KR102115158B1 - Formulation comprising thymosin beta 4 - Google Patents

Abstract

The present invention relates to an ophthalmic disease treatment agent containing thymosin beta 4.
The present invention is characterized by being more effective in improving dry eye syndrome than eye drops containing cyclosporin A.

Description

Formulation containing thymosin beta 4 {FORMULATION COMPRISING THYMOSIN BETA 4}

The present invention relates to an ophthalmic disease treatment agent containing thymosin beta 4.

There are basic tears and reflective tears. Basic tears are constantly constant secretions, and reflective tears are secreted temporarily by reflex reactions to any stimulus, such as pain, dust, spiciness, smoke, smell, glad or sad emotions, or irritation due to lack of tears. Say things.

The secreted tears are present in the surface layer of the cornea, which gently covers the surface of the eye to protect and comfort the eyes, to smooth the surface of the cornea to secure the optical field, and to prevent the wounds of the corneal and conjunctival epithelial cells. It plays a role of acting as an antibacterial effect from external bacteria and supplying nutrients to the cornea.

Tears are composed of 3 layers of mucus layer, moisture layer and fat layer, which are usually produced in 2 ~ 3 mL per day to perform the stomach function. However, if one of the three layers constituting tears lacks one layer, or if the tear layer changes or the amount of production of the tear itself decreases, the eyeballs dry and the tears fail to perform their role, resulting in dry eye syndrome. Can occur.

Dry eye refers to a condition in which the amount and quality of tears are abnormal regardless of keratoconjunctivitis disorder [Masakazu YAMADA et al., Nippon Ganka Kiyo, 43, 1289-1293 (1992)]. In addition, dry eye refers to any disease associated with pathological changes in the tear layer or secretion of less tears, and lack of tears, excessive evaporation of tears, or an unbalanced composition of tears causes damage to the eye surface It includes irritation symptoms such as irritation, foreign body sensation or dry feeling, and keratitis or membrane epithelial wounds resulting from these symptoms. Typically, dry eye syndrome includes, but is not limited to, dry conjunctivitis or dry keratoconjunctivitis.

Dry eye syndrome is commonly found in middle-aged women and the elderly because of the lack of tears or decreased function as they age. However, in addition, it has been reported that it may be related to inflammation, medication (e.g., hypertension, hyperacidity, neurosis or depression drugs, etc.), trauma, chronic conjunctivitis, blepharopathy or prostate disease. I am not able. In addition, recently, it is known that dry eye syndrome may be caused by autoimmune disease due to an abnormal immune response.

Immunity refers to the action of defending the body from pathogens invading from outside, and autoimmune disease occurs when these immune functions become abnormal and the body's defense mechanisms, that is, the immune system fights against their body tissues and starts to damage it Immune hypersensitivity reaction. Autoimmune disease is a disease caused by excessive or improper control of the autoimmune response by inducing an inappropriate immune response as the body's cytokine (signaling material that controls and stimulates the body's defense system) changes against viruses or bacteria. Although it is reported, the exact cause of the disease is unknown, and it is discussed that it may be caused by environmental factors, genetic factors, or immunological factors.

Currently, most autoimmune diseases are treated or prevented by using steroids or immunosuppressive agents. Among them, a representative drug used as a treatment or prevention agent for dry eye syndrome in mammals, including humans, is RESTASIS ^TM , released by Elergan, and is 0.05% (w / v) of cyclosporin A eye drop (US Patent Publication) No. 5,474,979).

U.S. Patent No. 5,474,979 Republic of Korea Patent Publication No. 10-2008-0033939

Restasis ^TM is currently known to be unoptimized for bioavailability, despite being the most widely used as a treatment or prevention agent for dry eye syndrome. For example, it may take several weeks to see the full effectiveness of Restasis ^TM . Here, bioavailability means the percentage of drug detected in target eye tissue after administration, wherein detection of the drug is pharmacodynamic (quantification of the biological response to cyclosporin A) or pharmacokinetic (actual drug) Quantification).

Therefore, in the field of treatment or prevention of dry eye syndrome, there has been a continuing need for drugs with improved physicochemical stability and drugs with improved bioavailability. As a solution to this, recent studies on thymosin beta 4 Is going on.

Thymosin beta 4 is the first protein found in the thymus in 1981, consisting of 41 to 43 amino acids, and a protein with an isoelectric point of 5.1. It was discovered that thymosin beta 4, first discovered as an actin-sequestering molecule in animal cells by Riva et al. In 1991, also acts as an immunoregulatory and neuroendocrine. In addition, it not only performs the function of terminal deoxynucleotide transferase in thymocytes, but also increases macrophage migration and macrophage antigens, and is caused by hypothalamic explants. It is also known to increase the secretion of luteinizing hormone. In addition, thymosin beta 4 inhibits the cell cycle of hematopoietic stem cells in order to remove the toxicity of cytosine arabinoside and increase the adhesion and migration of the endothelium. It is also known to do. Furthermore, Korean Patent Publication No. 10-2008-0033939 discloses the use of eye drops containing thymosin beta 4 and a bactericidal activity preservative for the treatment of eye inflammation, eye infections (bacteria, fungi or viruses) and glaucoma. have.

However, the combination and content of components optimized as eye drops and their most preferred daily dosage is not yet known.

Accordingly, the present inventor intends to provide an optimal daily dosage of an optimal eye drop solution that improves the disadvantages of drugs and formulations that have been proposed for the treatment or prevention of dry eye syndrome.

The present invention solves the aforementioned problems through the following means.

(1) A pharmaceutical composition for the treatment or prevention of dry eye syndrome containing thymosin beta 4, wherein the pharmaceutical composition contains 0.05 to 0.5% (w / v) of thymosin beta 4 based on the total composition, and total daily 0.08 to A pharmaceutical composition, characterized in that it is present in a unit dose form capable of providing a dose of 2.0 ml.

(2) The pharmaceutical composition according to (1), wherein the pharmaceutical composition further contains sodium chloride, potassium chloride, calcium chloride hydrate and magnesium chloride hexahydrate.

(3) In the above (1) or (2), the pharmaceutical composition is 0.5 to 0.7% (w / v) of sodium chloride, 0.05 to 0.09% (w / v) of potassium chloride, and 0.03 to calcium chloride hydrate, based on the total composition. A pharmaceutical composition comprising 0.06% (w / v) and 0.01 to 0.05% (w / v) of magnesium chloride hexahydrate.

(4) The pharmaceutical composition according to any one of (1) to (3), wherein the pharmaceutical composition is present in a unit dosage form of 0.01 to 10 ml for disposable or multiple use so as to be suitable for providing a total dose of 0.08 to 2.0 ml per day. Pharmaceutical composition characterized by.

(5) The eye drop container according to any one of (1) to (4) above, wherein the pharmaceutical composition is present in an eye drop container having a dropping capacity of 0.01 to 2.0 ml per drop to be suitable for providing a total dose of 0.08 to 2.0 ml per day. Pharmaceutical composition, characterized in that.

The present invention is more effective in improving dry eye syndrome than eye drops containing cyclosporin A, and has less irritation to the eyes and the like compared to conventional eye drops.

1 is a result of observing the amount of corneal surface fluorescence staining for each dosing period of Example 2, Example 4, Example 5 and placebo control group.
Figure 2 is a result of observing the amount of corneal surface fluorescence staining for each dosing period compared to the baseline of Example 2, Example 4, Example 5 and the placebo control group.
3 is a result of observing the amount of change in fluorescence staining for each dosing period compared to Baseline for the lower region (inferior region) of the cornea of Example 2, Example 4, Example 5 and placebo control.
Figure 4 is a result of observing the amount of corneal surface fluorescence staining changes 6 and 13 days after the administration of Example 1, Example 2 and Example 3.
Figure 5 is a result of observing the amount of corneal surface fluorescence staining after the administration of Example 2, Comparative Example 1 and placebo control 6 and 13 days.
Figure 6 is the result of observing the amount of corneal surface fluorescence staining 30 days after the administration of Example 2 and the placebo control group.

[Composition]

The composition according to the invention comprises the drug thymosin beta 4; It is characterized by containing sodium chloride, potassium chloride, calcium chloride hydrate, and magnesium chloride hexahydrate. According to one embodiment, the composition according to the invention is characterized in that it further comprises a pH adjusting agent hydrochloric acid and / or sodium hydroxide. 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 and the like within the scope of not impairing the desired effect of the present invention.

The composition according to the present invention is prepared as a solution, lotion, plaster, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, oil or foaming agent, and then dried directly by contacting it with eye tissue by direct application Can be prevented or treated.

In particular, the composition according to the present invention may be used as a prophylactic or therapeutic agent for dry eye after preparing the ophthalmic solution by including all or part of the following process.

1. Weighing process

Each raw material, reagent, and solvent is weighed.

2. Mixing process

Each individually weighed reagent is added to sterile preparation water and mixed until each reagent is dissolved, and then the drug thymosin beta 4 is added to the mixed solution as weighed and mixed until dissolved.

3. Adjustment process

The acidity is adjusted to 7.0 using sodium hydroxide and hydrochloric acid after the above mixing process.

4. Filtration process

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

5. Filling process

The mixed solution that has undergone the above filtration process is filled into a low-density polyethylene container and sealed.

[drug]

In terms of the expression of the desired effect 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 composition, particularly at a concentration of 0.1% (w / v) It is most preferably contained.

[pH regulator]

It is preferable to prepare the ophthalmic preparation similar to the physiological pH of tears from the viewpoint of comfort, resistance and stability in use.

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

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

[Other excipients]

In terms of the expression of the desired effect of the present invention, based on the total composition, sodium chloride is 0.5 to 0.7% (w / v), potassium chloride is 0.05 to 0.09% (w / v), and calcium chloride hydrate is 0.03 to 0.06% (w / v) And magnesium chloride hexahydrate is preferably contained in the composition at a concentration of 0.01 to 0.05% (w / v). In particular, based on the total composition, sodium chloride is 0.6 to 0.7% (w / v), potassium chloride is 0.07 to 0.08% (w / v), calcium chloride hydrate is 0.04 to 0.05% (w / v), and magnesium chloride hexahydrate is 0.02. It is most preferably contained in the composition at a concentration of ~ 0.04% (w / v).

According to one embodiment, the composition according to the invention in terms of better expression of the desired effect of the invention is 0.2 to 0.5% (w / v) of sodium acetate hydrate and / or 0.1 to 0.3% based on the total composition. (w / v) sodium citrate hydrate.

[Usage, capacity]

The composition according to the present invention containing 0.05 to 0.5% (w / v) of thymosin beta 4 is irritating to the eye and other side effects when the total amount of 0.08 to 2 ml per day is contacted with the eye tissue over 1 to several times. While minimizing, the expression of the desired effect can be optimized. To this end, the composition according to the present invention may exist in a unit dose form of 0.01 to 10 ml for disposable or multiple use, and furthermore, the unit dose form of 0.01 to 2.0 ml per drop is suitable to provide the total daily dose of the stomach. It may be present in an eye drop container having a dropping capacity.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to them.

[Examples 1 to 5 and Comparative Example 1]-Preparation of eye drops

An eye drop was prepared at the composition ratio shown in Table 1 below. For reference, in Table 1 below, the unit of the numerical value, unless otherwise indicated, is% (w / v), which is the concentration based on the total composition. That is, 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), and the thi in Example 3 The concentration of mosin beta 4 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).

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 achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Sodium hydroxide Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Add as needed to achieve pH 7.0 Sterile preparation water Sufficient amount to make 100% (w / v) Sufficient amount to make 100% (w / v) Sufficient amount to make 100% (w / v) Sufficient amount to make 100% (w / v) Sufficient amount to make 100% (w / v)

Meanwhile, hereinafter, Comparative Example 1 refers to commercially available Restasis ^TM of cyclosporine 0.05% (w / v).

[Experimental Example 1]-Experiment for confirming improvement of dry eye symptoms

In order to confirm the improvement effect of dry eye treatment by concentration of thymosin beta 4 (placebo 2, Example 4 and Example 5) compared to a placebo control group (a formulation that does not contain thymosin beta 4) in a dry eye mouse model Byo experiment was performed. To induce dry eye symptoms, mice were tested in 8 groups of 8 female C57BL / 6 strains under each group under CAE (Controlled Adverse Environment), inhibiting tear formation and exacerbating the signs of dry eye disease. To do this, 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected 4 times a day and then injected between Day 5 and Day 10. The drug was administered 0.005 ml 4 times a day for 14 days, and corneal surface fluorescence staining was performed on the 9th, 12th, and 14th days after administration to confirm the therapeutic effect. For reference, the corneal surface fluorescence stain is an indicator for confirming the signs of dry eye syndrome, and the lower the score, the better the dry eye symptoms.

The results were as in FIGS. 1, 2 and 3.

For reference, in FIG. 1 and FIG. 3, Baseline refers to the corneal surface fluorescence staining score in healthy mice before induction of dry eye syndrome, and prior to dosing, corneal surface fluorescence staining score in mice before drug administration after induction of dry eye syndrome. Means

Looking at the results of Figure 1, in the placebo group, Example 4, Example 5, the corneal surface fluorescence staining score increased, but showed a low corneal surface fluorescence staining score on Day 14, but no improvement effect compared to baseline. However, surprisingly, in Example 2, a significant value was observed for improving dry eye syndrome. From this, it was confirmed that the dose of thymosin beta 4 and the expression of the effect are not in a proportional relationship with each other.

Looking at the results of Figure 2, Figure 2 is a graph showing the amount of change in the corneal surface fluorescence staining score for each dosing period compared to the baseline, it can be seen that only Example 2 shows an effect on improving dry eye syndrome.

Looking at the results of Figure 3, Figure 3 is a graph showing the results of observing the amount of change in the fluorescence staining score of the corneal surface only on the lower surface (inferior region), Examples 2 and 5 had the effect of improving dry eye syndrome, but only It can be seen that only in Example 2, symptoms of dry eye were improved without exacerbation of symptoms.

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

[Experimental Example 2]-Experiment for confirming the improvement of dry eye symptoms to set the dosage

A comparative experiment was conducted on a mouse model of dry eye syndrome to determine the effect of improving dry eye treatment by administering Examples 1 and 3 twice a day, focusing on Example 2. To induce dry eye symptoms, mice were tested in 5 groups of 8 week old females of the C57BL / 6 strain in each group under a CAE (Controlled Adverse Environment), inhibiting tear production and exacerbating signs of dry eye disease. In order to do this, 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected four times a day and then injected between Day 5 and Day 10. The drug was dosed 0.005 ml twice a day for 6 days in each experimental group (Example 1, Example 2 and Example 3), and fluorescence of the corneal surface was observed on the 6th and 13th days before and after dosing to confirm the treatment effect. A staining test was conducted to observe the amount of fluorescence staining on the corneal surface.

The results were as in FIG. 4.

Looking at the results of Figure 4, compared to the pre-dose compared to Example 2 and Example 3 in the experimental group dosing twice a day compared to the experimental group dosing Example 1 twice a day, it can be seen that the corneal surface fluorescence staining degree is reduced. In particular, it can be seen that the score tends to decrease slightly more in Example 2 than in Example 3.

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

[Experimental Example 3]-Comparative experiment of the improvement pattern of dry eye symptoms with cyclosporine 0.05% (w / v) (Comparative Example 1)

Example 2, Comparative Example 1, which is an active control group, and an experiment comparing the improvement of dry eye symptoms between the placebo control group were tested using a dry eye mouse model. To induce dry eye symptoms, mice were tested in 5 groups of 8 week old females of the C57BL / 6 strain in each group under CAE (Controlled Adverse Environment), inhibiting tear formation and exacerbating the signs of dry eye disease. To do this, 0.5 mg / 0.2 ml of scopolamine hydrobromide was injected 4 times a day and then injected between Day 5 and Day 10. The drug was dosed 0.0054 ml twice a day for 6 days for each experimental group, and to confirm the therapeutic effect, corneal surface fluorescence staining was performed on the 6th and 13th days after dosing and after the administration to observe the change in fluorescence staining on the corneal surface.

The results were as shown in FIG. 5.

Looking at the results of Figure 5, it can be seen that the degree of fluorescence staining of the corneal surface is significantly reduced in Example 2 than in Comparative Example 1, which is the active control, compared to before administration. More specifically, when Example 2 was administered, the degree of corneal surface fluorescence staining was significantly reduced until the 5th day, and from day 6, the responsiveness was observed while administering scopolamine to see the improvement in the condition of exacerbation of dry eye syndrome. The response to the exacerbation of dryness was significantly reduced.

[Experimental Example 4]-Clinical trial (Phase 2)

Based on the above non-clinical results, clinical trials were conducted to compare the safety and effectiveness of Example 2 with placebo control for signs and symptoms of dry eye syndrome. The above trials were performed by double-blinding and evaluating the safety and effectiveness of 0.1% (w / v) thymosin beta 4 eye drops compared to placebo for signs and symptoms of dry eye under a controlled adverse environment (CAE). The experiment was conducted in a single institution by randomization. Specifically, 1 to 2 drops were administered twice a day using a squeeze bottle with a dropping capacity of 40 µl per drop for placebo control group and Example 2 for 30 days for 36 subjects for each group, confirming the treatment effect For this, a corneal surface fluorescence staining test was performed before and 30 days after administration to observe the change in fluorescence staining of the corneal surface.

The results were as shown in FIG. 6.

Looking at the results of Figure 6, it can be seen that the difference in fluorescence staining of the corneal surface after dosing Example 2 twice a day appears clearly compared to the placebo control group.

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

Claims (5)

As a pharmaceutical composition for the treatment or prevention of dry eye syndrome containing thymosin beta 4,
0.05 to 0.5% (w / v) of thymosin beta 4,
0.5 to 0.7% (w / v) sodium chloride,
0.05 to 0.09% (w / v) potassium chloride,
0.03 to 0.06% (w / v) calcium chloride hydrate,
0.01 to 0.05% (w / v) magnesium chloride hexahydrate,
0.2 to 0.5% (w / v) sodium acetate hydrate and
A pharmaceutical composition comprising 0.1 to 0.3% (w / v) sodium citrate hydrate, and present in a unit dose form capable of providing a total dose of 0.08 to 2.0 ml per day.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises hydrochloric acid or sodium hydroxide.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is present in a unit dose form capable of providing a dosage of 0.01 to 10 ml.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is present in a unit dosage form of 0.01 to 10 ml for disposable or multiple use so as to be suitable for providing a total dose of 0.08 to 2.0 ml per day.
The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is present in an eye drop container having a dropping dose of 0.01 to 2.0 ml per drop to be suitable for providing a total dose of 0.08 to 2.0 ml per day.

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