KR20160047321A - Composition comprising thymosin beta 4 - Google Patents

Abstract

The present invention relates to an agent for treating ophthalmologic diseases comprising thymosin beta 4. The agent according to the present invention is more effective for improving the dry eye syndrome as compared to the eye drop solutions comprising cyclosporin A, is less irritating to eyes as compared to the conventional eye drop solutions, and is physicochemically stable.

Description

[0001] COMPOSITION COMPRISING THYMOSIN BETA 4 [0002]

The present invention relates to a therapeutic agent for ophthalmic diseases containing thymosin beta 4.

Tears have basic tears and reflex tears. Basic tears are those that are constantly secreted by constant reflexes such as pain, dust, spit, smoke, smell, pleasure or sad feelings, or irritation due to lack of tears. It says.

The secreted tears are present in the surface layer of the cornea, so that it smoothly covers the surface of the eyes to protect the eyes and make them comfortable. It smoothes the surface of the cornea to secure the optical field of vision, and protects the cornea and conjunctival epithelial cell Role, antimicrobial action from external germs, and role of supplying nutrition to the cornea.

Tears are composed of three layers of mucous layer, a water layer and a fat layer, and they are usually produced in the order of 2 to 3 mL per day. If one of the three layers that make up tears is lacking and changes in the tear layer or the amount of tear production is small, the eye becomes dry and the tear does not play a role, Lt; / RTI >

Ocular drying refers to the abnormal state of the amount and quality of tears, regardless of the conjunctivitis disorder [Masakazu YAMADA et al., Nippon Ganka Kiyo, 43, 1289-1293 (1992)]. In addition, dry eye syndrome refers to any disease associated with pathologic changes in the tear film or secretion of less tears. If the tear is insufficient, the tear is excessively evaporated, or the components of the tear are not in balance, Irritation symptoms such as irritation, foreign body, or dry feeling, as well as keratitis or membrane epithelial wounds resulting from such symptoms. Typically, the dry eye syndrome includes, but is not limited to, dry eye syndrome or dry eye conjunctivitis.

Dry eye syndrome is often found in middle-aged women and elderly people because of the frequent occurrence of ocular dryness due to a lack of tear or a decrease in function with age. However, it has also been reported that it may be associated with inflammation, drug use (eg, hypertension, gastric hyperplasia, neuropathy or depression), trauma, chronic conjunctivitis, eyelid dysfunction or prostate disease. In most cases, I can not. It is also known that dry eye syndrome may be caused by autoimmune disease due to immune reaction abnormality.

Immunity is the function of defending the body from the germs invaded from the outside. The autoimmune disease is caused by abnormalities in the immune function, which occurs when the body's defense mechanism, that is, the immune system, It refers to diseases caused by immune and hypersensitivity reactions. An autoimmune disease is a virus or a bacterium that causes the body's cytokine (a signaling substance that controls and stimulates the body's defense system) to change, causing an inappropriate immune response leading to an autoimmune reaction that is either excessive or inadequately controlled But the exact cause of the disease is unknown, and it is being discussed that it may be caused by environmental factors, genetic factors or immunological factors.

Currently, most autoimmune diseases are treated or prevented by the use of steroids or immunosuppressants. Among them, a typical drug used as a therapeutic or prophylactic agent for dry eye syndrome of mammals including humans is RESTASIS ^TM, which is a drug of 0.05% (w / v) cyclosporin A 5,474, 979).

U.S. Patent No. 5,474,979 Korean Patent Publication No. 1008189 Korean Patent Publication No. 10-2008-0033939

Cyclosporin A is an insoluble drug having a water solubility of about 20 ug / ml to 30 ug / ml, and Resistis ^TM is prepared in the form of a microemulsion comprising a liquid-liquid dispersion system. However, the microemulsion is thermodynamically unstable, so that phenomena such as flocculation, sedimentation, creaming, particle growth or adhesion occur frequently and the liquid-liquid dispersion is destroyed. In order to make the particles more stable, there have been attempts to reduce the particles into nano-sized particles in the form of nano emulsion (Korean Patent Registration No. 1008189), but the nano-emulsification process is a high-pressure homogenizer or a micro- It is not only necessary to have such an expensive machine as that, but also deteriorate during the process, which is disadvantageous in that a lot of effort and cost are required in the manufacturing process.

In addition, Restasis ^TM is an emulsion based on castor oil, which has a short storage period.

In addition, although Restasis ^™ is currently the most widely used treatment or prophylaxis of dry eye syndrome, it is known that the bioavailability is not optimized. For example, it can take several weeks to see the full effect of Restasis ^TM . Here, biological availability refers to the percentage of drug detected in the target eye tissue after administration, where detection of the drug is pharmacodynamic (quantification of the biological response to cyclosporine A) or pharmacokinetics ≪ / RTI >

Therefore, in the field of treatment or prevention of dry eye syndrome, there has been a demand for a drug having improved physico-chemical stability and a drug having improved bioavailability. As a solution to this problem, a recent study on thymosin beta 4 It is progressing.

Thymosin beta 4 is the first protein found in the thymus in 1981, consisting of 41 to 43 amino acids and an isoelectric point of 5.1. Tymosin beta4, first identified as an actin-sequestering molecule in animal cells by Riba et al. In 1991, has subsequently been shown to act as an immunoregulatory and neuroendocrine. In addition, thymosin beta 4 not only functions as a terminal deoxynucleotide transferase in thymocytes but also increases the migration of macrophages and the antigen of macrophages, It is also known to increase the secretion of luteinizing hormone by hypothalamic explants. In order to remove the toxicity of cytosine arabinoside and increase the attachment and migration of endothelium, It is also known to inhibit the cell cycle of hematopoietic stem cells. In addition, Korean Patent Laid-Open Publication No. 10-2008-0033939 discloses the use of eye drops containing thymosin beta 4 and a sterilizing activity preserving agent for treating eye inflammation, eye infections (bacteria, fungi or viruses) and glaucoma have.

However, the combination and content of each component optimized as an eye drop composition for treating dry eye syndrome to date, including Korean Patent Laid-Open Publication No, have not been disclosed.

Accordingly, the present inventor intends to provide an optimal ophthalmic solution for treatment or prevention of dry eye syndrome, which has been proposed for the treatment or prevention of dry eye syndrome, which has been improved in disadvantages of conventional drugs and preparations.

The present invention solves the above-described problems through the following means.

(1) A pharmaceutical composition for treating or preventing dry eye syndrome, which comprises thymosin beta 4, sodium chloride, potassium chloride, calcium chloride hydrate and magnesium chloride hexahydrate.

(2) The pharmaceutical composition according to the above (1), wherein the pharmaceutical composition further comprises hydrochloric acid or sodium hydroxide.

(3) The pharmaceutical composition according to the above (1) or (2), wherein the concentration of thymosin beta 4 is 0.05 to 0.5% (w / v) based on the total composition.

(4) The pharmaceutical composition according to any one of (1) to (3) above, wherein the pharmaceutical composition contains sodium chloride in an amount of 0.5 to 0.7% (w / v), potassium chloride in an amount of 0.05 to 0.09% (w / v) 0.03 to 0.06% (w / v) of magnesium chloride and 0.01 to 0.05% (w / v) of magnesium chloride hexahydrate.

(5) The pharmaceutical composition according to any one of (1) to (4) above, wherein the pharmaceutical composition further contains sodium acetate hydrate or sodium citrate hydrate.

(6) The pharmaceutical composition according to (5), wherein the concentration of sodium acetate hydrate is 0.2 to 0.5% (w / v) based on the total composition.

(7) The pharmaceutical composition according to (5), wherein the concentration of sodium citrate hydrate is 0.1 to 0.3% (w / v) based on the total composition.

The present invention is more effective in improving dry eye syndrome than eye drops containing cyclosporin A. Further, the present invention is less irritating to eyes as compared with conventional eye drops, is physically stable, and has excellent storage period. In particular, the present invention can have excellent stability while minimizing the use of additives compared to conventional eye drops.

FIG. 1 shows the results of observation of changes in the corneal surface fluorescence staining for each administration period of Example 2, Example 4, Example 5, and placebo control.
FIG. 2 shows the results of observing changes in the fluorescence staining of the cornea on the baseline versus baseline in the case of Example 2, Example 4, Example 5, and the placebo control group.
FIG. 3 shows the results of observing changes in fluorescence staining intensity according to baseline versus baseline for the inferior region of the corneas of Example 2, Example 4, Example 5, and the placebo control group.
FIG. 4 shows the results of observing changes in the fluorescence staining on the cornea on days 6 and 13 after administration of Example 1, Example 2, and Example 3. FIG.
FIG. 5 shows the results of observation of changes in the fluorescence staining on the corneal surface after 6 and 13 days of administration of Example 2, Comparative Example 1 and the placebo control group.
FIG. 6 shows the results of observing changes in the fluorescence staining on the surface of the cornea 30 days after the administration of Example 2 and the placebo control group.

[Composition]

The composition according to the present invention comprises a drug, thymosin beta 4; Sodium chloride, potassium chloride, calcium chloride hydrate and magnesium chloride hexahydrate. According to one embodiment, the composition according to the present invention further comprises hydrochloric acid and / or sodium hydroxide as a pH adjusting agent. According to another embodiment, the composition according to the present invention may further contain sodium acetate hydrate and / or sodium citrate hydrate. In addition, the composition according to the present invention may further contain other well-known additives within the range not hindering the object of the present invention.

The composition according to the present invention may be prepared as a solution, lotion, paste, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil or foaming agent and then contacted with the eye tissue by direct application to prevent dry eye syndrome Or treat it.

In particular, the composition according to the present invention may be used as a preventive or therapeutic agent for dry eye syndrome by making eye drops containing all or a part of the following steps, and then bringing them into contact with eye tissues by direct application.

1. Weighing process

Each raw material, reagent and solvent is weighed.

2. Mixing process

Add each reagent weighed individually to sterilized water, mix until all of the reagents are dissolved, add thymosin beta 4, the drug, to the mixed solution, and mix until all is dissolved.

3. Adjustment process

After the above mixing process, the solution is adjusted to pH 7.0 using sodium hydroxide and hydrochloric acid.

4. Filtration process

The solution that has undergone the above adjustment process is filtered through a 0.2 mu m filter.

5. Charging process

The mixture obtained through the above filtration process is filled in a low density polyethylene container and sealed.

[drug]

In view of the manifestation of the desired effect of the present invention, thymosin beta 4 is preferably contained in the composition at a concentration of 0.05 to 0.5% (w / v) based on the total composition, especially 0.1% (w / v) Most preferably in the composition.

[pH adjusting agent]

The ophthalmic preparation is preferably prepared similar to the physiological pH of the tears in terms of comfort, tolerance and stability in use.

The normal physiological pH of the tears is about 7.4. However, when eye secretion is stimulated or blinking, the pH of the tears decreases. Conversely, if the eyelid is open for an extended period of time, the tear film is alkalized by equilibrium with the partial pressure of CO _{2 in} the ambient air, resulting in a tear pH of 9 or higher.

Thus, the preparation for ophthalmic preparation is preferably prepared in the range of pH 3.5 to 11.5, preferably in the range of pH 3.5 to 9, more preferably in the range of pH 4.5 to 8.0, and most preferably in the range of pH 5.5 to 7.8 desirable. To this end, the composition according to the invention may contain hydrochloric acid and / or sodium hydroxide.

[Other excipients]

(W / v) of sodium chloride, 0.05 to 0.09% (w / v) of potassium chloride, 0.03 to 0.06% (w / v) of calcium chloride hydrate on the basis of the total composition in terms of expression of the desired effect of the present invention, v) and magnesium chloride hexahydrate are preferably contained in the composition at a concentration of 0.01 to 0.05% (w / v). In particular, sodium chloride, sodium chloride, potassium chloride and sodium chloride are 0.07-0.08% (w / v), 0.04-0.05% (w / v) and 0.02 To 0.04% (w / v).

According to one embodiment, the composition according to the present invention comprises 0.2-0.5% (w / v) sodium acetate hydrate and / or 0.1-0.3% (w / v) (w / v) sodium citrate hydrate.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Examples 1 to 5] - Preparation of eye drops

An eye drop was prepared at the composition ratios shown in Table 1 below. For reference, in the following Table 1, the unit of the numerical value without a separate designation is the concentration (% w / v) based on the total composition. That is, the concentration of thymosin beta 4 in Example 1 was 0.05% (w / v), the concentration of thymosin beta 4 in Example 2 was 0.1% (w / v) The concentration of thymosin beta 4 in Example 4 was 1% (w / v), the concentration of thymosin beta 4 in Example 5 was 2% (w / v) (w / v).

ingredient Example 1 Example 2 Example 3 Example 4 Example 5 Thymosin beta 4 0.05 0.1 0.5 One 2 Sodium chloride 0.64 0.64 0.64 0.64 0.64 Potassium chloride 0.075 0.075 0.075 0.075 0.075 Calcium chloride hydrate 0.048 0.048 0.048 0.048 0.048 Magnesium chloride hexahydrate 0.03 0.03 0.03 0.03 0.03 Sodium acetate hydrate 0.39 0.39 0.39 0.39 0.39 Sodium citrate hydrate 0.17 0.17 0.17 0.17 0.17 Hydrochloric acid Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Sodium hydroxide Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Add as needed to adjust pH 7.0 Sterilized water Amount to make 100% (w / v) Amount to make 100% (w / v) Amount to make 100% (w / v) Amount to make 100% (w / v) Amount to make 100% (w / v)

On the other hand, in the following, Comparative Example 1 refers to a commercially available Resistis ^TM of 0.05% (w / v) of cyclosporin.

[Experimental Example 1] - Storage stability test

In order to examine the stability after storage of Example 2 and Comparative Example 1, the ocular solutions were stored at room temperature (15 to 25 ° C) and refrigerated for 70 days, and the state of the composition and the activity of the drug in the composition were measured by HPLC. Immediately after the preparation, the content of thymosin beta 4 in Example 2 was 1 mg / ml, the content of cyclosporin in Comparative Example 1 was 0.5 mg / ml, and the operating conditions of HPLC were as follows.

HPLC operating conditions Column: Nova-Pack Phenyl 4 탆 (3.9 X 150 mm), flow rate 1 μl / min, pump pressure 700-800 psi, detection wavelength 205 nm, mobile phase: acetonitrile 65% M monobasic phosphate buffer (35%), injection volume: 30 μl

The results are shown in Table 2 below.

Example 2 Comparative Example 1 Appearance The potency of the drug Appearance The potency of the drug Immediately after manufacture Clear, clear solution 1 mg / ml White opaque or slightly translucent homogeneous liquid 0.5 mg / ml After storage at room temperature for 70 days Clear, clear solution 0.993 mg / ml Precipitation generation No potentiation After 70 days of refrigerated storage Clear, clear solution 0.998 mg / ml Precipitation generation No potentiation

That is, the composition of Example 2 is superior to that of Comparative Example 1 in storage stability.

[Experimental Example 2] - Test for confirming improvement of ocular dryness

In order to confirm the improvement effect of dry eye syndrome treatment by the concentration of thymosin beta 4 (Example 2, Example 4 and Example 5) in dry eye syndrome mouse model against placebo control (preparation not containing thymosin beta 4) A comparative experiment was performed. In order to induce ocular dryness, mice were treated with CAE (Controlled Adverse Environment) for 8 groups of 8 female C57BL / 6 strain in each group, and inhibited tear production and worsened the symptoms of dry eye disease , 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected four times a day between Day 6 and Day 10 after drug administration. The drug was administered 4 times with 0.005 ml per day for 14 days, and the change of the corneal surface fluorescence staining was observed on the corneal surface fluorescence staining test on the 9th, 12th and 14th day after the administration to confirm the therapeutic effect. For reference, the corneal surface fluorescence staining is an indicator of signs of dry eye syndrome, and the lower the score, the better the dry eye symptoms.

The results were as shown in Figs. 1, 2 and 3.

In FIG. 1 and FIG. 3, Baseline indicates the number of corneal surface fluorescence staining score in healthy mice before induction of dry eye syndrome. Before administration of the drug, the corneal surface fluorescence staining score .

1, in the placebo group, Example 4, and Example 5, the corneal surface fluorescence staining score was increased, but on Day 14, the corneal surface fluorescence staining score was shown, but the improvement effect on the baseline was not observed. Surprisingly, however, in Example 2, significant improvement was observed in dry eye syndrome. From this, it was confirmed that the dose and effect expression of thymosin beta 4 were not in proportion to each other.

FIG. 2 is a graph showing changes in the number of corneal surface fluorescence staining scores for each treatment period versus baseline. FIG. 2 shows that only Example 2 is effective in improving dry eye syndrome.

FIG. 3 is a graph showing the results of observing changes in the corneal surface fluorescence staining score only for the inferior region of the cornea. In Examples 2 and 5, , The ocular dryness symptom was improved only in Example 2 without worsening of symptoms.

That is, the composition of Example 2 is most effective for improving dry eye syndrome.

[Experimental Example 3] - Test for confirming improvement of eye dryness for setting the dose concentration

A comparative experiment to examine the improvement of treatment of dry eye syndrome caused by administration of Example 1 and Example 3 twice a day was carried out using a mouse model of dry eye syndrome. For the induction of eye dryness, mice were tested in CAE (Controlled Adverse Environment) under the control of a controlled adverse environment (C57BL / 6 strain) in 5 groups of 8 week old females for each group to inhibit tear production and worsen the symptoms of dry eye disease 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected four times a day between Day 6 and Day 10 after drug administration. The drug was administered twice in 0.005 ml per day for 6 days with each experimental group (Example 1, Example 2 and Example 3), and on the 6th day and the 13th day after the administration, Dyeing tests were performed to observe changes in the fluorescence staining on the cornea.

The results are shown in Fig.

The results of FIG. 4 show that the degree of fluorescence staining on the corneal surface of the experimental group administered with Example 2 and Example 3 twice a day was decreased compared to that of Example 1 administered twice a day In particular, it can be seen that the score tends to be slightly decreased in the second embodiment as compared with the third embodiment.

That is, the effective concentration of Example 2 is more effective than the effective concentration of the drug contained in Examples 1 and 3 in improving the dry eye syndrome.

[Experimental Example 4] Comparative experiment of eye dry symptom improvement with cyclosporin 0.05% (w / v) (Comparative Example 1)

Experiments comparing the ocular dry symptom improvement patterns between Example 2, the active control, Comparative Example 1, and the placebo control group were conducted using a dry eye mouse model. For the induction of eye dryness, mice were tested in CAE (Controlled Adverse Environment) under the control of a controlled adverse environment (C57BL / 6 strain) in 5 groups of 8 week old females for each group to inhibit tear production and worsen the symptoms of dry eye disease , 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected four times a day between Day 6 and Day 10 after drug administration. The drug was administered twice daily for 6 days at 0.005ml per day for 6 days. The corneal surface fluorescence staining was examined before and after the treatment on the 6th and 13th day of the experiment.

The results were as shown in Fig.

The results of FIG. 5 show that the degree of fluorescence staining on the cornea was significantly reduced in Example 2 as compared with Comparative Example 1, which is the active control group, compared with before administration. More specifically, the degree of fluorescence staining on the corneal surface was significantly decreased by day 5 when the administration of Example 2 was performed, and the response was observed while administering scopolamine to improve the degree of ocular dryness deterioration from day 6, The response to worsening of dryness was significantly reduced.

[Experimental Example 5] - Clinical Experiment (Phase 2)

Based on the non-clinical results, we conducted a Phase 2 clinical trial comparing the safety and efficacy of the placebo-controlled group with the symptom and symptoms of dry eye syndrome. To assess the safety and efficacy of 0.1% (w / v) thymosin beta 4 instillation compared to placebo, the above clinical signs and symptoms of dry eye under the Controlled Adverse Environment (CAE) We randomly assigned to a single institution. Specifically, 36 patients in each group were treated with 1 or 2 drops twice a day using a squeeze bottle with a dose of 40 μl per drop for 30 days in the placebo control group and Example 2, The corneal surface fluorescence staining test was performed before and 30 days after dosing to observe the changes in the corneal surface fluorescence staining.

The results are shown in Fig.

The results of FIG. 6 show that the difference in fluorescence staining on the corneal surface after administration of Example 2 twice a day is apparent compared to the placebo control group.

That is, Example 2, as in the non-clinical results, is effective in the treatment of dry eye syndrome.

[Experimental Example 6] - Irritation test

To compare the irritation (or lining) between Example 2 and Comparative Example 1 during the above clinical study, the degree of stimulation (or lining) was measured by dividing the degree of the stimulus by the following criteria.

Evaluation criteria - The degree of feeling discomfort due to tingling of the eyes after eye drops or disturbance of the visual field for a certain period of time on the scale of 5 points is very unsatisfactory, 2 is dissatisfied, 3 is normal, 4 is Satisfaction, and 5 points are measured as very satisfied.

The results are shown in Table 3 below.

Example 2 Comparative Example 1 Irritability 4.5 2.5

That is, the feeling of use of Example 2 is significantly superior to that of Comparative Example 1.

Claims (7)

A pharmaceutical composition for treating or preventing dry eye syndrome comprising thymosin beta 4, sodium chloride, potassium chloride, calcium chloride hydrate and magnesium chloride hexahydrate. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises hydrochloric acid or sodium hydroxide. 3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of thymosin beta 4 is 0.05 to 0.5% (w / v). The method according to claim 1 or 2, 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% w / v) and the concentration of magnesium chloride hexahydrate is 0.01 to 0.05% (w / v). 3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition further comprises sodium acetate hydrate or sodium citrate hydrate. The pharmaceutical composition according to claim 5, wherein the concentration of sodium acetate hydrate is 0.2 to 0.5% (w / v). The pharmaceutical composition according to claim 5, wherein the concentration of sodium citrate hydrate is 0.1 to 0.3% (w / v).

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