Eyedrop for inhibiting ocular pathological angiogenesis and preparation method therefor

Abstract

An eyedrop for inhibiting ocular pathological angiogenesis and a preparation method therefor. The eyedrop comprises the following components in weight-volume percentage: withaferin A, a solubilizer, a thickener, an antimicrobial agent, a tonicity adjuster, and a pH-adjusting agent, with the balance being water for injection. The specific preparation method comprises: mixing withaferin A with a solubilizer, and thoroughly stirring same until a uniform mixed solution is obtained for later use; heating a thickener, a pH-adjusting agent, a tonicity adjuster and an antimicrobial agent in a water bath at 60°C under stirring until a clear and transparent solution is obtained, and then cooling the solution to room temperature; adding the mixed solution to the solution, and making same up to the volume with water for injection; and sterilizing the prepared eyedrop by means of filtration through a 0.22 μm microporous filter membrane, and performing aseptic packaging to obtain the final product. The eyedrop has a good solubility and a prolonged retention time on the ocular surface, thus having a good therapeutic effect on pathological angiogenesis in the eye, particularly on the ocular surface. Moreover, the eyedrop has the advantages of a topical ocular formulation, including a good biocompatibility, a moderate viscosity and no irritation to the eyes; and also including a good permeability, ease of use and low systemic side effects.

Description

An eye drop for inhibiting ocular pathological angiogenesis and a preparation method thereof TECHNICAL FIELD

[0001] The present application relates to an eye drop, in particular to an eye drop for inhibiting ocular pathological angiogenesis and a preparation method thereof. BACKGROUND

[0002] Ocular pathological angiogenesis, i.e. abnormal growth of new blood vessels in the eye, among which ocular surface pathological angiogenesis, also known as corneal neovascularization, is a pathological change, rather than an independent corneal disease. Under normal circumstances, the cornea is colorless and transparent without blood vessels, when the cornea is covered with new blood vessels, these blood vessels will block part of the light, resulting in blurred or reduced vision. Ocular surface pathological angiogenesis can cause visual impairment, which is the second largest cause of blindness worldwide.

[0003] Trauma, infection, wearing low oxygen permeability contact lenses, corneal degeneration and malnutrition can cause ocular surface ischemia and inflammatory response, leading to increased vascular endothelial growth factor (VEGF) production and inducing pathological angiogenesis. Previous studies have found that Withaferin A, a small molecule compound extracted from Withania somnifera in South Africa, may prevent NF-κB activation by inhibiting the activation of IKKβ; it is believed to have anti-inflammatory and anti-tumor effects; our team's previous research has confirmed that Withaferin A has anti-hypoxic injury and anti-VEGF dual effects, and may become a potential eye drug raw material, Patent No.: 201910398536.6, discloses an "application of Withaferin A in preparation of drugs for treating ocular fundus ischemic diseases", but the invention uses a subcutaneous implant pump to give Withaferin A solution systemically to treat ocular fundus lesions. There are also related basic researches internationally that Withaferin A may have an inhibitory effect on corneal blood vessels, but all of them use the method of systemic administration of Withaferin A solution (such as subcutaneous injection). Mohan R, Bargagna-Mohan P et al. Academic Press, 2016, Investigative Ophthalmology & Visual Science, 2012, PLoS One, 2015, Investigative Ophthalmology & Visual Science, 2005 Chemistry & biology, 2007, 14(6): 623-634. All the above literatures are basic researches, which only simply dissolve Withaferin A and give it systemically, and never try to make it into related pharmaceutical preparations. A large number of researches by our research group have confirmed that Withaferin A is an anti-VEGF compound that inhibits angiogenesis, and if used systemically, it will inhibit the formation of collateral circulation in the cardiovascular system, which is easy to induce cardiovascular accidents, so it is obviously more harmful than beneficial to make it into a pharmaceutical preparation for systemic use. Therefore, our team turned to research to make it into an eye preparation for local use to avoid its cardiovascular side effects. In fact, Withaferin A has small molecular weight, stable structure and lipophilicity, and making it into eye drops undoubtedly has the advantages of direct effect, good penetration, convenience of use and less systemic side effects. However, so far, there has been no research report on Withaferin A eye drops. This is because there are technical barriers to making Withaferin A into eye drops: 1. Withaferin A is not soluble in water, and the organic solvents (such as methanol, DMSO) commonly recognized for Withaferin A have certain cytotoxicity, which will cause damage to the ocular surface and also cause eye irritation symptoms if used directly on the ocular surface. 2. Withaferin A needs to be made into eye preparations by adding excipients and other auxiliary materials to prolong the residence time on the ocular surface in order to achieve better therapeutic effect and less frequency of use. Therefore, solving these problems is the key to making the small molecule compound Withaferin A into eye drops which has not been used as a medicine so far.

[0004] The eye drop provided by the present application solves the technical barrier of preparing withering saponin A into eye drops, and has good therapeutic effect on eye, especially pathological angiogenesis of ocular surface. At present, there is no eye drop for inhibiting ocular angiogenesis by anti-VEGF, so it has clear innovation and market value. SUMMARY

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: an eye drop for inhibiting pathological angiogenesis of eye, containing the following components by weight volume ratio: withering saponin A 0.01-0.8 mg / L, a dissolving agent 1-25 g / L, a thickening agent 2-40 g / L, a bacteriostatic agent 0.04-0.2 g / L, an isotonicity adjusting agent and a pH adjusting agent in appropriate amount, and the balance being water for injection.

[0006] Further preferred scheme is that the dissolving agent is one or any mixture of Tween 80, propylene glycol, poloxamer, polyethylene glycol and beta cyclodextrin.

[0007] Further preferred scheme is that the thickening agent is one or any mixture of methyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium hyaluronate, povidone, hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose and carboxymethyl cellulose.

[0008] Further preferred scheme is that the pH adjusting agent is one or any mixture of boric acid, boric acid buffer, phosphate buffer and citrate buffer, and the amount of the pH adjusting agent is determined according to the pH value of the eye drop being 6.0-8.0.

[0009] Further preferred scheme is that the osmotic pressure adjusting agent is one or any mixture of sodium chloride, potassium chloride, boric acid, glucose and glycerol, and the amount of the osmotic pressure adjusting agent is determined according to the osmotic pressure molar concentration ratio being 0.9-1.1.

[0010] Further preferred scheme is that the bacteriostatic agent is one or any mixture of hydroxyphenyl ethyl ester, benzalkonium chloride, benzalkonium bromide and polyquaternary ammonium salt-1.

[0011] The method for inhibiting pathological angiogenesis of eye by the eye drop comprises the following steps:

[0012] Firstly, the corresponding weight volume ratio of withering saponin A and dissolving agent is weighed, mixed and stirred uniformly, and prepared for use;

[0013] Secondly, the corresponding weight volume ratio of thickening agent, pH adjusting agent, isotonicity adjusting agent and bacteriostatic agent is weighed, heated and stirred in 60℃ water bath until clear and transparent, and cooled to room temperature;

[0014] Thirdly, the mixture of the first step is added into the solution of the second step, and water for injection is used to make up the volume;

[0015] Fourthly, the prepared eye drops are filtered by a 0.22 μm microporous filter membrane to remove bacteria, and are sterilely packaged and distributed, thereby obtaining the eye drops.

[0016] The preparation method of the present application includes, but is not limited to, eye drops, eye washes, solid mixtures (dissolved before use), concentrated solutions (diluted before use), eye ointments, eye gels and other various ophthalmic preparations acceptable in the medical field.

[0017] The eye drops for inhibiting pathological angiogenesis of the eye are used for preparing a medicament for treating pathological neovascularization of the eye.

[0018] The eye drops of the present application have the following advantages:

[0019] (1) The eye drops of the present application make solubility of withaferin A better and prolong the residence time of withaferin A on the ocular surface. Compared with withaferin A dissolved by using a traditional solvent, the withaferin A dosage form disclosed in the present application can better exert the pharmacological effect of withaferin A, and has a definite therapeutic effect on pathological neovascularization of the eye. The withaferin A eye drops have good biocompatibility, moderate viscosity and no irritation to the eye. Therefore, the withaferin A eye drops have transformation value.

[0020] (2) The eye drops of the present application not only have the anti-inflammatory, anti-oxidative stress and anti-VEGF effects of withaferin A itself, but also have the advantages of a local eye preparation, such as direct effect, good permeability, convenient use and small systemic side effects.

[0021] (3) The effective component withaferin A in the eye drops disclosed in the present application has good solubility and permeability, and therefore can treat deeper neovascularization of the eye, such as iris neovascularization, angle neovascularization and fundus neovascularization, when the concentration of withaferin A in the preparation is high. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a graph showing that the withaferin A eye drops improve pathological neovascularization of the mouse cornea caused by alkali burn, while the use of withaferin A solution alone has no obvious effect;

[0023] Figure 2 is a graph further proving that the withaferin A eye drops significantly improve pathological neovascularization of the mouse cornea caused by alkali burn;

[0024] Figure 3 is a graph showing that the withaferin A eye drops improve corneal opacity caused by alkali burn in mice;

[0025] Figure 4 is a graph showing that the withaferin A eye drops improve pathological neovascularization of the mouse cornea caused by suturing, while the use of withaferin A solution alone has no obvious effect;

[0026] Figure 5 is an immunofluorescence further confirming that withaferin A eye drops significantly improve the pathological angiogenesis of the cornea of mice caused by suturing. DETAILED DESCRIPTION

[0027] The following examples and experimental examples are only further illustrations of the present application and should not be understood as limiting the present application. Example 1

[0028] The eye drops for inhibiting pathological angiogenesis of the eye of the present embodiment contain the following ingredients in the weight / volume ratio: withaferin A 0.01 mg / L, Tween 80 1 g / L, povidone K30 5.6 g / L, benzalkonium chloride 0.1 g / L, sodium chloride 6.2 g / L, sodium dihydrogen phosphate 4 g / L, disodium hydrogen phosphate 7.1 g / L, and the balance being water for injection.

[0029] The amount of sodium dihydrogen phosphate and disodium hydrogen phosphate is adjusted to a pH value of 6.8 of the eye drops.

[0030] The amount of sodium chloride is adjusted to a molar concentration ratio of 0.9.

[0031] The method for inhibiting pathological angiogenesis of the eye of the present embodiment comprises the following steps:

[0032] In the first step, the corresponding weight / volume ratio of withaferin A and Tween 80 is weighed and mixed, and then stirred thoroughly to obtain a mixture, which is ready for use.

[0033] In the second step, the corresponding weight / volume ratio of povidone K30, benzalkonium chloride, sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate is weighed and heated to be clear and transparent in a 60°C water bath, and then cooled to room temperature.

[0034] In the third step, the mixture of the first step is added to the solution of the second step, and then diluted with water for injection.

[0035] In the fourth step, the prepared eye drops are filtered with a 0.22 μm microporous filter to remove bacteria, and then aseptically packaged to obtain the eye drops.

[0036] The eye drops for inhibiting pathological angiogenesis of the eye of the present embodiment are used for treating pathological angiogenesis of the eye. Example 2

[0037] The eye drops for inhibiting pathological angiogenesis of the eye of the present embodiment contain the following ingredients in the weight / volume ratio: withaferin A 0.1 mg / L, Tween 80 5 g / L, povidone K30 5.6 g / L, benzalkonium chloride 0.1 g / L, sodium chloride 6.2 g / L, sodium dihydrogen phosphate 4 g / L, disodium hydrogen phosphate 7.1 g / L, and the balance being water for injection.

[0038] The amount of sodium dihydrogen phosphate and disodium hydrogen phosphate is adjusted to the pH value of 6.8 of the eye drops.

[0039] The amount of sodium chloride is adjusted to the osmotic pressure molar concentration ratio of 0.9.

[0040] The method for inhibiting pathological angiogenesis of the eye drops comprises the following steps:

[0041] First step, take the corresponding weight volume ratio of withering A and Tween 80, mix well and reserve;

[0042] Second step, take the corresponding weight volume ratio of povidone K30, benzalkonium chloride, sodium chloride, sodium dihydrogen phosphate and disodium hydrogen phosphate, heat and stir in a 60℃ water bath until clear and transparent, and cool to room temperature;

[0043] Third step, add the mixed solution of the first step to the solution of the second step, and add water for injection to constant volume;

[0044] Fourth step, filter the prepared eye drops with a 0.22μm microporous filter to remove bacteria, and sterilely package to obtain the eye drops.

[0045] The eye drops for inhibiting pathological angiogenesis of the eye are used for treating pathological angiogenesis of the eye. Example 3

[0046] The eye drops for inhibiting pathological angiogenesis of the eye of the present embodiment contain the following ingredients in weight volume ratio: withering A 0.8mg / L, Tween 80 8g / L, povidone K30 5.6g / L, benzalkonium chloride 0.1g / L, sodium chloride 6.2g / L, sodium dihydrogen phosphate 4g / L, disodium hydrogen phosphate 7.1g / L, and the rest is water for injection.

[0047] The amount of sodium dihydrogen phosphate and disodium hydrogen phosphate is adjusted to the pH value of 6.8 of the eye drops.

[0048] The amount of sodium chloride is adjusted to the osmotic pressure molar concentration ratio of 0.9.

[0049] The method for inhibiting pathological angiogenesis of the eye drops comprises the following steps:

[0050] First step, take the corresponding weight volume ratio of withering A and Tween 80, mix well and reserve;

[0051] Second step, take the corresponding weight volume ratio of povidone K30, benzalkonium chloride, sodium chloride, sodium dihydrogen phosphate and disodium hydrogen phosphate, heat and stir in a 60℃ water bath until clear and transparent, and cool to room temperature;

[0052] Third step, the mixed solution of the first step is added to the solution of the second step, and water for injection is used to constant volume;

[0053] Fourth step, the prepared eye drops are filtered by 0.22 μm microporous filter membrane to remove bacteria, and are sterilely packaged and divided, to obtain the eye drops.

[0054] The eye drops for inhibiting pathological angiogenesis of the eye are used for preparing a medicament for treating pathological angiogenesis of the eye.

[0055] The preparation method of the present application includes, but is not limited to, eye drops, eye wash, solid mixture (dissolved before use), concentrated solution (diluted before use), eye ointment, eye gel and other various ophthalmic preparations acceptable in the medical field.

[0056] The eye drops for inhibiting pathological angiogenesis of the eye are used for preparing a medicament for treating pathological angiogenesis of the eye.

[0057] The solubilizing agent can also be one of propylene glycol, poloxamer, polyethylene glycol, beta cyclodextrin, or a mixture of any of Tween 80, propylene glycol, poloxamer, polyethylene glycol and beta cyclodextrin.

[0058] The thickening agent can also be one of methyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium hyaluronate, hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose, carboxymethyl cellulose, or a mixture of any of methyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium hyaluronate, povidone, hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose and carboxymethyl cellulose.

[0059] The pH regulator can also be one of boric acid, boric acid buffer, citrate buffer, or a mixture of any of boric acid, boric acid buffer, phosphate buffer and citrate buffer, and the amount of the pH regulator is determined according to the pH value of the eye drops, which is 6.0-8.0.

[0060] The osmotic pressure regulator can also be one of potassium chloride, boric acid, glucose and glycerol, or a mixture of any of sodium chloride, potassium chloride, boric acid, glucose and glycerol, and the amount of the osmotic pressure regulator is determined according to the osmotic pressure molar concentration ratio, which is 0.9-1.1.

[0061] The bacteriostatic agent can also be one of hydroxyphenyl ethyl ester, benzalkonium bromide and polyquaternary ammonium salt-1, or a mixture of any of hydroxyphenyl ethyl ester, benzalkonium chloride, benzalkonium bromide and polyquaternary ammonium salt-1.

[0062] In order to further prove the effect and result of the present application, the in vivo experimental results of two animal models are shown as follows:

[0063] WFA eye drops significantly improved corneal pathological neovascularization in mice caused by alkali burn, while WFA solution had no obvious effect

[0064] In this experiment, we selected the classic mouse corneal alkali burn model to induce corneal pathological neovascularization. Healthy wild-type female C57 mice, 6-8 weeks old. All mice had bright hair color, no hair loss, no abnormalities in eye examination, clear refractive index, and normal fundus. Randomly divided into 1, control group; 2, alkali burn group; 3, alkali burn + WFA (WFA) solution (DMSO solution) group; 4, alkali burn + WFA eye drops group, 20 in each group. Among them, groups 3 and 4 have the same concentration of WFA, which are given 4 times a day, eye drops intervention. After 14 days of intervention, under a microscope, the corneal pathological neovascularization was scored according to the following method: 0 = no neovascularization, no new blood vessels from the limbus; 1 = mild neovascularization, new blood vessels from the limbus; 2 = moderate neovascularization, blood vessels originating from the limbus, growing towards the corneal center; 3 = severe neovascularization, blood vessels starting from the limbus and reaching and / or penetrating the corneal center. As shown in Figure 1, WFA eye drops significantly improved corneal pathological neovascularization in mice caused by alkali burn, while WFA solution had no obvious effect, Figure 1 NS = no difference, n = 5-6. ** p <0.01.

[0065] Immunofluorescence further confirmed that WFA eye drops significantly improved corneal pathological neovascularization in mice caused by alkali burn

[0066] To further clarify the experimental results and reduce errors caused by visual observation, we euthanized the mice on the 14th day of medication, dissected and removed the eyeballs, labeled the new blood vessels with cd31 (red fluorescence) and the cell nuclei with DAPI (blue fluorescence) using conventional immunofluorescence staining, and imaged the flat corneas using a confocal microscope; As shown in Figure 2, immunofluorescence further confirmed that WFA eye drops significantly improved corneal pathological neovascularization in mice caused by alkali burn, and the results were statistically significant, Figure 2 n = 5-6. ** p <0.01.

[0067] WFA eye drops improved corneal opacity in mice caused by alkali burn

[0068] The experiment still chooses the classic mouse corneal alkali burn model to induce ocular corneal pathological angiogenesis. Healthy wild-type male C57 mice, 6-8 weeks old. All mice have bright hair color, no hair loss, no abnormalities in eye examination, clear refractive medium, normal fundus, and are randomly divided into: 1, control group; 2, alkali burn; 3, alkali burn + WFA eye drops group, 20 in each group. After 14 days of intervention, the cornea was observed under a dissecting microscope, and the haze after burning was scored: 0 = no haze, cornea transparent; 1 = mild haze, iris and pupil area slightly blurred, iris and pupil easily visible; 2 = moderate haze, iris and pupil almost invisible; 3 = severe haze, iris or pupil invisible; 4 = cornea opaque, iris and pupil invisible. As shown in the results in Figure 3: the Withaferin A eye drops significantly improved the corneal haze caused by alkali burn in mice, Figure 3 n = 5-6. *** p <0.001.

[0069] Withaferin A eye drops improve suture-induced pathological angiogenesis in the cornea of mice, while using Withaferin A solution has no obvious effect

[0070] The experiment selects the classic suture method to induce ocular corneal pathological angiogenesis. Healthy wild-type female ICR mice, 6-8 weeks old. All mice have bright hair color, no hair loss, no abnormalities in eye examination, clear refractive medium, normal fundus, and are randomly divided into: 1, control group; 2, suture group; 3, suture + WFA (DMSO dissolved) solution group; 4, suture + WFA eye drops group, 20 in each group. Among them, groups 3 and 4 have the same concentration of Withaferin A, both given 4 times / day, eye drops intervention. After 14 days of intervention, under a microscope, the area of neovascularization was counted to compare the angiogenesis of each group. As shown in the results in Figure 4: Withaferin A eye drops significantly improved suture-induced pathological angiogenesis in the cornea of mice, while Withaferin A DMSO solution did not significantly improve ocular surface angiogenesis in mice, Figure 4 NS = no difference, *** p <0.001.

[0071] Withaferin A eye drops improve suture-induced pathological angiogenesis in the cornea of mice

[0072] In order to further clarify the experimental results and reduce errors caused by visual observation, we used conventional immunofluorescence staining to image the flat cornea using a confocal microscope, as in Example 3; As shown in the results in Figure 5: immunofluorescence further confirmed that Withaferin A eye drops significantly improved suture-induced pathological angiogenesis in the cornea of mice, the results were statistically significant, Figure 5 n = 5-6. ** p <0.01.

[0073] The in vivo experimental results of the above two animal models show that the solution of withaferin A alone has no effective treatment effect on the pathological angiogenesis of the eye, and the eye drops containing the effective component as described in the present patent have good solubility of withaferin A and prolong the residence time of withaferin A on the ocular surface, and are used for treating the pathological angiogenesis of the eye, especially the pathological angiogenesis of the ocular surface, and have a remarkable effect.

Claims

1. An eye drop for inhibiting pathological angiogenesis in the eye, characterized by comprising: The eye drop contains the following components by weight volume ratio: withanolide A 0.01-0.8 mg / L, a dissolving agent 1-25 g / L, a thickening agent 2-40 g / L, a bacteriostatic agent 0.04-0.2 g / L, an isotonicity adjusting agent and a pH adjusting agent in proper amount, and the balance is water for injection.

2. The eye drops for inhibiting ocular pathological angiogenesis according to claim 1, characterized by: The dissolving agent is one or a mixture of any of Tween 80, propylene glycol, poloxamer, polyethylene glycol and β-cyclodextrin.

3. The eye drops for inhibiting ocular pathological angiogenesis according to claim 1, characterized by: The thickening agent is one or a mixture of any of methyl cellulose, polyvinyl alcohol, polyethylene glycol, sodium hyaluronate, povidone, hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose and carboxymethyl cellulose.

4. The eye drops for inhibiting ocular pathological angiogenesis according to claim 1, characterized by: The pH adjusting agent is one or a mixture of any of boric acid, boric acid buffer, phosphate buffer and citrate buffer, and the amount of the pH adjusting agent is determined according to the pH value of the eye drop being 6.0-8.

0.

5. The eye drops for inhibiting ocular pathological angiogenesis according to claim 1, characterized by: The osmotic pressure adjusting agent is one or a mixture of any of sodium chloride, potassium chloride, boric acid, glucose and glycerol, and the amount of the osmotic pressure adjusting agent is determined according to the osmotic pressure molar concentration ratio being 0.9-1.

1.

6. The eye drops for inhibiting ocular pathological angiogenesis according to claim 1, characterized by: The bacteriostatic agent is one or a mixture of any of hydroxyphenyl ethyl ester, benzalkonium chloride, benzalkonium bromide and polyquaternary ammonium salt-1.

7. Process for the preparation of eye drops for inhibiting pathological vascularization in the eye according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: In the first step, the withanolide A and the dissolving agent are weighed according to the above-mentioned weight volume ratio, mixed and stirred uniformly, and prepared for use; In the second step, the thickening agent, the pH adjusting agent, the isotonicity adjusting agent and the bacteriostatic agent are weighed according to the above-mentioned weight volume ratio, heated and stirred in a 60℃ water bath until clear and transparent, and then cooled to room temperature; In the third step, the mixed solution in the first step is added to the solution in the second step, and then diluted with water for injection; In the fourth step, the prepared eye drop is filtered and sterilized by a 0.22 μm microporous filter, and then aseptically packaged to obtain the eye drop.

8. Use of the eye drop for inhibiting pathological angiogenesis in the eye according to any one of claims 1-6 in the preparation of a medicament for treating pathological angiogenesis in the eye.

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