KR101558477B1 - Compositions for Prevention or Treating Autoimmune Thyroid Diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of autoimmune thyroid diseases comprising biguanide derivatives, more particularly metformin as an active ingredient.
The composition of the present invention can be used for the treatment or prevention of various pathological conditions caused by thyroid autoantibodies, for example, thyroid ophthalmopathy, by effectively inhibiting the production of hyaluronan and the differentiation of fat, Can be improved.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating autoimmune thyroid disease,

The present invention relates to a composition for the prevention or treatment of autoimmune thyroid disease, including thyroid ophthalmopathy comprising a biguanide derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Thyroid-related diseases include hyperthyroidism, a disease in which the thyroid gland functions excessively and thyroid hormone is excessively produced and the metabolism of the body is abnormally increased, and thyroid gland function, which is a disease in which the metabolism of the whole body is slowed by the deficiency of thyroid hormone There is hypopnea. In the case of hyperthyroidism, if left untreated for a long time, it may cause cardiac complications, resulting in arrhythmia or heart failure, and it causes aggravation of osteoporosis in postmenopausal women. Hypothyroidism is divided into primary hypothyroidism due to hypothyroidism and hypothyroid hypothyroidism due to pituitary hypothyroidism. In the case of hypothyroid hypothyroidism, genetic cause, thyroidectomy, radioiodine administration, chronic lymphocytic Thyroid tissue is destroyed by thyroid inflammation and secondary hypothyroidism occurs when hypothyroid function that stimulates secretion of thyroid hormone is lost. This decrease in thyroid function caused the metabolism to become too slow to cause constipation, weakness, swelling, fatigue, memory loss, excessive menstruation, muscle aches, poor resistance to the cold, poor sweating, decreased appetite, .

Recently, the number of patients with thyroid-related diseases has been steadily increasing. According to the National Health Insurance Corporation data, from 2002 to 2009, the number of hypothyroidism was 2.3 times from 128,000 to 289,000, and the number of hyperthyroidism was 173,000 To 233,000. The number of patients with autoimmune thyroid disease is expected to be much higher, even if there are no abnormalities.

Thyroid-associated opththalmopathy is a chronic inflammatory disease of the extraocular muscle and connective tissue induced by autoimmune thyroid disease. About 90% of thyroid ophthalmopathy is found in patients with Graves' disease, also called Graves' ophthalmopathy (Graves' ophthalmopathy), and Hashimoto's thyroiditis also causes thyroid ophthalmopathy. Unlike hyperthyroidism, which removes thyroid gland or inhibits hormone production, thyroid gland is also treated with immunotherapy for medical treatment, but the effect is still at a level expected to alleviate symptoms. In addition, the use of a non-selective thyroid hypotensive agent or surgical removal of the thyroid tissue may cause recurrence, and it is not only necessary to continuously administer thyroid hormone but also exhibits abnormal body homeostasis.

Metformin, developed in Europe in the 1920s, is a compound that has been administered to the human body as an oral diabetes drug, which has been shown to improve blood sugar and prevent cardiovascular disease. By activating AMP-activated protein kinase (AMPK) It has been reported that it inhibits the metabolic syndrome by inhibiting biosynthesis and promoting glucose uptake into cells. However, the therapeutic or ameliorative effect of autoimmune thyroid disease in relation to metformin is not known.
As a background of the present invention, there is Korean Patent Publication No. 10-2013-0129496 (Feb.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to find effective natural therapeutic compositions for autoimmune thyroid disease. As a result, the bivalent guanidine derivative compound represented by the above formula (1) significantly reduces the expression level of major indicator / pathogenic agent for autoimmune thyroid disease, thereby preventing and preventing autoimmune thyroid disease including thyroid ophthalmopathy The present invention has been completed.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of autoimmune thyroid diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for the prevention or treatment of autoimmune thyroid disease comprising, as an active ingredient, a biguanide derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof: do:

Formula 1

In the above formulas, R ₁ to R ₄ are each independently hydrogen or C ₁ -C ₃ alkyl.

The present inventors have made extensive efforts to find effective natural therapeutic compositions for autoimmune thyroid disease. As a result, the bivalent guanidine derivative compound represented by the above formula (1) significantly reduces the expression level of major indicator / pathogenic agent for autoimmune thyroid disease, thereby preventing and preventing autoimmune thyroid disease including thyroid ophthalmopathy And that it is effective for treatment.

As used herein, the term " alkyl " means a straight or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl or isopropyl. C ₁ -C ₃ alkyl means an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the number of carbon atoms of the substituent is not included.

According to a specific embodiment of the present invention, R ₁ and R ₂ in formula (1) of the present invention are hydrogen, and R ₃ and R ₄ are each independently C ₁ -C ₃ alkyl. More specifically, R ₃ and R ₄ in the above formula (1) are methyl.

The compound wherein R ₁ and R ₂ are hydrogen and R ₃ and R ₄ are methyl is metformin (N, N-dimethylimidodicarbonimidic diamide). Metformin is currently the first oral diabetic drug prescribed for patients with type 2 diabetes and has been shown to improve blood glucose and prevent cardiovascular disease. One of the mechanisms of action is activation of AMP-activated protein kinase (AMPK) It has been reported that grape inhibits biosynthesis, promotes the uptake of glucose into cells, and inhibits the metabolic syndrome. However, the therapeutic or ameliorative effect of autoimmune thyroid disease in relation to metformin is not known.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used.

Preferably, the pharmaceutically acceptable salts of the compounds of the present invention are selected from the group consisting of hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, gluturonate, ebonate, glutamate, or aspartate salts, And salts formed using various inorganic acids and organic acids. The compounds of the present invention may also exist in the form of solvates (e.g., hydrates).

The composition of the present invention may be provided in the form of a pharmaceutical composition for the prevention or treatment of autoimmune thyroid disease. When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The term " thyroid gland disorder " in the present invention means a disorder in which the secretion of thyroid hormone is excessive or deficient due to abnormal thyroid function. Accordingly, the thyroid disease treated with the composition of the present invention includes both hyperthyroidism and hypothyroidism.

Hyperthyroidism is a disorder in which the body's metabolism is abnormally increased due to abnormally activated thyroid function and excessive production of thyroid hormone. Hypothyroidism is caused by the deficiency of thyroid hormone, It is a disease that slows down the process.

As used herein, the term " Autoimmune Thyroid Diseases " refers to a disorder in which the thyroid hormone is excessively secreted or deficient by an immune response mediated by autoantibodies to the thyroid. When thyroid stimulating hormone receptor antibody (TRAb) is produced, the thyroid hormone is overproduced and progresses to hyperthyroidism. In contrast, thyroid stimulating blocking antibody (TSBAb), which destroys thyroid cells, When it is produced, it decreases hormone secretion and develops hypothyroidism.

The composition of the present invention can effectively treat or prevent autoimmune thyroid disease by controlling the autoimmune response by inhibiting or inactivating the production of autoantibodies.

As used herein, the term " treatment " includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. The term " treatment " or " therapeutic agent " is used herein to refer to a therapeutic adjuvant or " therapeutic adjuvant " ≪ / RTI >

As used herein, the term " prophylactic " means inhibiting the development of a disease or disease in a subject who has never been diagnosed as having a disease or disease, but is likely to suffer from such disease or disease.

According to a specific embodiment of the present invention, the autoimmune thyroid disease to be treated or prevented with the composition of the present invention is thyroid-associated ophthalmopathy, Graves' disease, Hashimoto's thyroiditis, atrophic thyroiditis atrophic thyroiditis, painless thyroiditis, and postpartum thyroiditis.

According to a more specific embodiment of the present invention, the autoimmune thyroid disease which is treated or prevented with the composition of the present invention is thyroid-associated ophthalmopathy.

As used herein, the term " thyroid-associated ophthalmopathy " refers to a disorder in which the orbital orbital fibroblast cell is infiltrated with inflammatory reaction and lipidation due to autoimmune thyroid disease. According to the present invention, the composition of the present invention inhibits the production of hyaluronan, which is one of the most important pathological mechanisms of thyroid ophthalmopathy, in a concentration-dependent manner, and significantly inhibits differentiation of fat and increases PPARγ , C / EBP? And C / EBP?. By this multilateral experimental evidence, the present inventors have found that the composition of the present invention can be effectively used as an effective therapeutic agent for thyroid ophthalmopathy.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The term " administering " or " administering " as used herein refers to the administration of an effective amount of a composition of the present invention directly to a subject so that the same amount is formed in the body of the subject.

As used herein, the term " subject " includes without limitation humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Preferably, the subject of the present invention is a human.

As used herein, the term " effective amount " means an amount of a composition that is effective enough to treat, or at least ameliorate, a condition in which a subject is expected to suffer or suffer from pain when administered to the subject.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the pharmaceutical composition of the present invention can be manufactured as a sustained-release preparation so as to maintain the therapeutically effective amount continuously, thereby reducing the number of times the medicament is used to increase the compliance of the medicament. The sustained-release preparation may be formulated to include a sustained-release carrier and other adjuvants in addition to the active ingredient of the present invention. The sustained release carrier used in the present invention may be any of various sustained release carriers known in the art, and specifically a hydrophilic polymer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a pharmaceutical composition for the prevention or treatment of autoimmune thyroid diseases comprising the biguanide derivative of formula (1), more specifically metformin as an active ingredient, to provide.

(b) The composition of the present invention has a low cytotoxicity, but significantly inhibits the production of hyaluronan and the differentiation of fat, thereby preventing various pathological states caused by thyroid autoantibodies, for example, thyroid ophthalmopathy Can be effectively treated or improved.

FIG. 1 is a graph showing the results of MTT analysis confirming that the composition of the present invention has no toxicity to fibroblasts.
FIG. 2 is a graph showing the effect of the composition of the present invention on hyaluronan production, which is one of pathological mechanisms of thyroid ophthalmopathy. Metformin was treated with 10 ng / ml of TNF-α for 24 h in concentrations of 10, 50, 100, 500, 1000 and 5000 μg / ml, and the production of hyaluronic acid was analyzed by ELISA kit. Metformin significantly inhibited hyaluronan production even at 10 μM.
FIG. 3 shows the effect of the composition of the present invention on lipid differentiation, which is one of pathological mechanisms of thyroglobulinemia, in the fat formation (10, 100 and 1000 μg / ml) without special staining. It can be confirmed that the fat formation of the orbital fibroblast is suppressed with increasing concentration.
FIG. 4 is a graph showing the effect of the composition of the present invention on lipid differentiation, one of pathological mechanisms of thyroid ophthalmopathy, by oil-red o staining at concentration (10, 100 and 1000 μg / ml) . It is confirmed that the lipid formation of the orbital fibroblast is suppressed with increasing concentration (X40).
FIG. 5 shows the results of FIG. 4 at high magnification (X400). The effect of the composition of the present invention on lipid differentiation, which is one of the pathological mechanisms of thyroglobulinemia, is shown by concentration (10, 100 and 1000 μg / ml) oil-red o staining.
FIG. 6 shows that the composition of the present invention exhibited a decrease in the expression of PPARg, C / EBP? And C / EBP? Increased upon fat differentiation under metformin at different concentrations (10, 100 and 1000 占 퐂 / ml) Of the total amount of water.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Orbital fibroblast differentiation from orbital adipose tissue of thyroid ophthalmopathy

Orbital decompression was performed in patients with thyroid ophthalmopathy and primary orbital fibroblasts were cultured from orbital fat tissue. For comparative experiments, normal orbital fibroblasts were obtained from the orbital fat tissue through upper eyelid surgery or orbital surgery in normal subjects without thyroid ophthalmopathy. The culture was performed by shaking the tissue with wescott scissors and then shaking collagenase II (3 mg / ml in HBSS, 1.5% BSA) for 1 hour at 37 ° C, The floating fat cells and fat globule were removed by centrifugation. The cells were washed twice with PBS and incubated in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS) and incubated in a CO ₂ incubator (5% CO ₂ , 37 ° C). The fused cells were detached with trypsin-EDTA solution and subcultured while the culture medium was changed at intervals of 2-3 days.

Adipogenesis

After orbital fibroblasts were divided into 6- well plates, when the cells reached confluence, lipid differentiation of the lipid precursor cells was induced. (10 mM, Cayman, Ann Arbor, MI, USA), 33 mM biotin, 17 mM pantothenic acid, 10 mg / ml transferrin and 10 mg / ml transferrin were added to the DMEM supplemented with 10% , 0.2 nM T3, 1 mM insulin (Boehringer-Mannheim, Mannheim, Germany) and 0.2 mM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA), 1 mM dexamethasone and 0.1 mM isobutyl methylxanthine. The lipid differentiation lasted for 10 days and the medium and drug were replaced every 2-3 days.

Cytotoxicity test

The cytotoxicity test of metformin was performed using MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-di phenyltetrazolium bromide) analyzer (CellTiter 96 Aqueous nonreactive cell proliferative assay, Promega). MTT assays were performed by culturing orbital fibroblasts in each well and treating the metformin of the present invention by concentration (10-5000 [mu] M) for 24 hours. After incubation for 48 hours, 20 μl of MTT solution was added. After 4 hours, the optical density (OD) was measured at 490 nm wavelength in an ELISA plate reader to compare cell growth inhibition. The dehydrogenase enzyme found in metabolically active cells converts MTS to formazan and the degree of absorbance measured at 490 nm ultraviolet light is directly proportional to the number of living cells in culture. The experiment was repeated three times and the average values were compared. As a result, it was confirmed that metformin was not toxic to the orbital fibroblast (Fig. 1).

Hyaluronan ELISA

After orbital fibroblasts were divided into 12-well plates, metformin was treated with 10-5000 μg / ml concentration for 24 hours, and cells were stimulated with TNF-α. The cell culture was collected and the HA concentration was determined using a hyaluronan ELISA kit (Echelon, Salt Lake City, UT, USA). Absorbance was measured at 405 nm and the concentration was calculated. ELISA analysis confirmed that metformin efficiently inhibited hyaluronan production even at a low concentration (10 μM) (FIG. 2).

Oil Red O staining of differentiated adipocytes

A 0.5% (w / v) oil red O solution was prepared, diluted with sterilized distilled water at a ratio of 3: 2, and used as a staining reagent for adipocytes. After orbital fibroblasts were divided into 6-well plates, when the cells reached confluence, lipid differentiation of the lipid precursor cells was induced for 10 days. After the lipolysis was completed, the medium was removed, washed twice with PBS, and fixed with 3.7% (w / v) formalin for 1 hour. After fixing, the Oil Red O Walking solution was treated for 1 hour, washed twice with distilled water, and then confirmed using a microscope (Olympus, Melville, NY, USA). As a result of observation, it was confirmed that metformin inhibited the differentiation of fat in a concentration-dependent manner (Figs. 3 to 5)

Western blotting

In this study, we investigated the inhibitory effect of metopamine on lipid differentiation in the orbital adipose tissue of thyroid ophthalmoplegia by inducing the differentiation of metformin in the protein mixture using an antibody reacting with the antigen epitope. The orbital adipose tissue cells were treated with extracts of soft bran extract (50, 100μg / ml) for 10 days. After obtaining cell lysate, proteolytic enzyme inhibitor and phosphatase inhibitor were added to the cell lysis buffer and then kept on ice for 20 minutes. After centrifugation at 12,000 rpm to obtain the supernatant, protein quantification was performed to make the same amount of protein in each experimental group. Proteins were separated using 10% SDS-PAGE, and the separated proteins were transferred to a PVDF membrane (Millipore, MA, USA). All non-specific binding sites in the membrane were blocked using 5% skim milk. After incubation for 24 h at 4 ° C, the secondary antibodies were incubated with chromogenic substrate for 1 h. The primary antibodies were incubated at 37 ° C for 1 h. The primary antibody, PPARγ, C / EBPα, C / EBPβ, p-P65, P65, HO- Respectively. When 10, 100 and 1000 μM of metformin was treated, it was confirmed that the expression of PPARγ, C / EBPα and C / EBPβ, which are lipogenic differentiation factors, was reduced (FIG. 6).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

A composition for preventing or treating thyroid-associated ophthalmopathy comprising metformin or a pharmaceutically acceptable salt thereof as an active ingredient.
delete delete delete delete 2. The composition of claim 1, wherein the composition inhibits hyaluronan production.
2. The composition of claim 1, wherein the composition reduces the expression of PPARy, C / EBP < alpha > and C / EBP [beta].

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