KR20170021667A - A pharmaceutical composition for treating neurotrophic keratopathy - Google Patents

Abstract

The present invention relates to a composition for treating neurotrophic keratopathy, containing thymosin 4 as an active ingredient. Since the composition shows excellent therapeutic effects on neurotrophic keratopathy which has been hard to cure due to absence of treatment methods, the composition of the present invention is expected to be highly applicable in industries as treatment agent for neurotrophic keratopathy or treatment agents for ameliorating or relieving symptoms of neurotrophic keratopathy.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating neurotrophic keratopathy comprising thymosin beta4 as an active ingredient,

The present invention relates to a therapeutic agent for neurotrophic keratitis comprising thymosin beta 4 as an active ingredient.

The convex region covering the black zone of our eyes is called the cornea. The cornea is transparent like glass, and the inner iris appears to be black or brown through the cornea. Cornea is a disease that causes inflammation in the cornea, causing pain, redness, decreased vision, and corneal opacity.

The causes of keratitis can be divided into infectious and noninfectious. Infectious keratitis is caused by infection of the cornea by various pathogens such as bacteria, viruses, fungi (fungus). Bacteria causing infections are most commonly caused by Staphylococcus and Pseudomonas aeruginosa, and infections caused by fungi such as herpes simplex virus and fusarium are common. Non-infectious factors include exposure keratitis caused by continuous exposure of the cornea to the outside air, toxic keratitis caused by drugs, and neurotrophic keratitis caused by damage to the corneal nerve. Bilateral neurotrophic keratitis is difficult to treat, and is treated with artificial tears, contact lenses, amniotic membrane transplantation, etc. Unless the problem of corneal nerve injury is solved, it is difficult to cure.

On the other hand, thymosin β4 was the first protein found in the thymus in 1981, consisting of 41 to 43 amino acids and an isoelectric point of 5.1. Thymosin β4, first identified as an actin-sequestering molecule in animal cells by Riba et al. In 1991, has subsequently been found to be involved in immunoregulatory and neuroendocrine processes. Recently, Korean Patent Laid-Open Publication No. 10-2008-33939 discloses an eye drop composition comprising thymosin beta 4 and an amino acid stabilizer, and discloses an eye drop composition further comprising a bulking agent, a buffering agent, and a pH adjusting agent. However, the Korean Laid-Open Publication does not disclose that thymosin? 4 is effective for neurotrophic keratitis.

The present inventors have found that thymosin beta4 inhibits corneal damage caused by neurotrophic keratitis while trying to find a therapeutic effect on the cornea with impaired thymosin beta4 and an ingredient that may affect the activity of thymosin beta4 In particular, it has been confirmed that the specific organic acid can maintain or enhance the activity of thymosin beta 4, and that the composition can be utilized in the treatment of neurotrophic keratitis, thereby completing the present invention.

KR 10-2008-33939 A

It is an object of the present invention to provide a composition for treating neurotrophic keratitis comprising thymosin beta4.

In order to accomplish the object of the present invention, there is provided a composition for treating neurotrophic keratitis including thymosin beta 4 or treating corneal damage caused thereby. The present invention also provides a composition comprising thymosin beta 4 and citric acid and a composition comprising thymosin beta 4, citric acid and acetic acid for the treatment of the above diseases. The present invention also provides a method for manufacturing an eye drop containing the composition and an eye drops prepared thereby.

The composition containing thymosin beta 4 of the present invention is highly likely to be used medically by treating damaged cornea in patients with neurotrophic keratitis. In particular, in the case of a composition containing thymosin beta 4 and citric acid and a composition containing thymosin beta 4, citric acid and acetic acid Since it has better therapeutic effect than thymosin? 4 alone, it can be effectively used to treat damaged corneas in patients with neurotrophic keratitis.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention provides a composition for treating neurotrophic keratitis comprising thymosin beta 4 as an active ingredient.

As used herein, the term "thymosin beta4" is a polypeptide, also referred to as Thymosin beta-4 or T? 4, which is isolated from the thymus first and is composed of 43 amino acids of 4.9 kDa, The protein is a protein that is up-regulated during in vitro endothelial cell migration and differentiation. Many thymosin beta4 isoforms have been identified and have been found to be about 70%, or about 75%, or about 70% And has about 80% or more homology. The thymosin beta 4 of the present invention can be applied to the N-terminal variant of wild-type thymosin beta 4, and the C-terminal variant of T beta 4. Preferably a protein having the amino acid sequence of SEQ ID NO: 1. In addition, the concentration of thymosin beta 4 may be 0.01 to 1.0% (w / v), preferably 0.05 to 0.5% (w / v).

The term neurotrophic keratitis, a term used in the present invention, is a kind of keratitis caused by nerve injury, which is a degenerative disease characterized by reduced corneal sensitivity and low keratopathy. This may occur after infection with herpes zoster or herpes simplex and may be due to chemical or physical burns or by abuse or toxicity of drugs such as timolol, betaxolol, diclofenac sodium, or sulphacetamide 30% . It may also occur after surgery or laser treatment. It may also be caused by corneal incision, corneal injury, or infection.

In addition, the effect on neurotrophic keratitis is caused by nerve injury This may be interpreted as meaning that it is effective for ophthalmic diseases, and it can be interpreted as meaning that it can treat the damage occurring on the corneal surface or alleviate various symptoms of neurotrophic keratitis.

In addition, the composition may further contain citric acid or acetic acid in addition to the pharmaceutical composition.

In addition, citric acid used in the present invention is also referred to as citric acid, and is a compound having the chemical formula of C ₆ H ₈ O ₇ . In addition, the citric acid used in the present invention can be used in the form of a citric acid salt. The citric acid salt is a derivative of citric acid, and in one embodiment may be sodium citrate, and may be sodium citrate hydrate. Citric acid or a salt thereof is usually used as a buffer to alleviate the pH change. However, citric acid or a salt thereof used in the present invention should be used in a larger amount than the amount normally used. At this time, citric acid or a salt thereof may contain 0.01% (w / v) to 0.5% (w / v) in the whole composition. In addition, citric acid or a salt thereof may contain 0.05% (w / v) to 0.25% (w / v) in the total composition, preferably 0.1 to 0.3%.

Acetic acid used in the present invention is a weak acid having the formula CH ₃ COOH, also referred to as acetic acid. In addition, the acetic acid used in the present invention may be used in the form of an acetate salt. One embodiment of the acetic acid salt may be sodium acetate hydrate. At this time, citric acid or a salt thereof may contain 0.01% (w / v) to 1.5% (w / v) in the whole composition. In addition, citric acid or a salt thereof may contain 0.1% (w / v) to 0.8% (w / v) in the whole composition, preferably 0.2 to 0.5%.

Also, the concentration of sodium chloride may be 0.1-1.2% (w / v) and may be 0.3-1.0% (w / v). Preferably, it may be 0.5 to 0.7% (w / v). Also, the concentration of potassium chloride may be 0.01 to 0.15% (w / v), and may be 0.03 to 0.12% (w / v). Preferably, it may be 0.05 to 0.09% (w / v). In addition, the concentration of calcium chloride hydrate may be 0.01 to 0.12% (w / v), and may be 0.03 to 0.09% (w / v). Preferably, it may be 0.03 to 0.06% (w / v). The concentration of the magnesium chloride hexahydrate may be 0.01 to 0.12% (w / v), and preferably 0.01 to 0.05% (w / v).

In addition, the composition of the present invention may further include hydrochloric acid or sodium hydroxide. Hydrochloric acid and sodium hydroxide may be added in an appropriate amount to adjust the pH in the composition. The pH of the composition may also be 6.5 to 7.5 and may be 6.8 to 7.2. Preferably the pH of the composition may be 7.0.

In addition, the composition may be in a form that can be used for ophthalmic use. Specifically, the formulations may be in the form of solutions, lotions, lozenges, gels, creams, pastes, sprays, suspensions, dispersions, hydrogels, ointments, oils or foams. According to one embodiment, a composition comprising thymosin beta 4 and citric acid may be formulated by mixing with pharmaceutically acceptable, especially ophthalmically acceptable, non-toxic excipients or carriers. For example those of the types mentioned below, in particular carriers, stabilizers, solubilizers, buffering substances, preservatives, thickeners and other excipients are used. In addition, the solution is used adjusted to the desired pH.

Carriers which can be used according to the present invention are typically suitable for topical or general administration, for example water, mixtures of water and water-miscible solvents, for example C ₁ -C ₇ -alkanols, vegetable oils or Mineral oils, for example 0.5 to 5% by weight of hydroxyethylcellulose, ethyl oleate, carboxymethylcellulose, polyvinyl-pyrrolidone and other non-toxic water-soluble polymers for ophthalmic use, such as cellulose derivatives, For example, methylcellulose, alkali metal salts of carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylhydroxypropylcellulose and hydroxypropylcellulose, acrylates or methacrylates such as salts of polyacrylic acid or Ethyl acrylate, polyacrylamides, natural products such as gelatin, alginates, packings, Starch derivatives such as starch acetate and hydroxypropyl starch, and also other synthetic products such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, Polyvinyl methyl ether, polyethylene oxide, preferably crosslinked polyacrylic acid, such as neutral carbopol, or mixtures of these polymers. Preferred carriers are water, cellulose derivatives such as methylcellulose, alkali metal salts of carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylhydroxypropylcellulose and hydroxypropylcellulose, neutral carbopol, or their Mixtures.

Stabilizers that may be used in the ophthalmic compositions of the present invention include, for example, tyloxapol, fatty acid glycerol poly-lower alkylene glycol esters, fatty acid poly-lower alkylene glycol esters, polyethylene glycols, glycerol ethers, Mixtures. The added amount is typically sufficient to dissolve the active ingredient.

The buffers that may be used in the compositions of the present invention are borate, hydrogen carbonate / carbonate, gluconate, phosphate, propionate and TRIS (tromethamine) buffers. Tromethamine and borate buffers are preferred buffering agents. The amount of buffer substrate added is necessary, for example, to ensure and maintain a physiologically acceptable pH range. The pH range is typically 5 to 9, preferably 6 to 8.2, more preferably 6.8 to 8.1.

Preservatives that may be used in the compositions of the present invention include quaternary ammonium salts such as cetrimide, benzalkonium chloride or benzoin chloride, alkyl-mercury salts of thiosalicylic acid, such as thimerosal, phenylmercuric acid nitrate, For example, phenylmercuric acid acetate or phenylmercuric acid borate, parabens such as phenylparaben or propylparaben, alcohols such as chlorobutanol, benzyl alcohol or phenylethanol, guanidine derivatives such as chlorohexidine or polyhexamethylene Gt; acid, < / RTI > sorbic acid. Preferred preservatives are cetrimide, benzalkonium chloride, benzalkonium chloride and parabens. Adequate amounts of preservatives can be added to prevent secondary contamination during use caused by bacteria and fungi.

Tension agents that may be used in the compositions of the present invention are those among those enumerated herein that are used to control physiological stiffness (e.g., 0.9% saline). For example, sodium chloride, potassium chloride, calcium chloride, dextrose and / or mannitol may be added to the composition comprising thymosin beta 4 of the present invention. The amount of the tensing agent varies depending on the particular agent to be added. Generally, certain inventive compositions can be added with a tonic to have the final composition have an ophthalmic acceptable osmolality of preferably 150-450 mOsm and most preferably 250-350 mOsm. Preferred tranquilizers are sodium salts, and potassium salts, especially sodium chloride and potassium chloride. The most preferred tensioning agent is sodium chloride.

In addition, the following formulations may be used to maintain adequate viscosity in ophthalmic formulations, but are not limited to: (a) monomeric polyols such as tyloxapol, glycerol, propylene glycol, ethylene glycol; (b) polymeric polyols such as polyethylene glycol (e.g., PEG 300, PEG 400); (c) cellulose derivatives (polymers of the class of cellulose), such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose; Carboxymethylcellulose sodium, hydroxylpropylcellulose; (d) dextrans, such as dextran 70; (e) water-soluble proteins such as gelatin; (f) vinyl polymerized products such as polyvinyl alcohol; Polyvinylpyrrolidine; (g) other polyols such as polysorbate 80; Povidone; (h) carbomers such as carbomer 934P, carbomer 941, carbomer 940 and carbomer 974P, and (i) polysaccharides / glycosaminoglycans such as hyaluronan / Hyaluronate), chondroitin sulfate. In addition, one or more viscosity enhancing agents may be added to the compositions of the present invention to increase the viscosity of the carrier (vehicle).

The amount and type of excipient (s) added may vary depending on the specific requirements, but may generally be in the range of about 0.0001 to about 90% by weight, and may be used within the scope of ordinarily used by those skilled in the ophthalmic arts. In addition, the pH range of ophthalmic formulations may range from 3.5 to 9, preferably from 4.5 to 8, and most preferably from pH 5.5 to 7.8.

In another aspect of the present invention, there is provided a method of treating neurotrophic keratitis comprising administering thymosin beta4 to a subject.

The present invention provides a therapeutic method comprising the step of contacting an eye tissue with an effective amount of a composition containing thymosin beta 4 as an active ingredient. Examples of direct administration include, for example, by direct application with solutions, lozenges, pastes, gels, creams, pastes, sprays, suspensions, dispersions, hydrogels, ointments, oils or foams containing the peptide agonists as disclosed herein And contacting the tissue.

In addition, the composition may further comprise citric acid and its salts. In addition, the composition may additionally comprise citric acid, acetic acid and salts thereof.

At this time, thymosin beta 4 and citric acid or a salt thereof may be administered jointly or sequentially, and may be administered in appropriate amounts divided into several times a day. However, the most preferred form of administration is simultaneous administration of thymosin beta 4 and citric acid or a salt thereof.

Thymosin beta 4 in the composition may be 0.05 to 0.5% (w / v), or 0.1 to 0.4% (w / v), based on the total composition, and administered in a total dose of 0.08 to 2.0 ml per day. It may be administered once or several times a day, preferably two to five times a day. In addition, citric acid or a salt thereof may include from 1.5% (w / v) to 5% (w / v) based on the total composition and may include from 2.5% (w / v) to 4% (w / v) . It may also be dosed in a total dose of 0.1 to 4.0 ml per day. May be administered once a day or divided into several times, preferably two to five times a day

In addition, acetic acid or a salt thereof may be administered concurrently or sequentially with thymosin beta4 and citric acid or a salt thereof, but preferably the acetic acid or a salt thereof may be administered simultaneously with thymosin beta4 and citric acid or a salt thereof. (W / v) to 1.5% (w / v) of citric acid or a salt thereof may be contained in an amount of 0.1% (w / v) to 0.8% (w / v), and preferably 0.2 to 0.5% may be included. It may also be dosed in a total dose of 0.15 to 6.0 ml per day. May be administered once a day or divided into several times, preferably two to five times a day

Examples of routes of administration of the pharmaceutical compositions of the present invention include parenteral administration such as intravenous, intradermal, subcutaneous, oral, intranasal (such as inhalation), transdermal (such as topical) ≪ / RTI >

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments.

Example  One. Thymosin β4  Preparation of compositions containing

Each reagent was added to the sterilized auxiliary water in the same amount as shown in Table 1 below and mixed until all the reagents were dissolved. Then, thymosin beta 4 (Bachem, US, SEQ ID NO: 1) mg / ml, and mixed until they were completely dissolved. After the above mixing process, the pH of the solution was adjusted to 7.0 using sodium hydroxide and hydrochloric acid. Thereafter, the solution that had undergone the above adjustment process was filtered through a 0.2 mu m filter, and the mixed solution obtained through the above filtration process was filled in a low density polyethylene container and then sealed.

reagent Addition amount (mg / ml) %  (w / v) function Thymosin beta 4 One 0.1 Active ingredient Sodium Chloride 6.4 0.64 An emerging topic Potassium Chloride 0.75 0.075 An emerging topic Calcium Chloride Dihydrate 0.48 0.048 Electrolyte Magnesium Chloride Hexahydrate 0.3 0.03 Electrolyte Citric Acid 1.7 0.17 Buffer Hydrochloride Acid Add as needed to adjust pH 7.0 pH adjusting agent Sodium Hydroxide Add as needed to adjust pH 7.0 pH adjusting agent Sterile Water for Injection Amount to make 100% (W / V) solvent

Experimental Example  One. Neurotrophic keratitis  Clinical trial to confirm treatment effect

In nine patients with neurotrophic keratitis who were not healed with chronic corneas for more than 6 to 10 years, the composition of Example 1 was administered four times a day for 28 days or 49 days to improve the treatment of neurotrophic keratitis And the prognosis was observed up to about 30 days after the administration. This trial was conducted in patients who were not treated with conventional therapies or treatments other than thymosin β4.

To confirm the treatment, 5 ml of 2% sodium fluorescence solution without preservative was slowly injected into each conjunctival region under each eye, and the wound size was changed after fluorescent staining for 3 to 5 minutes. The wound size is expressed by the width X lengthwise dimension (mm ² ). Table 2 shows changes in the size of damaged cornea in patients with neurotrophic keratitis administered with the composition of Example 1 for 49 days. In Table 2, although neurotrophic keratitis was not cured during the dosing period, each patient was cured within 29 days after the prognosis. In the case of patient A, the conjunctival treatment with Vandy Contact Lens at the end of dosing showed a noticeable decrease in corneal wound size and cure on the 29th day of prognosis. B patients did not show cure results at the end of dosing, but corneas were smaller in size than before treatment and cured at 29 days of prognosis. These results indicate that even after the administration of the composition of Example 1, the therapeutic effect of neurotrophic keratitis is maintained, and it is effective even when the composition is used in combination with the conventional treatment method.

Wound size ( mm ² ) Case Day 0 Day 14 Day 28 Day 49 Day 78 A 15.0 18.0 28.0 36.0 0.0 B 12.4 13.2 12.4 7.2 0.0

Table 3 also shows the change in the size of the damaged cornea in patients who were administered the composition of Example 1 for 28 days. As shown in Table 3, all the patients were cured within 28 days of the administration of the composition of Example 1, and the cured results were maintained even on the 30th day of the prognostic observation.

Wound size ( mm ² ) Case Day 0 Day 14 Day 28 Day 58 C 12.2 3.0 0.0 0.0 D 10.5 5.5 0.0 0.0 E 2.5 2.0 0.0 0.0 F 1.0 1.0 0.0 0.0

Taking the results of Table 2 and Table 3 into consideration, it was confirmed that the composition of Example 1 containing thymosin? 4 is excellent in the treatment of neurotrophic keratitis.

The results for three patients with Punctate defects in nine patients with neurotrophic keratitis are shown in Table 4. These patients were treated with the composition of Example 1 for 49 days and observed for prognosis for 29 days. The degree of treatment of neurotrophic keratitis was evaluated by corneal fluorescence staining in the same manner as the above-mentioned method, Grade). The higher the grade level, the greater the degree of corneal damage.

Grade 1 = 2-10

Grade 2 = 11-30

Grade 3 = 31-60

Grade 4 ≥ 60

Case Day 0 Day 49 Day 78 G Grade 3 Grade 2 Grade 2 H Grade 4 Grade 3 Grade 3 I Grade 4 Grade 4 Grade 4

As shown in Table 4, in the case of neurotrophic keratitis patients with Punctate defect, the therapeutic effect of Example 1 including thymosin β4 was insignificant.

These results indicate that the composition of Example 1 containing thymosin beta4 has a better therapeutic effect on neurotrophic keratitis without punctate defect.

&Lt; 110 > G-treeBNT Co., Ltd. <120> A pharmaceutical composition for treating neurotrophic          keratopathy <130> FPD201507-0010 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> thymosine beta 4 <400> 1 Ser Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys Ser Lys   1 5 10 15 Leu Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Ser Lys Glu              20 25 30 Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser          35 40

Claims (12)

A composition for treating neurotrophic keratitis comprising thymosin beta 4 as an active ingredient. 2. The composition for treating neurotrophic keratitis according to claim 1, wherein the thymosin beta 4 is an amino acid sequence of SEQ ID NO: 1. The composition for treating neurotrophic keratitis according to claim 1, wherein the concentration of thymosin beta 4 is 0.05 to 0.5% (w / v). The composition for treating neurotrophic keratitis according to claim 1, wherein the composition further contains acetic acid or citric acid. 5. The therapeutic composition according to claim 4, wherein the citric acid is sodium citrate hydrate, and the acetic acid is sodium acetate hydrate. The composition for treating neuritic keratitis according to claim 5, wherein the concentration of the sodium citrate hydrate is 0.1 to 0.3% (w / v). The composition for treating neurotrophic keratitis according to claim 5, wherein the concentration of the sodium acetate hydrate is 0.2 to 0.5% (w / v). 8. The composition for treating neurotrophic keratitis according to any one of claims 1 to 7, which is effective for treatment of damaged cornea or symptom improvement caused by neurotrophic keratitis. 8. The composition according to any one of claims 1 to 7, wherein the composition further comprises sodium chloride, potassium chloride, calcium chloride hydrate and magnesium chloride hexahydrate. The method of claim 9, wherein the concentration of sodium chloride is 0.5 to 0.7% (w / v), the concentration of potassium chloride is 0.05 to 0.09% (w / v), the concentration of calcium chloride hydrate is 0.03 to 0.06% / v), and the concentration of the magnesium chloride hexahydrate is 0.01 to 0.05% (w / v). 10. A method for treating neurotrophic keratitis comprising administering to a subject a composition of any one of claims 1 to 10. 12. The method according to claim 11, wherein thymosin beta 4 in the composition is 0.05 to 0.5% (w / v) based on the total composition and is dosed in a total dose of 0.08 to 2.0 ml per day.