Composition for treating thyroid eye disease comprising ulinastatin

Abstract

The present invention relates to a composition for treating thyroid eye disease comprising ulinastatin. Specifically, the composition of the present invention reduces the degree of ocular protrusion by inhibiting PAPP-A associated with thyroid eye disease, and reduces the expression of PAPP-A increased in intraocular tissue, thereby being effective in treating thyroid eye disease.

Description

Composition for treating thyroid eye disease containing urinasatin

[0001] The present invention relates to a composition for treating thyroid eye disease comprising urinasatin. Specifically, the composition of the present invention inhibits PAPP-A, which is associated with thyroid eye disease, thereby reducing the degree of proptosis and reducing the expression of PAPP-A, which is increased in intraocular tissues, so as to be effective in treating thyroid eye disease.

[0002]

[0003] Thyroid eye disease (TED) is an autoimmune eye disease associated with thyroid disease that can lead to vision loss due to damage to the optic nerve, as well as functional problems such as proptosis, hypertrophy of the extraocular muscles, and diplopia. More than 20% of patients with hyperthyroidism have proptosis, and since a significant number of patients experience external changes, their Health-related Quality of Life (HRQOL) is low.

[0004] Thyroid eye disease can be described by Rundle's curve, which is divided into an active period, characterized by a predominant inflammatory response, and an inactive period (fibrotic period), where inflammatory responses and changes in clinical manifestations subside, depending on the nature of the disease. During the active period, inflammation such as ocular pain and edema occurs. Conversely, during the inactive period, the infiltration of inflammatory cells decreases, and fibrosis of the extraocular muscles develops. Since the condition does not fully recover to normal after the stabilization phase, permanent proptosis and motor dysfunction of the extraocular muscles persist, necessitating the development of treatments for this condition.

[0005] Loss of immune tolerance to the thyroid-stimulating hormone receptor (TSHR) and the production of activating antibodies against the protein are major mechanisms of TED. Interactions between TSHR and the insulin-like growth factor 1 receptor (IGF-1R) promote the pathogenesis of TED. IGF-1R mediates the effects of IGF ligands on cell proliferation, differentiation, and survival, and has been found to be overexpressed in TED patient tissues.

[0006] IGF-1 exists in an inactive state bound to IGFBP-4, and PAPP-A (Pregnancy Associated Plasma Protein-A) is a metalloprotease that degrades the insulin-like growth factor-binding protein (IGFBP).

[0007] PAPP-A is a secreted protein that anchors to the cell surface via autocrine and adrenal secretion. Under normal conditions, IGF-1 exists in the form of a complex bound to IGFBP-4, which prevents the activation of IGF-1R. However, in thyroid eye disease, overexpressed PAPP-A cleaves the complex, releasing IGF-1 from the complex into the cellular environment and initiating IGF-1R signaling.

[0008] The inventors discovered that PAPP-A can play an important role in promoting TED and investigated a method to treat TED using a substance that inhibits PAPP-A. Accordingly, they developed an animal model of TED and confirmed that urinastatin has the effect of reducing proptosis, a clinical symptom of thyroid eye disease, and treating thyroid eye disease by inhibiting the activity of PAPP-A in orbital tissues, thereby completing the present invention.

[0009]

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] (Patent Document 0001) Korean Registered Patent No. 10-1686881

[0013] (Patent Document 0001) Korean Registered Patent No. 10-2638210

[0014]

[0015] The present invention aims to provide a pharmaceutical composition for the treatment or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof. The composition of the present invention aims to treat thyroid eye disease by inhibiting PAPP-A.

[0016] The present invention aims to provide an animal feed composition for improving or preventing thyroid eye disease comprising urinastatin or a pharmaceutically acceptable salt thereof.

[0017]

[0018] The present invention relates to a pharmaceutical composition for the treatment or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

[0019] The composition of the present invention can reduce the length of proptosis. Additionally, the composition of the present invention can reduce thyroid-stimulating hormone receptor (TSHR) Ab and PAPP-A in the blood. Additionally, the composition of the present invention can reduce PAPP-A and IGF-1R in orbital tissue. Furthermore, the composition of the present invention can reduce the expression of PAPP-A, IGF-1R, and TNF-α genes in orbital tissue.

[0020] In addition, the composition of the present invention can reduce adipose tissue within the orbital tissue.

[0021] The present invention relates to an animal feed composition for improving or preventing thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

[0022] The present invention relates to a method for treating thyroid eye disease by administering urinasatin or a pharmaceutically acceptable salt thereof to a patient with thyroid eye disease.

[0023] The present invention relates to the use of a pharmaceutical composition for the treatment or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

[0024] The present invention relates to a pharmaceutical composition for the treatment or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

[0025]

[0026] The urinastatin of the present invention has the effect of treating thyroid eye disease by inhibiting PAPP-A. In addition, the urinastatin of the present invention can effectively treat, prevent, and improve thyroid eye disease by reducing TSHR Ab, IGF-1R, and TNF-α. Furthermore, the urinastatin of the present invention can be used to treat thyroid eye disease that may occur due to thyroid disease by reducing the length of proptosis that may occur due to thyroid eye disease.

[0027]

[0028] Figure 1 shows a specific schematic diagram for inducing a mouse model of thyroid eye disease.

[0029] Figure 2 shows the results of ocular photography in an animal model of thyroid eye disease.

[0030] Figure 3 shows the results of measuring the length of the degree of exophthalmos in an animal model of thyroid eye disease.

[0031] Figure 4 shows the results of measuring changes in TSHR Ab in the blood of an animal model of thyroid eye disease.

[0032] Figure 5 shows the results of measuring changes in PAPP-A in the blood of an animal model of thyroid eye disease.

[0033] Figure 6 shows the results of measuring changes in PAPP-A in orbit tissue of an animal model of thyroid eye disease.

[0034] Figure 7 shows the results of measuring changes in IGF-1R in orbital tissue of an animal model of thyroid eye disease.

[0035] Figure 8 shows the results of measuring changes in PAPP-A gene expression in orbital tissue of an animal model of thyroid eye disease.

[0036] Figure 9 shows the results of measuring changes in IGF-1R gene expression in orbital tissue of an animal model of thyroid eye disease.

[0037] Figure 10 shows the results of measuring changes in TNF-α gene expression in orbital tissue of an animal model of thyroid eye disease.

[0038] Figure 11 shows the results of imaging adipose tissue in orbital tissue of an animal model of thyroid eye disease.

[0039] Figure 12 shows the results of measuring the change in size of adipose tissue in orbital tissue of an animal model of thyroid eye disease.

[0040]

[0041] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0042] In this invention, "Ulinastatin" is a protein synthesized and secreted in human urine, which primarily acts as a protease inhibitor. It is a component that is clinically used because it is effective in suppressing inflammation and alleviating tissue damage, mainly in acute pancreatitis and acute renal failure.

[0043] In the present invention, "thyroid eye disease" refers to an autoimmune disease related to the thyroid gland, in which autoantibodies attack the muscles and fatty tissues around the eyes, causing abnormal reactions. Thyroid eye disease may occur in patients with thyroid disease.

[0044] In the present invention, "pharmaceuticalally acceptable salt" is used to refer to an acid addition salt or a base addition salt that is suitable or compatible for the treatment of a patient. Exemplary inorganic acids that form a suitable salt include hydrochloric acid, hydrobromide, sulfuric acid, and phosphoric acid, as well as metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form a suitable salt include mono-, di-, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvate, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Mono- or di-acid salts may be formed, and such salts may exist in a hydrated, solvated, or substantially anhydrous form. In general, the acid addition salt of the compound of the present invention is more soluble in water and various hydrophilic organic solvents compared to its free base form and generally exhibits a higher melting point. The selection of a suitable salt is known to those skilled in the art.

[0045] In the present invention, "prevention" refers to any act of suppressing or delaying the onset of a disease by administering a composition, "treatment" refers to any act of improving or beneficially changing the symptoms of an individual suspected of or suffering from a disease by administering a composition, and "improvement" refers to any act of at least reducing parameters related to the condition, such as the degree of symptoms, by administering a composition.

[0046] In the present invention, the "animal feed composition" can be fed to all non-human animals, for example, non-human primates, sheep, dogs, cattle, horses, etc.

[0047]

[0048] [Test Example 1]

[0049] Induction of thyroid eye disease mouse model

[0050]

[0051] A mouse model of thyroid eye disease was induced by administering Zymosan A to SKG mice and simultaneously inducing pTriEx-1.1 Neo A-subunit plasmid electroporation.

[0052] Specifically, 3 mg of zymosan A was injected once intraperitoneally into 8-week-old female SKG mice, followed by the injection of pTriEX neo 1.1 A-subunit plasmid DNA into the thigh muscle, and immunization was performed using an electroporator once every 3 weeks for a total of 5 times. The vector group was injected with an empty vector without the plasmid, and The military is a normal military.

[0053] A specific schematic diagram for inducing a mouse model of thyroid eye disease is shown in Figure 1.

[0054]

[0055] [Test Example 2]

[0056] Urinastatin drug administration and measurement of proptosis length

[0057]

[0058] After inducing the thyroid eye disease animal model in Test Example 1, the drug was administered once every two weeks for a total of four times starting from 16 weeks of age via retro-orbital injection. 5000 IU of urinastatin was administered once per mouse.

[0059] In the above-mentioned animal model of thyroid eye disease, ocular photography was performed to confirm changes in orbital fat and muscle tissue along with inflammation in the periorbital region, and the results are shown in Fig. 2. In addition, the length of the degree of proptosis was measured from the inflammation in the periorbital region to the cornea using Image J, and the results are shown in Fig. 3.

[0060] As shown in Figures 2 and 3 above, it was confirmed that the length of proptosis significantly decreased after four administrations in the drug administration group. Specifically, in an animal model group administered Zymosan A and pTriEX neo 1.1 A-subunit plasmid DNA to induce thyroid eye disease, the normal group ( Clinically, proptosis was observed along with a peripheral orbital inflammatory response compared to the vector. In addition, when the length from the peripheral inflammation to the cornea was measured, the length of proptosis increased by approximately 1.3 ± 0.1 mm in the animal model group compared to the normal group. However, the length of proptosis decreased by approximately 0.3 ± 0.2 mm in the drug administration group.

[0061] In conclusion, it can be seen that urinastatin has the effect of reducing exophthalmos in thyroid eye disease.

[0062]

[0063] [Test Example 3]

[0064] Analysis of Thyroid-Stimulating Hormone Receptor (TSHR) Ab and PAPP-A in Animal Model Blood

[0065]

[0066] In cases of hyperthyroidism, the expression of TSHR Ab in the blood increases. Therefore, changes in the expression of TSHR Ab in the blood were observed using blood obtained through cardiac blood sampling in an animal model, and the results are shown in Fig. 4. As shown in Fig. 4, in the animal model group, the normal group ( Compared to Vector and Vector, TSHR Ab increased, but it was confirmed that TSHR Ab decreased in the drug administration group.

[0067] In addition, blood was isolated after euthanizing the animal model, and changes in PAPP-A, the pathogenic mechanism of thyroid eye disease, were observed, and the results are shown in Fig. 5. As shown in Fig. 5, in the animal model group, the normal group ( Compared to Vector, PAPP-A increased, but it was confirmed that PAPP-A decreased in the drug administration group.

[0068]

[0069] [Test Example 4]

[0070] Analysis of PAPP-A and IGF-1R expression in intraocular tissues of an animal model

[0071]

[0072] ELISA was performed using samples obtained after extracting and homogenizing intraocular tissues (fat and muscle tissues) from an animal model. Through the above experiment, changes in the expression of PAPP-A and IGF-1R in orbital tissue were observed, and the results are shown in Figures 6 and 7. As shown in Figures 6 and 7, in the animal model group, the normal group ( Compared to Vector, PAPP-A and IGF-1R increased, but it was confirmed that PAPP-A and IGF-1R decreased in the drug administration group.

[0073]

[0074] [Test Example 5]

[0075] Analysis of gene expression in intraocular tissues of animal models

[0076]

[0077] mRNA was extracted by homogenizing intraocular tissue of an animal model using a kit. The extracted mRNA was quantified using a NanoDrop spectrophotometer, and cDNA was synthesized using RNA with a purity of 1.8 or higher. Polymerization of the synthesized cDNA with SYBR Green Master Mix and primer genes was performed. The concentration of each gene, corrected by dividing by beta-actin, was calculated, and the sequences of each gene are shown in Table 1 below.

[0078]

[0079]

[0080] As shown in FIGS. 8 to 10, in the animal model group, the normal group ( Compared to Vector, PAPP-A, IGF-1R, and TNF-α gene expression increased, but it was confirmed that PAPP-A, IGF-1R, and TNF-α gene expression decreased in the drug administration group.

[0081] Ultimately, from the above test results, it can be seen that urinasatin inhibits the expression of TSHR Ab, PAPP-A, IGF-1R, and TNF-α, which are associated with thyroid eye disease.

[0082]

[0083] [Test Example 6]

[0084] Analysis of adipose tissue in intraocular tissue of animal models

[0085]

[0086] To observe changes in intraocular tissue, the orbital tissue was stained with H&E to measure the size of the adipose tissue. Specifically, the intraocular tissue was fixed with 4% paraformaldehyde for 3 days, and then a paraffin block was prepared using a tissue process. H&E staining was performed after sectioning. Changes in the size of the adipose tissue were analyzed using Image J, and the results are shown in Figures 11 and 12.

[0087] As shown in Figs. 11 and 12, in the animal model group, the normal group ( Compared to Vector, the size of adipose tissue increased, but it was confirmed that the size of adipose tissue decreased in the drug administration group.

[0088] Through this study, we confirmed that PAPP-A expression increases in an animal model of thyroid eye disease by analyzing blood and intraocular tissues. Furthermore, we verified that urinastatin, which reduces PAPP-A expression, is effective in treating thyroid eye disease by decreasing the degree of proptosis and reducing the elevated PAPP-A expression in intraocular tissues.

Claims

1. A pharmaceutical composition for the treatment or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition according to claim 1, wherein the composition reduces the length of the protrusion of the eyeball.

3. A pharmaceutical composition according to claim 1, wherein the composition reduces thyroid-stimulating hormone receptor (TSHR) Ab and PAPP-A in the blood.

4. A pharmaceutical composition according to claim 1, wherein the composition reduces PAPP-A and IGF-1R in orbital tissue.

5. A pharmaceutical composition according to claim 1, wherein the composition reduces PAPP-A, IGF-1R, and TNF-α gene expression in orbital tissue.

6. A pharmaceutical composition according to claim 1, wherein the composition reduces adipose tissue within orbital tissue.

7. An animal feed composition for the improvement or prevention of thyroid eye disease comprising urinasatin or a pharmaceutically acceptable salt thereof.

8. An animal feed composition according to claim 7, wherein the composition reduces the length of the protrusion of the eyeball.

9. An animal feed composition according to claim 7, wherein the composition reduces thyroid-stimulating hormone receptor (TSHR) Ab and PAPP-A in the blood.

10. An animal feed composition according to claim 7, wherein the composition reduces adipose tissue within orbital tissue.

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