WO2009088119A1

WO2009088119A1 - Composition for preventing or treating dry eye syndrome comprising poly-gamma-glutamic acid

Info

Publication number: WO2009088119A1
Application number: PCT/KR2008/001055
Authority: WO
Inventors: Moon-Hee Sung; Seon Lyang Bae; Chul Joong Kim; Haryoung Poo; Ji Won Hong; Jae-Chul Choi; So Hyun Yoon
Original assignee: Bioleaders Corporation; Catholic University Industry Academic Cooperation Foundation; The Industry and Academic Cooperation in Chungnam National University; Korea Research Institute Of Bioscience And Biotechnology; Kookmin University Industry-Academic Cooperation Foundation
Priority date: 2008-01-07
Filing date: 2008-02-22
Publication date: 2009-07-16

Abstract

The present invention relates to a composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid as an active ingredient. The composition for preventing or treating dry eye syndrome, which comprises poly- gamma-glutamic acid as an active ingredient, is effective for preventing or treating dry eye syndrome by retaining ocular moisture to relieve dry eye syndrome based on its high moisturizing capacity and its ability to activate natural moisturizing factors, and inhibiting a hyaluronidase enzyme activated upon the development of inflammation to relieve the inflammation.

Description

Composition for Preventing or Treating Dry Eye Syndrome Comprising Poly-Gamma-Glutamic Acid

TECHNICAL FIELD

The present invention relates to a composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid (γ-PGA).

BACKGROUND ART

The U.S. National Eye Institute defined dry eye syndrome as ocular surface diseases which cause symptoms of grittiness or foreign body sensation due to tear deficiency or excessive tear evaporation that causes damage to the interpalpebral ocular surface (i.e. exposed eye surface). Additionally, the range of dry eye syndrome has expanded to include a deficiency in the quantity and quality of tears without relation to ocular surface diseases, symptoms caused by neurogenic factors such as dry eye symptoms occurring after LASIK surgery, and environmental factors such as severe pollution or a decrease in humidity.

Keratoconjunctivitis is caused by abnormal tear production due to abnormalities in any one of the lipid, aqueous and mucin layers of the trilaminar tear film. Particularly, a mucin deficiency may lead to the risk of developing corneal epithelial abrasions, corneal epithelial disorders, corneal ulcers and infectious ocular diseases resulted from the ocular surface diseases referred to in the above definition of dry eye syndrome.

Dry eye syndrome occurs often in pregnant women or post-menopausal women, and it is known that about ten million Americans suffer from dry eye syndrome, and particularly, it is estimated that nearly 75% of people over 65 years old i experience dry eye syndrome. In Korea, survey results show that three out of ten women in their twenties, thirties and forties are affected by moderate dry eye syndrome, whereas the dry eye syndrome patient population rapidly increases up to six out often women in their fifties and over 50.

Methods for treating dry eye syndrome include drug therapy (treatment of inflammation and administration of artificial tears) and surgical therapy. Drug therapy comprises administering artificial tears or ointments to the eyes of a subject in order to supplement the normal tears. Examples of artificial tear solutions include an ophthalmic solution (commercially available under the trademark Hyalein from Taejoon Pharmaceutical Co., Ltd., Korea) containing hyaluronic acid as an active ingredient and restasis (cyclosporine A) for relieving dry eye syndrome due to invasion of T cells, developed by Allergan, USA, which became commercially available in May, 2003, and in March, 2006 in Korea. Other examples of artificial tear solutions include an ophthalmic solution, Diquafosol which is a P2Y2 receptor against that stimulates tear and mucin secretion on the ocular surface, and autologous serum eye drops for improving tear stability, and the like.

However, such therapeutic agents only temporarily relieve symptoms through tear supplements, and cannot treat the fundamental causes of dry eye syndrome caused by corneal epithelial wounds, inflammation, etc. In addition, medical expenses may increase due to long-term abuse of therapeutic agents, and side effects can occur due to the components of ophthalmic solution.

Meanwhile, poly-gamma-glutamic acid is a viscose polymer consisting of D,L- glutamic acid, which is produced from the genus Bacillus, strain. The present inventors obtained a patent relating to poly-gamma-glutamate with high molecular weight and a method for using the same (Korean Patent Registration No. 10- 0399091), and a patent relating to a method for producing γ-PGA using a halophilic Bacillus subtilis var. chungkookjang that produces γ-PGA with high molecular weight (Korean Patent Registration No. 10-0500796). Also, we obtained patents relating to an anticancer composition, an immune adjuvant, and an immune enhancing agent, which contain γ-PGA (Korean Patent Registration Nos. 10- 0496606; 10-0517114; and 10-0475406). In addition, we have identified the effects of γ-PGA by continuously developing applications for γ-PGA, including studies on the use of γ-PGA in medical applications, such as development of a hyaluronidase inhibitor containing poly-gamma-glutamic acid (Korean Patent Registration No. 10-0582120).

Accordingly, the present inventors have made extensive efforts to develop new applications for γ-PGA, a natural amino acid polymer, and as a result, confirmed that γ-PGA can prevent or treat dry eye syndrome by retaining ocular moisture to relieve dry eye syndrome based on its high moisturizing capacity and its ability to activate natural moisturizing factors, and inhibiting a hyaluronidase enzyme activated upon the development of inflammation to relive the inflammation.

SUMMARY OF INVENTION

It is an object of the present invention to provide a composition for preventing or treating dry eye syndrome, which comprises γ-PGA as an active ingredient.

It is another object of the present invention to provide a quasi-drug composition for preventing or treating dry eye syndrome, which comprises γ-PGA.

To achieve the above objects, the present invention provides a composition for preventing or treating dry eye syndrome, which comprises γ-PGA as an active ingredient. In addition, the present invention provides a quasi-drug composition for preventing or treating dry eye syndrome, which comprises γ-PGA.

Other features and embodiments of the present invention will be more apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a graph showing the tear secretion rate measured after applying γ-PGA to the eyes of a rabbit dry eye model.

Fig. 2 is a graph showing the difference in the number of goblet cells observed under a microscope by performing impression cytology in order to examine the treatment effect of γ-PGA on dry eye syndrome, after applying γ-PGA to the eyes of a rabbit dry eye model.

Fig. 3 is a photograph observed under a microscope by performing impression cytology in order to examine the treatment effect of γ-PGA on dry eye syndrome, after applying γ-PGA to the eyes of a rabbit dry eye model (magnification X 200).

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In the present invention, it was confirmed that γ-PGA, a natural amino acid polymer, which is derived from foods, has a high moisturizing capacity, activates natural moisturizing factors, and inhibits a hyaluronidase enzyme, which is activated upon the development of inflammation to alleviate inflammation and thus it is effective to prevent or treating dry eye syndrome. Therefore, in one aspect, the present invention relates to a composition for preventing or treating dry eye syndrome, which comprises γ-PGA as an active ingredient.

In the present invention, the γ-PGA may be produced by chemical synthesis or microbial fermentation, preferably by microbial fermentation, and more preferably by fermentation of Bacillus subtilis var. chungkookjang. In addition, the poly-γ- glutamic acid preferably has an average molecular weight of 1-15,000 kDa.

The γ-PGA according to the present invention has a high moisturizing capacity, and activates natural moisturizing factors, thus making it possible to effectively retain ocular moisture.

In addition, the γ-PGA inhibits a hyalurqnidase enzyme, which is activated upon the development of the inflammation to relieve the inflammation. Blepharitis is one cause making dry eye syndrome worse. Blepharitis is a cyst in the eyelid that is caused by inflammation of a blocked meibomian gland, and it results in a thin lipid layer to form an unstable tear film and thus tears dry out quickly. Therefore, the γ- PGA according to the present invention inhibits a hyaluronidase enzyme which is activated upon the development of the inflammation to relieve inflammation, thus preventing or treating dry eye syndrome.

In the present invention, in order to secure the safety of γ-PGA, an ocular irritation test of γ-PGA was performed by Biotoxtech Co., Ltd., an institute approved as a Good Laboratory Practice (GLP) facility. As a result, it was proven safe for ocular use.

In the inventive composition for preventing or treating dry eye syndrome, which comprises γ-PGA as an active ingredient, the γ-PGA can be contained at a therapeutically effective concentration, which is suitably determined depending on the patients' age, disease severity or the degree of treatment. For example, the γ- PGA may be added thereto at a concentration of 0.001 to 5 % by weight, and preferably 0.01 to 3 % by weight. If the concentration of the γ-PGA is less than 0.001 wt% on a dry weight basis, the pharmacological action of γ-PGA cannot be expected, and if it exceeds 5 wt% on a dry weight basis, a further increase in pharmacological action will not be expected, and the viscosity of the resulting composition can be excessively increased.

The composition for preventing or treating dry eye syndrome can be formulated into an ophthalmic liquid formulation by a conventional method of adding to the γ- PGA, additives including an isotonic agent such as sodium chloride or potassium chloride, a buffer such as sodium hydrogen phosphate or sodium dihydrogen phosphate, a stabilizer such as sodium ether, a preservative such as ethyl paraben, butyl paraben, benzalkonium chloride, a pH adjusting agent such as sodium hydroxide and diluted hydrochloric acid, an ointment base such as white Vaseline or liquid paraffin, and the like. Examples of the ophthalmic liquid formulation include ophthalmic solution, eye lubricating drops and the like.

The composition for preventing or treating dry eye syndrome according to the present invention is useful for preventing or treating tear deficiency, Sjogren's syndrome, Keratoconjunctivitis sicca, Stevens-Johnson syndrome, ocular pemphigoid, blepharitis, failure of eyelid closure and facial sensory nerve paralysis, dry eye such as dry eye associated with allergic conjunctivitis, dry eye associated with viral conjunctivitis, dry eye after cataract surgery, dry eye associated with VDT work, and dry eye associated with contact lens wear. The composition for preventing or treating dry eye syndrome according to the prevent invention is also effective for treating conjunctival and corneal epithelial defects, corneal epithelial abrasions, corneal ulcers, infectious ocular diseases and the like, which are caused by dry eye syndrome. In another aspect, the present invention relates to a quasi-drug composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid.

Examples of the quasi-drug composition according to the present invention may include contact lens solutions, ocular wash solutions, an ophthalmic saline solution and the like. The quasi-drug composition can be prepared by a conventional method of adding to the γ-PGA, additives, including a reducing irritation agent such as polyethyleneglycol-120 methyl glucose dioleate, polyvinylalcohol, polyvinylpyrrolidone etc., a non-ionic surfactant such as polyethylene oxide-co- polypropylene oxide, polyoxyethylene sorbitan monolaurate, polyethyleneglycol p- isoocty phenylehter, polyoxyethylene monooleate, polyoxyethylene lauryl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene lauryl ether, polyethyleneglycol p-isoocty phenylehter etc., an amphoteric surfactant such as alkyl betaine, propyl betaine, 2-alkyl-N-carboxymethyl-N- hydroxyethyl imidazolinium betaine, N-lauryl-N-methyl-β-alanine sodium, lauryl dimethylamino acetic acid betaine etc., a polymeric preservative such as lauryldimonium hydroxypropyl hydrolyzed collagen, cocodimonium hydroxypropyl hydrolyzed collagen, stearyldimonium hydroxypropyl hydrolyzed collagen, poly [oxyethylene(dimethylimino) ethylene(dimethylimino)ethylene dichloride], polyquaternium- 16 etc., and the like, but not limited thereto.

As described above, since γ-PGA has high moisturizing capacity and the ability to inhibit a hyaluronidase enzyme, when γ-PGA is added to contact lens solutions, ocular wash solutions, ophthalmic saline solutions and the like, dry eye syndrome can be prevented. The γ-PGA is preferably added to the quasi-drug composition for preventing dry eye syndrome at a concentration of 0.001 to 2.0 % by weight. Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1: Production of poly-gamma-glutamic acid

3 L of a basal medium for γ-PGA production (GS medium containing 5% L- glutamic acid: 5% glucose, 1% (NH₄)₂SO₄, 0.27% KH₂PO₄, 0.42% Na₂HPO₄- 12H₂O, 0.05% NaCl, 0.3% MgSO₄-TH₂O, lml/1 vitamin solution, pH 6.8) was inoculated with 1% culture broth of Bacillus subtilis var chungkookjang (KCTC 0697BP) and then cultured at a stirring speed of 150 rpm, an air injection rate of 1 vvm and a temperature of 37 ^°C for 72 hours. Then, the cells were removed from the culture broth after completion of the culture using a filter press, thus obtaining a γ-PGA-containing sample solution.

2N sulfuric acid solution was added to the γ-PGA-containing sample solution and left to stand at 10°C for 12 hours to collect a γ-PGA precipitate. The collected precipitate was washed with a sufficient amount of RO water to obtain γ-PGA using a Nutsche filter. Molecular weight of the obtained poly-gamma-glutamic acid was measured using GPC (gel permeation column), and as a result, it was confirmed that poly-gamma-glutamic acid having a molecular weight of 1- 15,00OkDa was produced, and then separated according to molecular weight to collect poly-gamma-glutamic acid having an average molecular weight of 5,000 kDa. The collected γ-PGA was used in the following examples.

Example 2: Investigation of effect of poly-γ- glutamic acid on relief from dry eye syndrome An experiment was performed on 15 brown and white rabbits weighing 2.0-3.0 kg, and all the rabbits were handled in accordance with the principles of the Helsinki Declaration. All rabbits were intramuscularly injected with 1 mg/kg of 1% atropine sulfate for 10 days to induce dry eye syndrome, and rabbits with dry eyes were randomly divided into two groups, a test group and a control group. 0.5% γ-PGA having a molecular weight of 5,000 kDa was dropped into the eyes of the test animals 4 times per day, but was not dropped into the eyes of the control animals.

To measure tear secretion in rabbits with dry eyes, all rabbits were anesthetized by intramuscular injection of 30 mg/kg of ketamine and 5 mg/kg of xylazine, and then a standard Schirmer strip was inserted into the lower conjunctival sac at the junction of the middle and temporal thirds of the lower lid. After 5 minutes, the length of the strip that is wet from tears was measured in millimeters. To eliminate the variation of tear secretion per eye, values obtained by dividing the value measured after atropine sulfate injection by the value measured before atropine sulfate injection, were expressed as the rates of tear secretion. As a result, as shown in Fig.l, the rates of tear secretion were statistically significantly decreased in the γ-PGA-treated group and the control group compared to baseline values (p<0.05) during the period ranging from day 2 to day 10, and there was no statistically significant difference between the groups.

At day 10, impression cytology was carried out on all rabbits. For this purpose, a 5 x 5 mm filter paper (Millpore, Billerica, USA) was placed onto the superior bulbar conjunctival surface, approximately 2-3 mm apart from the limbus, and then pressed with a 1 ml curved tip syringe for 10 seconds. Then, the samples were fixed in 10% formalin solution and subjected to periodic acid Schiff (PAS) staining and hematoxylin staining. The number of goblet cells in each specimen was counted at 20Ox magnification, and five repeated measurements were averaged. As a result, as shown in Figs. 2 and 3, the numbers of goblet cells in the γ-PGA-treated group and the control group were 44.5±15.2 and 12.5±7.2, respectively, suggesting that the number of goblet cells was significantly increased in the γ-PGA-treated group compared to the control group. Goblet cells produce a gel-like mucin, which forms the deepest of the three layers of tear film and thus protect the cornea from damage caused by dry eye syndrome. From the experiment, it was confirmed that rapid restoration of goblet cells was induced by instillation of poly-γ-glutamic acid into rabbit eyes, suggesting that poly-γ-glutamic acid is very effective in the treatment of dry eye syndrome.

Example 3 : Ocular irritation test on poly-γ-glutamic acid

An ocular irritation test on poly-γ-glutamic acid was carried out using three 16- week-old male NZW rabbits. As a test substance, 10% poly-γ-glutamic acid solution was applied to the rabbits. In a test group, 0.1 ml of the test substance was applied into the conjunctival sac of the right eye, and at 1 hr, 24 hr, 48 hr, 72 hr and 96 hr after applying the test substance, the ocular defects of the cornea, the iris and the conjunctiva were observed. Ocular irritancy was scored according to the Draize criteria and the ocular irritation scores were classified according to the method of Kay and Calandra.

As a result, as shown in Table 1 below, ocular irritation in the cornea, the iris and the conjunctiva was not shown in all the animals when observed at 1 hr, 24 hr, 48 hr, 72 hrs and 96 hr after the application of poly-γ-glutamic acid. The mean total score (MTS) was zero (no irritability). During the observation period, there was no death of animals, and abnormal changes in general conditions and body weight caused by the application of the test substance was not shown. From the above results, it was concluded that the test substance, poly-γ-glutamic acid did not irritate the ocular tissue of the rabbits under the test conditions.

Table 1

MMTS: maximum mean total score (¹⁾ hour after application

Formulation Examples 1 to 4: Preparation of ophthalmic solution containing poly- γ-glutamic acid

As shown in Table 2 below, ophthalmic solution was prepared by mixing poly-γ- glutamic acid with isotonic agents (sodium chloride and potassium chloride), buffers (sodium hydrogen phosphate and sodium dihydrogen phosphate), a stabilizer (sodium ether), a preservative (benzalkonium chloride), a pH adjusting agent (sodium hydroxide) and sterile purified water.

Table 2

(2) based on 100 ml of ophthalmic solution

Formulation Examples 5 and 6: Preparation of contact lenses solution containing poly-γ-glutamic acid As shown in Table 3 below, contact lenses solution was prepared by mixing poly-γ- glutamic acid with sodium chloride, polyoxy ethylene sorbitan monolaurate , alkyl betaine, polyethylene glycol- 120 methyl glucose dioleate, boric acid, borax and sterile purified water.

Table 3

(3) based on 100 ml of contact lenses solution

INDUSTRIAL APPLICABILITY

As described above, the composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid as an active ingredient, can prevent or treat dry eye syndrome by retaining ocular moisture to relieve dry eye syndrome based on its high moisturizing capacity and its ability to activate natural moisturizing factors, and inhibiting a hyaluronidase enzyme activated upon the development of inflammation to relieve the inflammation. In addition, poly- gamma-glutamic acid, which is contained as an active ingredient, has little toxicity and side effects, so that the inventive composition is safe for long-term use.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMSWhat is Claimed is:

1. A composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid as an active ingredient.

2. The composition for preventing or treating dry eye syndrome according to claim 1, wherein the average molecular weight of poly-gamma-glutamic acid is 1-15,00O kDa.

3. The composition for preventing or treating dry eye syndrome according to claim 1, wherein it is formulated into an ophthalmic liquid formulation.

4. A quasi-drug composition for preventing or treating dry eye syndrome, which comprises poly-gamma-glutamic acid.

5. The quasi-drug composition for preventing or treating dry eye syndrome according to claim 4, wherein the quasi-drug composition is selected from the group consisting of contact lens solutions, ocular wash solutions and ophthalmic saline solutions.