method according to claim 3, wherein a concentration of the solubilizing agent is 0.1 w/v % to 25.0 w/v %.

10. The method according to claim 1, wherein the solubilizing agent is at least one selected from the group consisting of surfactants, solvents, and mixtures thereof.

11. The method according to claim 2, wherein the solubilizing agent is at least one selected from the group consisting of surfactants, solvents, and mixtures thereof.

12. The method according to claim 3, wherein the solubilizing agent is at least one selected from the group consisting of surfactants, solvents, and mixtures thereof.

13. The method according to claim 1, wherein the solubilizing agent comprises at least one of surfactants.

14. The method according to claim 2, wherein the solubilizing agent comprises at least one of surfactants.

15. The method according to claim 3, wherein the solubilizing agent comprises at least one of surfactants.

16. The method according to claim 1, wherein the eye disease is a posterior segment eye disease, and the posterior segment eye disease is at least one selected from the group consisting of diabetic retinopathy (DR), diabetic macular edema, age-related macular degeneration, acute macular neuroretinopathy, retinopathy of prematurity (ROP), polypoidal choroidal vasculopathy, ischemic proliferative retinopathy, retinitis pigmentosa, cone dystrophy, Behcet's disease, retinal disorders, proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal vein occlusion, retinitis, uveitis, Leber's hereditary optic neuropathy, retinal detachment, retinal pigment epithelial detachment, neovascular glaucoma, retinal neovascularization and choroidal neovascularization (CNV), trauma of the posterior segment, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic nerve disorder, non-retinopathy diabetic retinal dysfunction, and glaucoma.

17. The method according to claim 2, wherein the eye disease is a posterior segment eye disease, and the posterior segment eye disease is at least one selected from the group consisting of diabetic retinopathy (DR), diabetic macular edema, age-related macular degeneration, acute macular neuroretinopathy, retinopathy of prematurity (ROP), polypoidal choroidal vasculopathy, ischemic proliferative retinopathy, retinitis pigmentosa, cone dystrophy, Behcet's disease, retinal disorders, proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal vein occlusion, retinitis, uveitis, Leber's hereditary optic neuropathy, retinal detachment, retinal pigment epithelial detachment, neovascular glaucoma, retinal neovascularization and choroidal neovascularization (CNV), trauma of the posterior segment, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic nerve disorder, non-retinopathy diabetic retinal dysfunction, and glaucoma.

18. The method according to claim 3, wherein the eye disease is a posterior segment eye disease, and the posterior segment eye disease is at least one selected from the group consisting of diabetic retinopathy (DR), diabetic macular edema, age-related macular degeneration, acute macular neuroretinopathy, retinopathy of prematurity (ROP), polypoidal choroidal vasculopathy, ischemic proliferative retinopathy, retinitis pigmentosa, cone dystrophy, Behcet's disease, retinal disorders, proliferative vitreoretinopathy (PVR), retinal artery occlusion, retinal vein occlusion, retinitis, uveitis, Leber's hereditary optic neuropathy, retinal detachment, retinal pigment epithelial detachment, neovascular glaucoma, retinal neovascularization and choroidal neovascularization (CNV), trauma of the posterior segment, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic nerve disorder, non-retinopathy diabetic retinal dysfunction, and glaucoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIGS. 1 to 3 are graphs showing an amount of related substances produced depending on the pH of the ophthalmic composition measured according to Test Example 1.

[0048] FIG. 4 is a graph showing the result of observing and digitizing the size of CNV lesions after performing a retinal imaging evaluation using FFA after administration of the composition of the present invention (Example 13) and the control group according to Test Example 3.

[0049] FIG. 5 is a picture showing the comparison of the sizes of CNV lesions by performing a retinal imaging evaluation using FFA after administration of the composition of the present invention (Example 13) and the control group according to Test Example 3.

[0050] FIG. 6 is a graph showing the result of observing and digitizing the cross-sectional area of CNV lesions after performing a retinal imaging evaluation using OCT after administration of the composition of the present invention (Example 13) and the control group according to Test Example 3.

[0051] FIG. 7 is a picture showing the comparison of the cross-sectional areas of CNV lesions by performing a retinal imaging evaluation using OCT after administration of the composition of the present invention (Example 13) and the control group according to Test Example 3.

[0052] FIG. 8 is a graph showing the result of observing and digitizing the size of CNV lesions after performing a retinal imaging evaluation using FFA after administration of Example 19 among the compositions of the present invention, as well as Comparative Example 2 and the control group according to Test Example 4.

MODE FOR INVENTION

[0053] Hereinafter, the present invention will be described in more detail through exemplary embodiments. These exemplary embodiments are provided only for the purpose of illustrating the present invention in more detail, and thus it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Examples 1 to 9: Preparation of 0.1 w/v % to 0.5 w/v % APX-115 Eye Drops at pH 7.2

[0054] A composition containing 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol hydrochloride (APX-115) was prepared with the addition of a solubilizing agent and a buffer agent as shown in table 1 below.

[0055] According to the following examples, the compositions were prepared by heating or adjusting pH depending on a concentration of the active ingredient, if necessary.

TABLE-US-00001 TABLE 1 (Composition of 0.1 w/v % to 2.0 w/v % APX-115 eye drops at pH 7.2) Component Example name 1 2 3 4 5 6 7 8 9 Active APX-115 0.1 0.1  0.25  0.25  0.25  0.25 0.5 1.0 2.0 ingredient Solubilizing Ethanol 0.2 — — — 0.5 0.5 1.0 — 2.0 agent Propylene — — — 0.2 0.5 0.2 — 1.0 1.5 glycol Castor oil — 0.2 — — — — 1.0 1.5 1.5 Polyoxyl 35 — 2.5 — 2.0 — — — 20.0  castor oil Polysorbate 80 2.0 — — 5.0 — 5.0 — 10.0 — Sodium lauryl — 2.0 — — — — 5.0 — — sulfate Buffer solution q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. pH 7.2

[0056] In Examples 1 to 9, anhydrous sodium dihydrogen phosphate and anhydrous sodium phosphate were used as the buffer solution, and the pH was adjusted with 0.5 N sodium hydroxide solution, if necessary.

[0057] Example 1: 0.2 g of ethanol and 2 g of polysorbate 80 were mixed and homogenized. After the resulting mixed solution was heated to 40° C. or higher, 0.1 g of APX-115 was added and sonicated to dissolve and homogenize the resulting mixture, and then the buffer solution was added to adjust a final volume to 100 mL, and then filtered through a 0.22 um sterile filter.

[0058] Example 2: 0.1 g of APX-115 was added to 0.2 g of castor oil, heated to 40° C. or higher, and sonicated to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL by using the buffer solution, in which 2 g of sodium lauryl sulfate was dissolved in the buffer solution of Example 1, to the mixed solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter.

[0059] Example 3: 0.5 N sodium hydroxide solution was added to 25 mL of 10.0% polyoxyl 35 castor oil solution to adjust the pH to 9.0 or higher, and then 0.25 g of APX-115 was added to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL with the addition of the buffer solution to the mixed solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter.

[0060] Example 4: 0.5 N sodium hydroxide solution was added to the mixed solution, in which 0.2 g of propylene glycol and 5 g of polysorbate 80 were mixed and homogenized, so as to adjust the pH to 9 or higher, and then 0.25 g of APX-115 was added to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL with the addition of the buffer solution to the mixed solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter.

[0061] Examples 5 to 6: After homogeneously mixing each of the corresponding amounts of solubilizing agents specified in table 1, 0.25 g of APX-115 was added, heated to 40° C. or higher, and mixed with shaking to dissolve and homogenize the resulting mixture. The buffer solution was added to the mixed solution to adjust a final volume to 100 mL, and then filtered through a 0.22 μm sterile filter.

[0062] Example 7: After homogeneously mixing 1 g of ethanol and 1 g of caster oil, 0.25 g of APX-115 was added, heated to 40° C. or higher, and sonicated to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL by adding the buffer solution (5% sodium lauryl sulfate buffer solution), in which 5 g of sodium lauryl sulfate was dissolved in the buffer solution of Example 1, to the mixed solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter.

[0063] Example 8: 0.5 N sodium hydroxide solution was added to the mixed solution, in which 1 g of propylene glycol and 1.5 g of caster oil were mixed and homogenized, so as to adjust the pH to 9 or higher, and then 1.0 g of APX-115 was added to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL by adding and mixing 10 g of polysorbate 80 to the mixed solution having APX-115 dissolved therein and adding the buffer solution, and then filtered through a 0.22 μm sterile filter.

[0064] Example 9: A mixed solution obtained by mixing and homogenizing 2 g of ethanol, 1.5 g of propylene glycol, and 1.5 g of castor oil was heated to 40° C. or higher, after which 2.0 g of APX-115 was added to dissolve and homogenize the resulting mixture. A final volume was adjusted to 100 mL by adding and mixing 20 g of polyoxyl 35 castor oil to the mixed solution having APX-115 dissolved therein and adding the buffer solution, and then filtered through a 0.22 μm sterile filter.

Examples 10 to 14: Preparation of 0.5 w/v % APX-115 Eye Drops at pH 3.5 to 8.5

[0065] Eye drops containing APX-115 and solubilizing agents and having different pHs were prepared according to table 2 below.

TABLE-US-00002 TABLE 2 (Composition of 0.5 w/v % APX-115 eye drops at pH 3.5 to 8.5) Example Component name 10 11 12 13 14 Active APX-115 0.5 0.5 0.5 0.5 0.5 ingredient Solubilizing Ethanol 1.0 1.0 1.0 1.0 1.0 agent Propylene glycol 0.5 0.5 0.5 0.5 0.5 Castor oil 1.0 1.0 1.0 1.0 1.0 Polyoxyl 35 castor oil 5.0 5.0 5.0 5.0 5.0 Hydroxypropylbetadex 5.0 5.0 5.0 5.0 5.0 Buffer solution q.s. q.s. q.s. q.s. q.s. pH 3.5 4.5 5.5 7.2 8.5

[0066] Examples 10 to 14: After weighing each of the corresponding amounts of solubilizing agents except hydroxypropylbetadex according to table 2 and homogeneously mixing the resulting solution, 0.5 g of APX-115 was added and mixed with shaking to dissolve and homogenize the resulting mixture. In order to prepare an eye drop adjusted to the corresponding pH, each buffer solution suitable for the pHs of Examples 10 to 14 was prepared, and then 5 g of hydroxypropylbetadex was added and dissolved. A volume was adjusted to 100 mL by adding the prepared buffer solution containing hydroxypropylbetadex to the mixed solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter. In order to adjust pH suitable for each eye drop of Examples 10 to 14, the following buffer agent was used to prepare the buffer solution. Specifically, an ammonium acetate buffer solution at pH 3.5 was prepared with ammonium acetate and hydrochloric acid, an acetate buffer solution at pH 4.5 was prepared with acetic acid and sodium acetate, a citrate buffer solution at pH 5.5 was prepared with citric acid and disodium hydrogen phosphate, a phosphate buffer solution at pH 7.2 was prepared with sodium dihydrogen phosphate and sodium phosphate, and a borate buffer solution at pH 8.5 was prepared with borax.

Examples 15 to 22: Preparation of 1.0 w/v % or 0.5 w/v % APX-115 Eye Drops at pH 4.5 to 8.5

[0067] Eye drops containing APX-115 and solubilizing agents and having different pHs were prepared according to table 3 below.

TABLE-US-00003 TABLE 3 (Composition of 1.0 w/v % or 0.5 w/v % APX-115 eye drops at pH 4.5 to 8.5) Example Component name 15 16 17 18 19 20 21 22 Active APX-115 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 ingredient Solubilizing Castor oil — — — — 1.0 1.0 1.0 1.0 agent Capmul MCM NF 0.5 0.5 0.5 0.5 — — — — Ethanol 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Propylene glycol 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Polyoxyl 35 castor oil 5.0 5.0 5.0 5.0 — — — — Polysorbate 80 — — — — 5.0 5.0 5.0 5.0 Hydroxypropylbetadex 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Buffer solution q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. pH 4.5 6.0 7.2 8.5 4.5 6.0 7.2 8.5

[0068] After weighing each of the corresponding amounts of solubilizing agents except hydroxypropylbetadex according to table 3 and homogeneously mixing the resulting solution, 0.5 g of APX-115 was added and mixed with shaking to dissolve and homogenize the resulting mixture. In order to prepare an eye drop adjusted to the corresponding pH, each buffer solution suitable for the pHs of Examples 15 to 22 was prepared, and then 5 g of hydroxypropylbetadex was added and dissolved. A volume was adjusted to 100 mL by adding the buffer solution containing hydroxypropylbetadex prepared in accordance with each pH to the solution having APX-115 dissolved therein, and then filtered through a 0.22 μm sterile filter. The buffer solutions at pH 4.5, 7.2, and 8.5 used were prepared by the same method as the buffer solutions used in Examples 10 to 14, and the buffer solution at pH 6.0 was prepared with disodium hydrogen phosphate and citric acid (pH adjusted with phosphoric acid).

Examples 23 to 31: Preparation of 0.5 w/v % APX-115 Eye Drops at pH 7.2

[0069] The 0.5 w/v % APX-115 eye drop at pH 7.2 containing APX-115 and solubilizing agents was prepared according to table 4 below.

TABLE-US-00004 TABLE 4 (Composition of 0.5 w/v % APX-115 eye drops at pH 7.2) Example Component name 23 24 25 26 27 28 29 30 31 Active APX-115 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ingredient Solubilizing Castor oil 1.5 — — — — — — — — agent Capmul MCM 2.0 — — — — — — — NF Glycerin — — 2.5 — — — — — — Lanolin oil — — — 1.0 — — — — — Mineral oil — — — — 0.5 — — — — Peanut oil — — — — — 0.5 — — — Ethanol — — — — — — 1.0 1.0 — Propylene glycol — — — — — — — — 0.5 Phenyl ethyl — — — — — — 2.0 — 2.0 alcohol Propylene glycol — — — — — — — 3.0 — diacetate Polyoxyl 40 5.0 5.0 — 5.0 — 5.0 5.0 — — hydrogenated castor oil Polyoxyl 40 — — 10.0 — — — — 10.0 — stearate Polypropylene — 5.0 2.0 — 2.5 — — — 10.0 glycol Nonoxynol — — — — 0.1 — 0.1 — — Octoxynol 0.05 — — — — 0.05 — 0.05 — Tyloxapol — — — 5.0 — — — — — Polyoxyl 35 5.0 — — — — 5.0 — — — castor oil Buffer solution q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. pH 7.2

[0070] The solubilizing agents corresponding to each of Examples 23 to 31 were homogeneously dissolved and mixed, and then heated (40° C. or higher). After adding 500 mg of APX-115 to each composition in which the solubilizing agents were mixed, the resulting mixture was sufficiently dissolved by stirring, and then a final volume was adjusted to 100 mL with a buffer solution at pH 7.2 so as to prepare 0.5 w/v % APX-115 eye drops. The buffer solution at pH 7.2 used was prepared by the same method as the buffer solution used in the preparation of Examples 1 to 9.

Examples 32 to 40: Preparation of 0.5 w/v % APX-115 Eye Drops at pH 6.0

[0071] The 0.5 w/v % APX-115 eye drop at pH 6.0 containing APX-115 and solubilizing agents was prepared according to table 5 below.

TABLE-US-00005 TABLE 5 (Composition of 0.5 w/v % APX-115 eye drops at pH 6.0) Component Example name 32 33 34 35 36 37 38 39 40 Active APX-115 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 ingredient Solubilizing Castor oil 1.5 — — — — — — — — agent Capmul MCM 2.0 — — — — — — — NF Glycerin — — 2.5 — — — — — — Lanolin oil — — — 1.0 — — — — — Mineral oil — — — — 0.5 — — — — Peanut oil — — — — — 0.5 — — — Ethanol — — — — — — 1.0 1.0 — Propylene glycol — — — — — — — — 0.5 Phenyl ethyl — — — — — — 2.0 — 2.0 alcohol Propylene glycol — — — — — — — 3.0 — diacetate Polyoxyl 40 5.0 5.0 — 5.0 — 5.0 5.0 — — hydrogenated castor oil Polyoxyl 40 — — 10.0 — — — — 10.0 — stearate Polypropylene — 5.0 2.0 — 2.5 — — — 10.0 glycol Nonoxynol — — — — 0.1 — 0.1 — — Octoxynol 0.05 — — — — 0.05 — 0.05 — Tyloxapol — — — 5.0 — — — — — Polyoxyl 35 5.0 — — — — 5.0 — — — castor oil Buffer solution q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. pH 6.0

[0072] After adding 500 mg of APX-115 to a solution at pH 9 or higher (sodium hydroxide solution), the resulting mixture was sufficiently stirred and dissolved to adjust a final volume to 100 mL with a buffer solution containing additives according to the composition described in table 5 below, so as to prepare 0.5 w/v % APX-115 eye drops. The buffer solution at pH 6.0 was prepared with disodium hydrogen phosphate and citric acid, and the buffer solution used was the same as the buffer solutions used in the preparation of Examples 16 and 20.

Comparative Examples 1 to 8: Preparation of APX-115 Eye Drops at pH 3.0 or Lower or 9.0 or Higher

[0073] Compositions containing APX-115 were prepared as shown in table 6 below so that the compositions have the same ingredients as and different pHs from those of Examples 10 to 22.

TABLE-US-00006 TABLE 6 (Composition of APX-115 eye drops at pH 3.0 or lower or 9.0 or higher) Comparative Example Component name 1 2 3 4 5 6 7 8 Active APX-115 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.0 ingredient Solubilizing Castor oil 1.0 1.0 1.0 1.0 — — 1.0 1.0 agent Capmul MCM NF — — — — 0.5 0.5 — — Propylene glycol 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Ethanol 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Polyoxyl 35 castor oil 5.0 5.0 5.0 5.0 5.0 5.0 — — Polysorbate 80 — — — — — — 5.0 5.0 Hydroxypropylbetadex 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Buffer solution q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. pH 2.5 3.0 9.0 9.5 3.0 9.0 3.0 9.0

[0074] After weighing each of the corresponding amounts of solubilizing agents except hydroxypropylbetadex according to table 6 and homogeneously mixing the resulting solution, 0.5 g of APX-115 was added and mixed with shaking to dissolve and homogenize the resulting mixture. In order to prepare eye drops adjusted to the corresponding pHs, each buffer solution suitable for the pHs of Comparative Examples 1 to 8 was prepared, and then 5 g of hydroxypropylbetadex was added and dissolved. The buffer solution prepared in accordance with each pH was added to the solution in which APX-115 was dissolved, adjusted to a volume of 100 mL, and then filtered through a 0.22 μm sterile filter. Ammonium dihydrogen phosphate and phosphoric acid were used for the buffer solutions at pH 2.5 and pH 3.0, the buffer solution at pH 9.0 was prepared with sodium tetraborate and hydrochloric acid, and the buffer solution at pH 9.5 was prepared with 2-amino-2-hydroxymethyl-1,3-propanediol and hydrochloric acid.

Test Example 1: Related Substance Test

[0075] The solutions prepared in above Examples and Comparative Examples were stored under the condition of 40° C./75% RH. In four weeks later, the maximum amount of unknown related substances among APX-115 decomposition products was measured by an HPLC method under the conditions described below.

[0076] <Analysis Conditions> [0077] Column: Kromasil C18 (4.6×150 mm, 5 μm) [0078] Column temperature: 30° C. [0079] Mobile phase: 20 mM ammonium formate (pH 3.0)/acetonitrile=20/80 (v/v) [0080] Wavelength: 293 nm [0081] Injection amount: 10 μl

[0082] In the case of Examples 10 to 14 having the same composition of additives, but having pH ranges of 3.5 to 8.5 merely different from each other, as confirmed from FIG. 1, it can be understood that the related substances originating from the main ingredient are effectively suppressed, thereby improving the chemical stability of 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound. In contrast, in the case of Comparative Examples 1 and 2 having the same composition of additives, but having a pH of 3.0 or less, and in the case of Comparative Examples 3 and 4 having the same composition of additives, but having a pH of 9.0 or more, it was confirmed that 0.5% or more of impurities are generated within four weeks of storage after preparation, thereby not improving the chemical stability.

[0083] In addition, in the case of Examples 15 to 18 having the same composition of additives, but having pH ranges of 3.5 to 8.5 merely different from each other, as confirmed from FIG. 2, it seems that the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound shows an improvement in stability. In the case of Comparative Examples 5 (pH 3.0) and 6 (pH 9.0), it seems that the compositions thereof show stability lower than that of the compositions of Examples.

[0084] Furthermore, in the case of Examples 19 to 22 having a pH range of 3.5 to 8.5, as confirmed from FIG. 3, it seems that the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound shows an improvement in stability with less generation of impurities. In the case of Comparative Examples 7 (pH 3.0) and 8 (pH 9.0), it seems that the compositions thereof show stability lower than that of the compositions of Examples.

[0085] In other words, it was confirmed from the results of Test Example 1 that the eye drop of one embodiment containing the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound shows excellent stability in the pH range of 3.5 to 8.5.

[0086] According to the ICH guidelines, for a drug product with a maximum daily dose of 1 mg or more and 10 mg or less, a criterion for managing unknown impurities with an unidentified structure is set to 0.5% or less. Accordingly, it was confirmed that the compositions of Examples 10 to 22 having a pH range of 3.5 to 8.5 show a remarkable improvement in the stability of the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound compared to the compositions of Comparative Examples 1 to 8 having a pH range of 3.0 or lower or 9.0 or higher.

Test Example 2: Test on Eye Mucosal Irritation

[0087] After instilling 50 uL of each of the compositions of above Examples 10 to 14 and Comparative Examples 1 to 4 once into both eyes of a New Zealand white rabbit weighing 2.0 to 2.7 kg, the behavioral patterns of the experimental animal were observed. Examples 10 to 14 were used as a test group, Comparative Examples 1 to 4 were used as a Comparative group, and Diquas®, which is a commercially available eye drop, was used as a positive control group. Immediately after drug administration, the behavioral patterns of each rabbit were observed and shown in the table below.

TABLE-US-00007 TABLE 7 (Table 7: Results of eye mucosal irritation test evaluation) Irritation Comparative Irritation Example pH intensity Example pH intensity* 10 3.5 +(+) 1 2.5 +++ 11 4.5 + 2 3.0 +++ 12 5.5 + 3 9.0 ++ 13 7.2 + 4 9.5 +++ 14 8.5 + Diquas ® 7.3 + Irritation intensity* +++: Very serious, ++: Moderate, +: Mild, +(+): More serious than + and weaker than ++

[0088] As a result of evaluating an eye mucosal irritation using rabbits, Examples 10 to 14, which are compositions of the present invention, showed a degree of reaction (eye blinking) similar to that of Diquas® available on the market unlike Comparative Examples 1 to 4 with severe irritation intensity, and thus it was confirmed that the composition of the present invention having the pH 3.5 to 8.5 shows good results even for eye mucosal irritation. In particular, it was confirmed that the composition at pH 4.5 to 8.5 shows a more excellent degree of eye mucosal irritation.

Test Example 3. Test on Intraocular Tissue Distribution (Intravitreal PK Evaluation)

[0089] In order to confirm the effect of the composition of the present invention in which a drug thereof is delivered to the posterior segment through ophthalmic administration alone without injection administration, thereby showing a therapeutic efficacy, a concentration of the active ingredient in the vitreous body adjacent to the retina/choroid among the posterior segment tissues of the eyeball was confirmed through the test of Test Example 3. In addition, it was confirmed from a previous study on stability (Test Example 1) that there is a difference in stability of the composition depending on the pH of the composition, and a test on PK distribution for each pH was performed in order to evaluate an amount of the active ingredient delivered to the posterior segment tissues depending on pH of the composition, since the degree of ionization of the active ingredient varies according to the pH.

[0090] The evaluation method of this Test Example 3 is an efficient evaluation method capable of predicting the efficacy on posterior segment eye diseases by measuring the amount of the drug in the vitreous body inside the eyeball, which is delivered by instillation, and thus by comparing the amount of the drug that can reach the posterior segment, which is the target organ.

[0091] With regard to the compositions of Examples prepared to have the pH range of 3.5 to 8.5 and the compositions of Comparative Examples prepared to have the pH range of 3.5 or lower or 9.5 or higher among the compositions of the present invention, each of 40 uL of those compositions was instilled once into both eyes of a New Zealand white rabbit weighing 2.0 to 2.7 kg. In 30 minutes later, a vitreous humor was collected from the eye tissues of the experimental animal and the concentration of the compound in the sample was measured by using an LC/MS/MS equipment.

[0092] <Analysis Conditions> [0093] Column: Waters Acquity UPLC BEH C18 (50×2.1 mm, 1.7 mm) [0094] Column temperature: 40° C. [0095] Mobile phase: Acetonitrile/0.100 formic acid=70/30 (v/v) [0096] Injection amount: 5 μl [0097] Flux: 0.2 mL/min

TABLE-US-00008 TABLE 8 (Table 8: Concentration of active ingredient in vitreous humor after instillation of the composition of the present invention) Concentration of active Composition (concentration of ingredient in active ingredient in eye drops) pH vitreous humor (ng/mL) Comparative Example 2 3.0 0.8 ± 0.5 (0.5 w/v %) Example 10 (0.5 w/v %) 3.5 2.5 ± 1.0 Example 11 (0.5 w/v %) 4.5 4.8 ± 3.2 Example 16 (0.5 w/v %) 6.0 5.1 ± 3.4 Example 34 (0.5 w/v %) 6.0 4.3 ± 2.7 Example 1 (0.1 w/v %) 7.2 3.1 ± 1.6 Example 3 (0.25 w/v %) 7.2 4.0 ± 3.1 Example 13 (0.5 w/v %) 7.2 5.7 ± 2.9 Example 14 (0.5 w/v %) 8.5 3.5 ± 1.9 Comparative Example 4 9.5 0.3 ± 0.2 (0.5 w/v %)

[0098] As a result of the eyeball PK test using rabbits, it was observed that the concentration of the active ingredient in the vitreous humor is very low in Comparative Example 2 at pH 3.0 and Comparative Example 4 at pH 9.5 and thus it could be confirmed that the amount of the active ingredient delivered to the posterior segment after instillation is very small. In contrast, it was observed that the concentration of the active ingredient in the vitreous humor is remarkably high in Examples 1, 3, 10, 11, 13, 14, 16 and 34 at pH 3.5 to 8.5 compared to Comparative Examples. In particular, the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound has a maximum level of lipophilicity (hydrophobicity) from pH 5 or lower due to the properties of the drug. Thus, although all the biomembrane permeabilities were theoretically the same in an acidic state of pH 5 or less, it was confirmed that the delivery of the active ingredient to the posterior segment becomes very low, particularly at pH 3.0 or less.

[0099] From the above results, it was confirmed that the delivery power of the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazol-5-ol compound to the posterior segment after instillation is most excellent in the pH range of 3.5 to 8.5.

[0100] In addition, with regard to the ophthalmic composition of one embodiment, the concentration of the active ingredient in the vitreous humor shows the same degree of concentration even when using different solubilizing agents. Thus, it was confirmed that the solubilizing agents do not have a significant effect on the delivery of the active ingredient to the posterior segment, as long as the solubilizing agents are capable of solubilizing the active ingredient herein.

Test Example 4. Evaluation of Validity of Ophthalmic Composition Using Mouse CNV Model

[0101] It was confirmed from the previous test on distribution inside eyeball tissues (Test Example 3) that the active ingredient is distributed to the vitreous body adjacent to the retina, which is the posterior segment tissue, when administering the ophthalmic composition for each pH according to one embodiment. After that, in order to confirm the inhibitory effect of the ophthalmic composition of the present invention (Example 13) on choroidal neovascularization (CNV), the efficacy was evaluated with the mouse CNV model by selecting the composition of the present invention as a test material, a placebo material as a negative control material (G1), and Eylea®, which is a commercially available injection, as a positive control material (G2).

[0102] The negative control material (G1) and test materials (G3 and G4) were instilled at a dose of 5 uL once from the day after CNV induction, and the positive control material (G2) was directly injected once into the vitreous body at a dose of 1 uL (20 ug/uL) by using a 36 G syringe on the day after CNV induction. The negative control material was administered four times a day. The efficacy of the test material was compared and evaluated according to the number of daily administrations, which was divided into an administration group of four times (G3) and an administration group of eight times (G4).

[0103] Specifically, on the 12th day after CNV induction, the experimental animal was subjected to general anesthesia and to intraperitoneal injection of a fluorescent contrast agent. Then, an anesthetic eye drop was applied into the eyeball of the experimental animal to induce additional local anesthesia. After that, mydriatic was instilled to induce mydriasis and perform a retinal image evaluation. Thus, the results are shown in FIGS. 4 to 7.

[0104] As a result of the test, as can be confirmed from FIGS. 4 to 7, in the case of administering the composition of the present invention four times only a day (G3), the composition of the present invention showed an effect equal to or higher than that of the group dosed with Eylea® (positive control group, G2), which is directly injected into the vitreous body.

[0105] The results of the evaluation using fundus fluorescein angiography (FFA) during retinal imaging evaluation are shown in FIGS. 4 and 5. Referring to FIGS. 4 and 5, it was confirmed from the comparison between the negative control group (G1) and the test groups (G3 and G4) that there is a significant difference therebetween and the size of the lesion is reduced in the test group, thereby providing a therapeutic effect. In addition, it was confirmed that administration of four times only (G3) shows a similar therapeutic effect compared to Eylea, which is the positive control group (G2).

[0106] FIGS. 6 and 7 show the results of evaluation using an optical coherence tomography (OCT) during retinal imaging evaluation. Referring to FIGS. 6 and 7, it was confirmed that the groups dosed with the test material (G3, G4) show a therapeutic effect due to a decrease in the cross-sectional area of the lesion in the test groups, and also show a statistically significant difference even when comparing the negative control group with the test groups. In addition, it was confirmed that instillation of four times only a day (G3) shows a therapeutic effect equal to or higher than that of the group dosed with Eylea, which is the positive control group (G2).

[0107] As a result of evaluating the efficacy of the composition of the present invention through Test Example 4, it was confirmed that the composition of the present invention has an effect equal to or higher than that of the commercially available Eylea injection (the positive control group, G2) in the CNV animal model.

Test Example 5. Evaluation of Validity According to pH Change of Ophthalmic Composition

[0108] A validity evaluation was performed by the same method as Test Example 4 with each ophthalmic composition having the same composition, but having different pHs.

[0109] Among the ophthalmic compositions of the present invention, Example 19 (G3) showed excellent stability and excellent PK results, and Comparative Example 2 (G4) showed low stability and poor PK test results. Accordingly, to confirm the inhibitory effect of both Example 19 (G3) and Comparative Example 2 (G4) on choroidal neovascularization (CNV), Example 19 and Comparative Example 2 were selected as a test material out of the compositions of the present invention, a placebo material was selected as a negative control material (G1), and Eylea®, which is a commercially available injection, was selected as a positive control material (G2), thereby evaluating the efficacy of the mouse CNV model.

[0110] The negative control material (G1) and test materials (G3 and G4) were instilled at a dose of 5 uL four times a day from the day after CNV induction, and the positive control material (G2) was directly injected once into the vitreous body at a dose of 1 uL (20 ug/uL) by using a 36 G syringe on the day after CNV induction.

[0111] As a result of the test, as can be confirmed from FIG. 8 (evaluation using FFA), in the case of administering Example 19 four times only a day (G3) out of the compositions of the present invention, it was confirmed that Example 19 shows an efficacy equal to or higher than that of the group dosed with Eylea® (positive control group, G2), which is directly injected into the vitreous body, and shows a significant therapeutic effect compared to the group dosed with the negative control material. In contrast, Comparative Example 2 was found to have no significance compared to the group dosed with the negative control material, and thus it was confirmed that Comparative Example 2 has a low or almost no effect of inhibiting choroidal neovascularization.

[0112] It could be confirmed from Test Examples 1 to 5 of the present invention that the eye drop of the 3-phenyl-4-propyl-1-(pyridin-2-yl)-1H-pyrazole-5-ol compound containing a solubilizing agent and a buffer agent and maintaining the pH range of 3.5 to 8.5 secures stability and safety as a drug product and has a remarkable effect on delivering a drug to the posterior segment as well as a remarkable therapeutic effect.