COMPOSITION CONTAINING THYMOSIN BETA 4, AND PHARMACEUTICAL FORMULATION COMPRISING SAME

Abstract

The present invention relates to a therapeutic agent for ophthalmic disease containing thymosin beta 4. The present invention is more effective in reducing xerophthalmia than an ophthalmic solution containing cyclosporine A, is less irritant to eyes than conventional ophthalmic solutions and is physiochemically safe.

Claims

1. A pharmaceutical composition for treatment or prevention of dry eye syndrome, comprising; (a) thymosin beta 4 in a concentration of from 0.05 to 0.5% (w/v); (b) sodium chloride; (c) potassium chloride; (d) calcium chloride dihydrate in a concentration of from 0.03 to 0.06% (w/v); and (e) magnesium chloride hexahydrate in a concentration of from 0.01 to 0.05% (w/v).

2. The composition according to claim 1, further comprising hydrochloric acid or sodium hydroxide.

3. (canceled)

4. The composition according to claim 1, wherein the sodium chloride is present in a concentration ranging from 0.5 to 0.7% (w/v), and wherein the potassium chloride is present in a concentration ranging from 0.05 to 0.09% (w/v).

5. The composition according to claim 1, further comprising sodium acetate trihydrate or sodium citrate dihydrate.

6. The composition according to claim 5, wherein the sodium acetate trihydrate is present in a concentration ranging from 0.2 to 0.5% (w/v).

7. The composition according to claim 5, wherein the sodium citrate dihydrate is present in a concentration ranging from 0.1 to 0.3% (w/v).

8. The composition according to claim 1, wherein the composition is in a unit dosage form to provide a total daily dosage of 0.08 to 2.0 ml.

9. The composition according to claim 8, wherein the composition is administered by droplet.

10. The composition according to claim 9, wherein each droplet ranges from 0.01 to 2.0 ml.

11. A unit dosage form comprising the composition according to claim 1, wherein the unit dosage form has a total volume ranging from 0.01 to 10 ml.

12. The unit dosage form according to claim 11, wherein the unit dosage form is an eye drop container.

13. The composition according to claim 1, wherein the composition is a clear and transparent solution.

14. A method for preventing or treating dry eye syndrome in a mammal, which comprises administering the composition according to claim 1 to the mammal.

Description

DESCRIPTION OF DRAWINGS

[0016] FIG. 1 illustrates changes in corneal surface fluorescein staining scores in relation to the time after administration of the respective products of the respective Examples 2, 4 and 5 and placebo control;

[0017] FIG. 2 illustrates changes from the baseline in corneal surface fluorescein staining scores in relation to the time after administration of the respective products of Examples 2, 4 and 5 and placebo control;

[0018] FIG. 3 illustrates changes from the baseline in fluorescein staining scores in relation to the time after administration of the respective products of Examples 2, 4 and 5 and placebo control;

[0019] FIG. 4 illustrates changes in corneal surface fluorescein staining scores at Day 6 and Day 13 after administration of the respective products of Examples 1 to 3;

[0020] FIG. 5 illustrates changes in corneal surface fluorescein staining scores at Day 6 and Day 13 after administration of the respective products of Example 2, Comparative Example 1 and placebo control; and

[0021] FIG. 6 illustrates changes in corneal surface fluorescein staining scores at Day 30 after administration of the respective products of Example 2 and placebo control.

BEST METHOD

[0022] In order to accomplish the objects of the present invention, the present invention provides a pharmaceutical composition for the treatment or prevention of dry eye syndrome, which includes thymosin beta 4, sodium chloride, potassium chloride, calcium chloride dihydrate and magnesium chloride hexahydrate.

[0023] The term “thymosin β4” as used herein refers to a polypeptide composed of 43 amino acids having 4.9 kDa, which is occasionally called thymosin beta-4 or Tβ4. This protein is upregulated during in vitro migration and differentiation of endothelial cells. A number of thymosin β4 isotypes have been discovered so far and have homology of approximately 70% or more, approximately 75% or more or approximately 80% or more with the known amino acid sequences of thymosin β4. Thymosin 62 4 in accordance with the present invention may also be an N-terminal mutant or a C-terminal mutant of wild type thymosin β4. Preferably, thymosin β4 in accordance with the present invention is the protein having an amino acid sequence denoted by SEQ ID No. 1. A concentration of thymosin beta 4 may range from 0.01 to 1.0% (w/v) preferably from 0.05 to 0.5% (w/v).

[0024] Furthermore, a concentration of sodium chloride may range from 0.1 to 1.2% (w/v) or from 0.3 to 1.0% (w/v). Preferably, it ranges from 0.5 to 0.7% (w/v). Furthermore, a concentration of potassium chloride may range from 0.01 to 0.15% (w/v) or from 0.03 to 0.12% (w/v). Preferably, it ranges from 0.05 to 0.09% (w/v). Furthermore, a concentration of calcium chloride dihydrate may range from 0.01 to 0.12% (w/v) or from 0.03 to 0.09% (w/v). Preferably, it ranges from 0.03 to 0.06% (w/v). Furthermore, a concentration of magnesium chloride hexahydrate may range from 0.01 to 0.12% (w/v) but preferably from 0.01 to 0.05% (w/v).

[0025] Meanwhile, the pharmaceutical composition in accordance with the present invention may further include hydrochloric acid or sodium hydroxide. Hydrochloric acid or sodium hydroxide may be suitably added to adjust a pH of the composition. The pH of the composition may be pH 6.5 to 7.5, or pH 6.8 to 7.2. Preferably, the composition may have pH 7.0.

[0026] Additionally, the pharmaceutical composition in accordance with the present invention may further include acetic acid or citric acid.

[0027] Acetic acid used herein is a weak acid represented by formula CH.sub.3COOH. In the present invention, this may also be used in the form of acetate. One embodiment of the acetate may be sodium acetate trihydrate. Acetic acid or its salt may be included in an amount of 0.01% (w/v) to 1.5% (w/v) based on the total volume of the composition. Furthermore, acetic acid or its salt may be included in an amount of 0.1% (w/v) to 0.8% (w/v) but preferably 0.2% (w/v) to 0.5% (w/v).

[0028] Citric acid used herein is a compound represented by formula C.sub.6H.sub.8O.sub.7. In the present invention, this may also be used in the form of citrate. The citrate is a derivative of citric acid, and embodiments of the citrate may be sodium citrate and sodium citrate dihydrate. Citric acid and its salt are generally used as buffers to limit the change in pH; the present invention needs quite a larger amount of citric acid or its salt than that commonly used in the art. In this case, citric acid or its salt may be included in an amount of 0.01% (w/v) to 0.5% (w/v). Furthermore, citric acid or its salt may be included in an amount of 0.05% (w/v) to 0.25% (w/v) preferably 0.1% (w/v) to 0.3% (w/v).

[0029] The composition in accordance with the present invention may be prepared as a formulation available for ophthalmic application. More specifically, the formulation may include, for example, solution, lotion, plaster, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil, foaming agent, etc.

[0030] The composition may include 0.01% (w/v) to 1.5% (w/v) preferably 0.05% (w/v) to 0.5% (w/v) of thymosin beta 4.

[0031] Furthermore, the composition may be formulated into a unit-dosage form to provide a total daily dosage of 0.08 to 2.0 ml, and for that purpose it may be suitably dispensed into a container for ophthalmic use, which may facilitate quantitative administration of the composition. For this purpose, the composition may be formulated into a unit-dosage form with a dosage of 0.01 to 10 ml that can be used once or several times. Furthermore, in order to suitably provide the pharmaceutical composition in a total daily dosage of 0.08 to 2.0 ml, the composition may be contained in an eye drop container dropping 0.01 to 2.0 ml per droplet.

[0032] According to another aspect of the present invention, there is provided a method for the treatment of dry eye syndrome, which involves administration of the above composition to the subject.

[0033] More particularly, the present invention provides a treatment method, which comprises contacting an eye tissue with an effective amount of a composition including thymosin β4 as an active ingredient. An example of direct administration may include directly applying the composition in the form of, for example, a solution, lotion, plaster, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil or foaming agent to the subject in order to ensure contact with eye tissues.

[0034] Furthermore, the composition may be formulated into a unit-dosage form to provide a total daily dosage of 0.08 to 2.0 ml, and for that purpose it may be suitably dispensed into a container for ophthalmic use, which may facilitate quantitative administration of the composition. For this purpose, the composition may be formulated into a unit-dosage form that can be used once or several times with a dosage of 0.01 to 10 ml. Furthermore, in order to suitably provide the pharmaceutical composition in a total daily dosage of 0.08 to 2.0 ml, the composition may be contained in an eye drop container dropping 0.01 to 2.0 ml per droplet. Preferably, the composition may be administered in the amount described above b.i.d. to q.i.d.

[0035] According to another aspect of the present invention, there is provided an eye drop containing thymosin β4 prepared in the manner described above. Thymosin β4 formulated as described above is not exposed to oxygen or heat, and can therefore be stable and retain its activity for a long period of time. Because the activity of thymosin β4 is retained for a long time, the eye drop in accordance with the present invention may maintain its pharmacological effects for a long period of time.

MODE FOR INVENTION

[0036] Hereinafter, in order to better understand the present invention, it will be described in more details by the following examples. However, such examples may include different variations or modifications, so it should not be construed that the scope of the present invention is limited within the scope of the following examples.

Process of Eye Drop Manufacture

[0037] The eye drop of the present invention may be manufactured according to the following process:

[0038] 1-1. Weighing

[0039] The raw materials, reagents and a solvent are weighed, respectively.

[0040] 1-2. Mixing

[0041] The weighed reagents are respectively placed in sterile water for injection as a solvent and mixed to be completely dissolved therein. Next, a weighed amount of thymosin beta 4 is added to the mixture and mixed until it is completely dissolved therein.

[0042] 1-3. Adjustment of pH

[0043] After completing the mixing, the solution is adjusted to pH 7.0 by using sodium hydroxide and hydrochloric acid.

[0044] 1-4. Filtering

[0045] Subsequent to adjustment, the solution is filtered through a 0.2 μm filter.

[0046] 1-5. Filling

[0047] Subsequent to filtration, the mixture is dispensed into a low-density polyethylene container, followed by sealing the container.

[Drug]

[0048] For the sake of the desired effects of the present invention, thymosin beta 4 is preferably contained in a concentration of 0.05 to 0.5% (w/v) based on the total volume of the composition and, most preferably, in a concentration of 0.1% (w/v).

[pH Modifier]

[0049] The ophthalmic formulation may be manufactured to have a pH value similar to the physiological pH of tears in view of comfort, tolerance and stability in use.

[0050] The pH value of tears is approximately 7.4. However, when the eye is stimulated by its exudates or blinking, the pH of tears is reduced. Contrastingly, if the eyelids remain open for an extended time, a tear film is alkalized by equilibrium with a partial pressure of CO.sub.2 in the surrounding air, and thus the pH of the tear becomes pH 9.0 or more.

[0051] The ophthalmic formulation manufactured herein may have pH 3.5 to 11.5, preferably, pH 3.5 to 9.0, more preferably, pH 4.5 to 8.0 and most preferably pH 5.5 to 7.8. For this purpose, the composition of the present invention may further include hydrochloric acid and/or sodium hydroxide.

[Other Excipients]

[0052] For the sake of the desired effects of the present invention, the composition may include 0.5 to 0.7% (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 dihydrate and 0.01 to 0.05% (w/v) of magnesium chloride hexahydrate based on the total volume of the composition. Most preferably, the composition includes 0.6 to 0.7% (w/v) of sodium chloride, 0.07 to 0.08% (w/v) of potassium chloride, 0.04 to 0.05% (w/v) of calcium chloride dihydrate, and 0.02 to 0.04% (w/v) of magnesium chloride hexahydrate based on the total volume of the composition.

[0053] According to one embodiment, the composition of the present invention may further include 0.2 to 0.5% (w/v) of sodium acetate trihydrate and/or 0.1 to 0.3% (w/v) of sodium citrate dihydrate based on the total volume of the composition to produce more excellent effects of the present invention.

[0054] [Dosage and Administration]

[0055] The composition of the present invention including 0.05 to 0.5% (w/v) of thymosin beta 4 may exhibit optimal effects while minimizing stimulation on the eyes and other side effects when it contacts with the eye tissues in a total daily dosage of 0.08 to 2 ml once or several times a day. For this purpose, the composition of the present invention may be formulated in a unit-dosage form with a volume of 0.01 to 10 ml for use once or several times a day. Furthermore, such a unit-dosage form may be contained in an eye drop container dropping 0.01 to 2.0 ml per droplet, so as to suitably provide the above-described total daily dosage.

EXAMPLES 1 THROUGH 5

Preparation of the Eye Drops

[0056] The eye drops were prepared with the compositional ratios shown in Table 1 below. Unless otherwise indicated, numerical values in Table 1 refer to a concentration of % (w/v) based on the total volume of the composition. For instance, the concentration of thymosin beta 4 in Example 1 is 0.05% (w/v), the concentration of thymosin beta 4 in Example 2 is 0.1% (w/v), the concentration of thymosin beta 4 in Example 3 is 0.5% (w/v), the concentration of thymosin beta 4 in Example 4 is 1% (w/v) and the concentration of thymosin beta 4 in Example 5 is 2% (w/v).

TABLE-US-00001 TABLE 1 Component Example 1 Example 2 Example 3 Example 4 Example 5 Thymosin beta 4 0.05 0.1 0.5 1 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 0.048 0.048 0.048 0.048 0.048 dihydrate Magnesium chloride 0.03 0.03 0.03 0.03 0.03 hexahydrate Sodium acetate 0.39 0.39 0.39 0.39 0.39 trihydrate Sodium citrate 0.17 0.17 0.17 0.17 0.17 dihydrate Hydrochloric Added if Added if Added if Added if Added if acid required for required for required for required for required for adjustment to adjustment to adjustment to adjustment adjustment to pH 7.0 pH 7.0 pH 7.0 to pH 7.0 pH 7.0 Sodium Added if Added if Added if Added if Added if hydroxide required for required for required for required for required for adjustment to adjustment to adjustment to adjustment adjustment to pH 7.0 pH 7.0 pH 7.0 to pH 7.0 pH 7.0 Sterile water Amount Amount Amount Amount Amount for injection enough to enough to enough to enough to enough to make make make make make 100% (w/v) 100% (w/v) 100% (w/v) 100% (w/v) 100% (w/v)

[0057] Meanwhile, Comparative Example 1 was RESTASIS™ comprising 0.05% (w/v) of commercially available cyclosporine.

Experimental Example 1

Storage Stability Test

[0058] In order to investigate stability after storage of the products of Example 2 and Comparative Example 1, both eye drops were stored at room temperature (15 to 25° C.) and under refrigeration for 70 days. Then, the appearance of the compositions was observed and titers of the drug in the compositions were determined by HPLC. Immediately after the preparation, the content of thymosin beta 4 in Example 2 was 1 mg/ml, whilst the content of cyclosporine in Comparative Example 1 was 0.5 mg/ml. HPLC was performed under the following conditions:

[0059] Column: Waters Delta-Pak C185 μm (3.9×150 mm)

[0060] Mobile phase A: Water containing 0.1% TFA. B: acetonitrile containing 0.1% TFA

[0061] Flow rate: 1.0 mL/min

[0062] Detection: UV 205 nm

[0063] Column temperature: Room temperature

[0064] Input volume: 10 mL

TABLE-US-00002 TABLE 2 Gradient Time (min) % A % B 0.0 85% 15% 15.0 75% 25% 15.1 20% 80% 17.0 20% 80% 17.1 85% 15% 26.0 85% 15%

[0065] The results are shown in Table 3:

TABLE-US-00003 TABLE 3 Example 2 Comparative Example 1 Titer Titer Appearance of drug Appearance of drug Immediately Clear, 1 White, opaque 0.5 after transparent mg/ml or slightly mg/ml preparation solution translucent, and homo- geneous liquid 70 days after Clear, 0.993 Precipitate Not storage at room transparent mg/ml occurred measurable temperature solution 70 days after Clear, 0.998 Precipitate Not storage in transparent mg/ml occurred measurable refrigerator solution

[0066] As such, the composition of Example 2 exhibits more excellent storage stability compared with that of Comparative Example 1.

Experimental Example 2

Verification of Improvement in Dry Eye Symptom

[0067] Experiments were performed in the mouse model with dry eye syndrome to determine effects of treating dry eye syndrome by the compositions of Examples 2, 4 and 5 containing different concentrations of thymosin beta 4, compared with a placebo control (formulation without thymosin beta 4). In order to induce the dry eye symptoms, female mice aged 8 weeks C57BL/6 were used, and eight mice per each group were subjected to the experiment under controlled adverse environment (CAE). In order to inhibit the generation of tears and aggravate dry eye disease symptoms, 0.5 mg/0.2 ml of scopolamine hydrobromide was injected q.i.d. between Day 6 and Day 10 after administering the test drug. The test drug was administered in a total daily dosage of 0.005 ml four times a day for 14 days and, in order to determine the treatment effects, a corneal surface fluorescein staining test was carried out on days 9, 12 and 14, after administration of the test drug to observe a change in corneal surface fluorescein staining scores. For reference, the corneal surface fluorescein staining score is a method of quantifying the symptoms of dry eye syndrome, and a decrease in the score means an improvement in the symptoms.

[0068] Results of the experiments are shown in FIGS. 1, 2 and 3.

[0069] Referring to FIGS. 1 and 2, the baseline refers to a corneal surface fluorescein staining score in a healthy mouse before inducing dry eye syndrome. On the other hand, ‘before administration’ refers to a corneal surface fluorescein staining score in the mouse after inducing dry eye syndrome but before administering the test drug. From the results shown in FIG. 1, the placebo control, Example 4 and Example 5 had an increase in the corneal surface fluorescein staining scores, whilst showing low corneal surface fluorescein staining scores at Day 14. No improvement effects were observed in these groups, compared to the baseline. However, Example 2 surprisingly had a significant effect in improving dry eye symptoms. Accordingly, it was found that the effect of thymosin beta 4 is not proportionate to the dosage administered.

[0070] FIG. 2 shows changes from the baseline in corneal surface fluorescein staining scores in relation to the times after administration. According to the results shown in FIG. 2, only Example 2 has improved the dry eye syndrome.

[0071] FIG. 3 shows changes in corneal surface fluorescein staining scores determined only in the inferior region of the cornea. According to the results shown in FIG. 3, although Examples 2 and 5 improved the dry eye syndrome as compared to the placebo control, only Example 2 produced such improvement without aggravating the symptoms.

[0072] Consequently, the composition of Example 2 was most effective in improvement of the dry eye syndrome.

Experimental Example 3

Determination of Desired Concentration for Improvement of Dry Eye Symptoms

[0073] Effects of treating and improving the dry eye syndrome by administering the products of Examples 1 to 3 twice a day was tested in the dry eye syndrome mouse model. In order to induce symptoms of the dry eye syndrome, female mice aged 8 weeks C57BL/6 were used, and five mice per each group were subjected to the experiment under controlled adverse environment (CAE). In order to inhibit the generation of tears and aggravate symptoms of the dry eye, 0.5 mg/0.2 ml of scopolamine hydrobromide was injected q.i.d. between Day 6 and Day 10 after administering the test drug. Each test drug (Examples 1, 2 and 3) was administered in a total daily dosage of 0.005 ml twice a day for 6 days. In order to determine treatment effects, a corneal surface fluorescein staining test was carried out before administration and at Day 6 and Day 13 after administration, respectively, to observe a change in corneal surface fluorescein staining scores.

[0074] Results of the experiments are shown in FIG. 4.

[0075] From the results shown in FIG. 4, it can be seen that, as compared with the scores measured before administration, the experimental groups for b.i.d. administration of the products of Examples 2 and 3, exhibited a greater decrease in corneal surface fluorescein staining scores than the experimental group for b.i.d. administration of the product of Example 1. Particularly, the score of Example 2 was more decreased than that of Example 3.

[0076] Consequently, the drug concentration of Example 2 is more effective in improvement of dry eye syndrome, compared to those of Examples 1 and 3.

Experimental Example 4

Comparison with 0.05% (w/v) Cyclosporine (Comparative Example 1) in the Improvement of Dry Eye Symptoms

[0077] In the dry eye syndrome mouse model, the effect of improving dry eye symptoms was compared between Example 2, Comparative Example 1 as an active control and a placebo control. In order to induce symptoms of the dry eye syndrome, female mice aged 8 weeks C57BL/6 were used, and five mice per each group were subjected to the experiment under controlled adverse environment (CAE). In order to inhibit the generation of tears and aggravate the dry eye disease symptoms, 0.5 mg/0.2 ml of scopolamine hydrobromide was injected q.i.d. between Day 6 and Day 10 after administering the drug. The test drug was administered in a total daily dosage of 0.005 ml twice a day for 6 days. In order to determine the treatment effects, a corneal surface fluorescein staining test was carried out before administration and at Day 6 and Day 13 after administration the test drug, respectively, to observe a change in corneal surface fluorescein staining scores.

[0078] Results of the experiments are shown in FIG. 5.

[0079] From the results shown in FIG. 5, it can be seen that, as compared with the scores measured before administration, Example 2 exhibited a remarkably greater decrease in the corneal surface fluorescein staining score than Comparative Example 1 as the active control. More particularly, the administration of the product of Example 2 significantly decreased the corneal surface fluorescein staining till Day 5. Meanwhile from Day 6, scopolamine was administered in order to observe the effect of the test drug on the aggravation of the dry eye syndrome. As a result, it was found that administration of Example 2 remarkably inhibited the aggravation of the dry eye syndrome caused by scopolamine.

Experimental Example 5

Clinical Trial (Phase II)

[0080] Based on the non-clinical results described above, phase II clinical trial was conducted in order to compare safety and effectiveness between Example 2 and the placebo control, regarding the signs and symptoms of dry eye syndrome. This clinical trial was conducted under controlled adverse environment (CAE), in a single institute in both double-blind and random assignments, in order to compare 0.1% (w/v) thymosin beta 4 eye drop with the placebo control, regarding the test drug's safety and effectiveness. More specifically, 36 persons in each group were subjected to administration of the placebo control and the product of Example 2, respectively. A squeeze bottle for dropping 40 μl per droplet was used to administer 1 or 2 drops twice a day for 30 days. In order to determine the treatment effects, a corneal surface fluorescein staining test was carried out before administration and at Day 30 after administration, respectively, to observe a change in corneal surface fluorescein staining scores.

[0081] Results of the experiments are shown in FIG. 6.

[0082] From the results shown in FIG. 6, a marked difference in corneal surface fluorescein staining was found after administering the product of Example 2 twice a day, as compared with the placebo control. Thus, in agreement with the non-clinical results, the results of this clinical trial confirm that Example 2 is effective in treatment of dry eye syndrome.

Experimental Example 6

Stimulation Test

[0083] The extent of the irritation caused by the eye drop was compared between Example 2 and Comparative Example 1.

[0084] Test scales: After administering an eye drop, the extent of discomfort, such as tingling in the eye or blurred vision, was measured with reference to a scale of 1 to 5, as follows: 1—very unsatisfied; 2—unsatisfied; 3—moderate; 4—satisfied; and 5—very satisfied.

[0085] The results were measured, as shown in Table 4 below.

TABLE-US-00004 TABLE 4 Example 2 Comparative Example 1 Extent of irritation 4.5 2.5

[0086] Consequently, Example 2 allowed users to feel more comfortable with less irritation, as compared to Comparative Example 1.

Sequence Listing Free Text

[0087]

TABLE-US-00005 SEQ ID No. 1.: SDKPDMAEIE KFDKSKLKKT ETQEKNPLPS KETIEQEKQA GES