KR102132554B1 - Phamaceutical composition comprising raloxifene for preventing or treating systemic sclerosis - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating systemic sclerosis (SSc) containing raloxifene or an acceptable salt thereof as an active component. The composition of the present invention has effects of reducing the expression of fibrosis factors (α-SMA), reducing the degree of cell proliferation induced by TNF-β1, reducing the thickness of a fibroblast layer, and reducing the thickness of skin tissues, thereby being able to be usefully used as the composition for preventing or treating SSc.

Description

Pharmaceutical composition for the prevention or treatment of systemic sclerosis containing raloxifene as an active ingredient {PHAMACEUTICAL COMPOSITION COMPRISING RALOXIFENE FOR PREVENTING OR TREATING SYSTEMIC SCLEROSIS}

The present invention relates to a composition for preventing, improving or treating systemic sclerosis (SSc) containing raloxifene or an acceptable salt thereof as an active ingredient.

Systemic sclerosis (SSc) is a rare autoimmune disease, also referred to as scleroderma, due to the fact that activated fibroblasts excessively produce connective tissue components such as collagen. SSc is a complex and heterogeneous disease that causes defects in the function of skin and connective tissues such as blood vessels, gastrointestinal system, lungs, kidneys, and muscle joints. Based on the skin involvement pattern, SSc is a limited type (lcSSc) that occurs only in the hands below the elbow, the feet below the knee, and the face, depending on the site of the disease, and more areas, i.e. above the elbow, It is largely classified as a diffuse type (dcSSc) accompanied by internal organ involvement such as kidneys and lungs, with changes in skin over the knee and torso appearing as the skin hardens. Until now, all treatments that have been proven to be effective for systemic sclerosis are treatments for complications, and there are no clearly proven treatments for skin symptoms. For example, a calcium channel antagonist may be used for Raynaud's phenomenon, which is often combined in systemic sclerosis, but it does not affect the course of the disease other than the degree of symptom relief. Pulmonary arterial hypertension is also a major complication, and aggressive treatments for lowering pulmonary arterial hypertension are performed, but this is not related to the course of sclerosis itself. Pulmonary fibrosis is a common complication in patients with systemic sclerosis, and is thought to be associated with fibrosis of the skin, but there is no established treatment method, such as cyclophosphamide, for some rapidly progressing interstitial pneumonia. Some powerful immunosuppressants are also used. However, like other immunosuppressants, the effect on the progression of systemic sclerosis is not clear.

Raloxifene is a selective estrogen receptor modulator (SERM) for oral administration that has estrogen-like action on bone and anti-estrogen activity on the uterus and breast. Raloxifene is used to prevent osteoporosis in postmenopausal women. In addition, raloxifene has been reported to be as effective as tamoxifen in terms of reducing the incidence of breast cancer in women, and has been approved by the US FDA for reducing the risk of invasive breast cancer along with osteoporosis in postmenopausal women. In addition, raloxifene is known to be useful in the treatment of prostate cancer and the treatment of fibrocystic diseases of the prostate. However, studies on the prevention and treatment of systemic sclerosis of raloxifene have not been conducted.

The present inventors have made extensive efforts to develop a therapeutic agent for systemic sclerosis by preparing a model of a systemic sclerosis disease in which the human immune system is implemented using human dedifferentiated stem cells and screening about 800 drugs approved by the FDA.

[Advanced technical literature]
Delle et al., Antifibrotic effect of tamoxifen in a model of progressive renal disease.; J Am Soc Nephrol. 2012 Jan;23(1):37-48
Chang et al., Effects of raloxifene on portal hypertension and hepatic encephalopathy in cirrhotic rats; European Journal of Pharmacology 802 36-43 (2017.02.24.)

The present inventors show that raloxifene decreases the expression of fibrosis factor (α-SMA) in the systemic sclerosis model, induces a decrease in cell proliferation induced by TNF-β1, a decrease in fibroblast layer thickness and a decrease in skin tissue thickness. The present invention has been completed by confirming that sclerosis can be prevented or treated.

Accordingly, the present invention is to provide a composition capable of preventing or treating systemic sclerosis (SSc).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition can reduce the cell proliferation induced by TGF-β1 and reduce the thickness of the fibroblast layer or the skin tissue.

In one embodiment of the present invention, the composition can inhibit or reduce the expression of the fibrosis factor marker induced by TGF-β1.

In one embodiment of the present invention, the fibrosis factor marker may be COL1A1, COL3A1, ACTA2.

In one embodiment of the present invention, the systemic sclerosis may include systemic sclerosis of the lung, liver, kidney, cardiovascular system or skin.

In one embodiment of the present invention, the systemic sclerosis may be diffuse skin systemic sclerosis (dcSSc) or limited skin systemic sclerosis (IcSSc).

In one embodiment of the present invention, the composition may further include dactinomycin as an active ingredient.

The present invention also provides a health functional food for preventing or improving systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition according to the present invention has the effect of reducing the expression of fibrosis factor (α-SMA), reducing cell proliferation induced by TNF-β1, reducing fibroblast layer thickness and skin tissue thickness. Sclerosis can be usefully used as a composition for prevention or treatment.

1A is a diagram showing the overall experiment outline.
Figure 1B confirms the morphology after preparing iPSC by separating PBMC from the blood of patients with systemic sclerosis.
1C is a result of confirming the starch ability of iPSC by Alkaline phosphatase staining.
1D is a result of confirming the expression of iPSC markers SSEA4, OCT4, TRA-1-60, SOX2, TRA-1-81, and KLF4 with IFA.
1E is a result of confirming the expression of iPSC markers OCT4, SOX2, Nanog, and LIN28 at the gene level through qRT-PCR.
1F is a result confirming that iPSC can differentiate into endoderm, mesoderm, and ectoderm using teratoma formation.
Figure 2 is a result confirming the differentiation of systemic sclerosis specific iPSC into keratinocytes (Keratinocyte),
Figure 2A shows the keratinocyte differentiation protocol,
Figure 2B is the result of confirming the morphology of keratinocytes and keratinocyte markers KRT14 and Np63 with IFA,
Figure 2C is a result of confirming the expression of the iPSC marker OCT4 and the neuroectodermal markers PAX6 and SOX1, the expression of OCT4 expressed in iPSC decreases after differentiation, and the expression of the neuroectoderm marker which is not expressed during keratinocyte differentiation decreases Is confirmed,
2D is a result of confirming the expression of the keratinocyte markers Np63, KRT5, and KRT14 through qRT-PCR, confirming that the expression of the iPSC marker is decreased and the expression of the keratinocyte marker is increased.
3 is a result confirming the differentiation of systemic sclerosis specific iPSC into fibroblasts,
Figure 3A shows the fibroblast differentiation protocol,
Figure 3B is the result of fibroblast morphology and fibroblast markers Fibronectin and vimentin confirmed by IFA,
3C shows the expression of iPSC markers OCT4 and fibroblast markers (ECM markers) COL1A1, COL1A2, COL3A1, ACTA2, and Vimentin. Expression of OCT4 expressed in iPSC decreases after differentiation and expression of fibroblast marker Is a result of confirming an increase, and at this time, it is a result of confirming that the expression of all ECM markers is increased in iPSC-fibroblast of systemic sclerosis patients compared to normal people.
Figure 4 shows a method of manufacturing a 3D skin organoid and a humanized mouse model, (A) shows a 3D skin organoid production protocol, and (B) a schematic diagram of a humanized mouse model production (C) to (J) show the 3D skin transplant protocol.
Figure 5 shows the results of modeling systemic sclerosis in vitro and in vivo,
Figure 5A is a proliferation of iPSC, showing that there is no difference in cell proliferation between normal iPSC and systemic sclerosis iPSC,
5B is a cell proliferation diagram of iPSC-derived fibroblast (iPSC-F), confirming that cell proliferation of systemic sclerosis iPSC-fibroblast is increased compared to normal iPSC-fibroblast,
5C is a result of confirming the expression of the fibrosis marker α-SMA by Western blot, and confirming that the expression of α-SMA is higher in iPSC-fibroblast in patients with systemic sclerosis than normal iPSC-fibroblast,
5D shows that Western blot was quantified, indicating that the expression of α-SMA in systemic sclerosis is higher than that of normal persons (statistically significant),
5E shows that when the total collagen in the cells is quantified, the expression of collagen in iPSC-F of patients with systemic sclerosis is higher than that of normal iPSC-F,
5F is a schematic diagram of a 3D fibroblast layer using iPSC,
5G is a result of confirming that when the 3D fibroblast layer is formed, the thickness of the 3D fibroblast layer of the systemic sclerosis patient is increased compared to the normal person,
5H is a result of confirming the expression of α-SMA in iPSC-F of patients with systemic sclerosis compared to iPSC-F in normal people when expression of the fibroblast marker α-SMA is confirmed through IFA,
FIG. 5I shows the skin tissue of mice transplanted with ISO of systemic sclerosis compared to mice implanted with normal iSO when 3D skin organoid (iSO) was prepared and transplanted into mice using keratinocytes and fibroblasts differentiated from iPSC. It is the result confirming that the thickness is increased and the expression of Collagen 3 and α-SMA is increased.
Figure 6 shows an anti-fibrotic drug screening using a systemic sclerosis model derived from iPSC,
6A shows a schematic diagram of FDA approved drug screening,
Figure 6B shows the results of the primary screening of drugs that reduce the activated cell proliferation of iPSC-F by measuring the cell proliferation after treatment of the FDA approved 800 drugs in iPSC-F,
6C is a result of confirming a decrease in total collagen in dactinomycin and raloxifene when total collagen is measured using the selected drug,
6D-6F is a result of confirming that total collagen expression is reduced when two drugs are treated after giving TGF-β1b stimulation to induce fibrosis to iPSC-F of normal people and iPSC-F of patients with systemic sclerosis,
6G is a result of confirming a decrease in the expression of α-SMA after treating two drugs in iPSC-F of a systemic sclerosis patient,
6H-6I shows the expression of α-SMA quantified.
7 is an in vitro efficacy verification result of raloxifene using a systemic sclerosis model derived from iPSC,
7A is a result of confirming that cell proliferation decreases and wound healing increased by TGF-β1b decreases when a wound healing assay is performed after treatment with raloxifene on iPSC-F,
Figures 7B-7C are quantified Wound length,
7D is a graph showing the change in wound length according to the incubation time, and is a result of confirming that the reduction width of the wound length is reduced when treated with raloxifene,
7E is a result of confirming that the thickness of the layer increased by TGF-β1b is reduced when the 3D fibroblast layer is formed and then treated with raloxifene.
7F-7G is a quantification of the thickness and area of the 3D fibroblast layer,
7H shows that when treated with raloxifene by concentration, the expression of α-SMA decreases with concentration, using Western blot,
7I is a quantitative expression of α-SMA when treated with concentrations of raloxifene,
Figure 7J confirms that when treated with raloxifene by concentration, the expression of total collagen decreases with concentration.
8 is a result of verifying the efficacy of raloxifene in the Bleomycin model, an animal model of systemic sclerosis,
Figure 8A shows a Bleomycin mouse model construction and drug administration protocol,
8B is a histological analysis result of the Bleomycin mouse model, confirming that the skin thickness increased by Bleomycin induction is reduced by drug treatment,
8C-E confirms that expression of COL1A1, COL3A1, and ACTA2 is reduced in the drug treatment group when expression of the fibrosis related gene is confirmed using qRT-PCR,
Figure 8F is a quantitative representation of the skin thickness increased by Bleomycin induction decreased by drug treatment,
8G is a histological analysis result of the Bleomycin mouse model lung tissue, and observes the antifibrotic effect of the lung tissue.

One aspect of the present invention comprises the steps of differentiating induced pluripotent stem cells (iPSCs) derived from patients with systemic sclerosis into keratinocytes or fibroblasts; And forming a 3D skin organoid by three-dimensional culture of the keratinocytes and fibroblasts.

In addition, the present invention comprises the steps of differentiating induced pluripotent stem cells (iPSCs) derived from patients with systemic sclerosis into keratinocytes or fibroblasts; 3D culture of the keratinocytes and fibroblasts to form a 3D skin organoid; And implanting the 3D skin organoid into the skin tissue of the mouse.

In the present invention, the term, “induced pluripotent stem cells (iPSCs) derived from patients with pre-systemic sclerosis” refers to the entire process through an artificial reprogramming process from peripheral blood cells (PBMCs) of patients with systemic sclerosis. Refers to cells induced to have pluripotency.

In the present invention, the "reprogramming (reprogramming)" is a differentiation cell that is present in different aspects, such as a cell with no differentiation capacity or a cell with a certain differentiation capacity, and finally is restored or converted to a state having a different type of differentiation potential. Means a process that can. In addition, in the present invention, the reprogramming may be used in the same sense as dedifferentiation. The reprogramming mechanism of these cells means establishing a different set of epigenetic marks after the epigenetic marks in the nucleus (the DNA state associated with causing genetic changes in function without changes in the nucleotide sequence) are deleted. However, while multicellular organisms differentiate and grow, different cells and tissues acquire different gene expression programs.

The reprogramming is not limited as long as it is possible to make induced pluripotent stem cells from PBMCs of systemic sclerosis patients, and methods known in the art can be used.

Specifically, in the present invention, the artificial reprogramming process is performed by the introduction of a virus-mediated or non-insertable non-viral vector using a non-insertable virus, a non-virus-mediated reprogramming factor using a protein and cell extract, or the like, It may include a reprogramming process using stem cell extracts, compounds, and the like. Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, it shows a similar cell shape, has similar gene and protein expression patterns, has starch differentiation ability in vitro and in vivo , forms teratomas, and blastocysts of mice ( blastocyst), chimera mice can form, and germline transmission of the gene is possible. Induced pluripotent stem cells of the present invention may be all derived from humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice or rabbits, and specifically humans.

In the present invention, the "reprogramming factor" is a substance that induces finally differentiated cells to be reprogrammed into Pluripotent stem cells having a new type of differentiation potential. The reprogramming factor may include, without limitation, any substance that induces reprogramming of the finally differentiated cell, and may be selected according to the type of cells to be differentiated. Specifically, the reprogramming factor may be at least one protein selected from the group consisting of Oct4, Sox2, Klf4, c-Myc, Nanog, Lin-28, and Rex1, or a nucleic acid molecule encoding the protein, but is not limited thereto.

In the present invention, the "nucleic acid molecule encoding a protein" may be in a form operably linked to a promoter or the like so as to express the protein itself when delivered into a cell.

In the present invention, the term "differentiation" refers to a phenomenon in which cells divide and proliferate and the structure or function of cells is specialized while the entire individual is growing. That is, it refers to a process in which cells, tissues, etc. of an organism are transformed into a suitable form and function in order to perform a role given to each, for example, a process in which starch-potential stem cells such as embryonic stem cells are transformed into ectodermal, mesodermal and endoderm cells. In addition, differentiation of hematopoietic stem cells into red blood cells, white blood cells, platelets, etc., that is, progenitor cells expressing a specific differentiation trait may be included in differentiation.

In a specific embodiment of the present invention, induced pluripotent stem cells (iPSC) were prepared through reprogramming from PBMC of the blood of patients with systemic sclerosis (FIG. 1).

In addition, in a specific embodiment of the present invention, in order to prepare keratinocytes and fibroblasts having the potential to reproduce the phenotype of systemic sclerosis-specific disease, iPSCs derived from patients with systemic sclerosis were differentiated into keratinocytes or fibroblasts, respectively. It was found that keratinocytes and fibroblasts derived from iPSC were prepared by confirming expression of keratinocytes or fibroblast markers, proteins and cell surface markers (FIGS. 2 and 3 ).

Specifically, the manufacturing method of the present invention may include the step of differentiating induced pluripotent stem cells derived from patients with systemic sclerosis into keratinocytes or fibroblasts, and producing 3D skin organoids by culturing the keratinocytes and fibroblasts in three dimensions. have.

The term “3D organoid” of the present invention means a cell mass having a 3D conformation, and means a reduced and simplified version of an organ manufactured through an artificial culture process that is not collected and obtained from animals or the like. The origin of the cells constituting it is not limited. Organoids can be derived from tissue, embryonic stem cells or induced pluripotent stem cells, and can be cultured in three dimensions due to their ability to regenerate and differentiate themselves. The organoid may have an environment that is allowed to interact with the surrounding environment during the cell growth process. Accordingly, in the present invention, "3D skin organoid" can be an excellent model for observing the development and treatment process of a therapeutic agent for a disease by almost simulating a skin layer that actually interacts in vivo.

In the present invention, 3D skin organoids prepared by three-dimensional culture of keratinocytes and fibroblasts derived from iPSC in patients with systemic sclerosis are spatial and temporal in revealing the accumulation and decomposition of collagen related to systemic sclerosis that occurs when using the existing 2D cell culture system. Limitations can be overcome. It is easy to check whether collagen accumulation or the like is stained, and further, it is further transplanted into the mouse skin to produce a humanized mouse model, which is a systemic sclerosis disease model, similar to the environment in the human body, and is capable of accurately reproducing.

Therefore, the humanized animal model in which the 3D skin organoid or 3D skin organoid of the present invention is implanted can be used as a model of systemic sclerosis disease.

In a specific embodiment of the present invention, in the 3D skin organoid produced through the above process, the expression rate of α-SMA and collagen, which is a fibrosis marker, is higher than that of normal iPSC-derived fibroblasts, and the thickness of the 3D fibroblast layer is increased compared to normal. Was confirmed. Furthermore, when the 3D skin organoid (iSO) of the present invention was transplanted into a mouse, it was confirmed that the thickness of the skin tissue was increased and the expression of collagen and α-SMA was increased compared to a mouse implanted with normal iSO. Could (Fig. 5). Through this, it was found that the 3D skin organoid of the present invention or a humanized animal model implanted with the same can be used as a new disease model for developing disease mechanisms and new therapeutic agents.

Another aspect of the present invention, (a) systemic sclerosis (Systemic sclerosis; SSc) patient induced induced pluripotent stem cells (iPSC) after differentiation into keratinocytes and fibroblasts, the keratinocytes and fibroblasts 3D culture to prepare a 3D skin organoid; (b) treating the 3D skin organoid with a candidate substance that inhibits collagen accumulation; (c) measuring collagen accumulation level in the 3D skin organoid treated with the candidate substance; And (d) when the level of collagen accumulation measured in step (c) is reduced compared to 3D skin organoids in which the candidate substance is not treated, determining the candidate substance as a therapeutic agent for systemic sclerosis. It relates to a screening method.

The term "collagen accumulation" of the present invention refers to abnormal accumulation of collagen matrix due to injury or inflammation that changes the structure and function of various tissues. It is naturally occurring in organs and tissues under normal circumstances, but it can occur excessively, and may be accompanied by disease or cause disease. Most etiology of systemic sclerosis involves excessive accumulation of collagen matrix replacing normal tissue.

The term "test agent" as used herein includes any substance, molecule, element, compound, entity or combinations thereof. For example, but not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound, or a combination of two or more substances.

In the systemic sclerosis disease model of the present invention, the expression or activity of α-SMA is reduced in fibroblasts, keratinocytes, or 3D skin organoids, and fibrosis is inhibited when collagen accumulation decreases. Therefore, an agent that reduces the expression level of mRNA of α-SMA or mRNA of its gene in fibroblasts, keratinocytes or 3D skin organoids of the present invention and inhibits collagen accumulation can be used as an agent for the prevention or treatment of systemic sclerosis. .

As used in the present invention, the term "measurement of protein expression level" refers to the process of determining the expression level of α-SMA that affects the accumulation of collagen in fibrotic diseases and measures the amount of protein. Specifically α-SMA It may be an antibody or aptamer that specifically binds to a protein expressed from a gene, but is not limited thereto. Such antibodies are polyclonal antibodies, monoclonal antibodies, or fragments of the antibodies, as long as they have antigen binding properties, are also included in the antibodies of the present invention. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies, human antibodies, and the like, and in addition to novel antibodies, antibodies already known in the art may also be included. The antibody is α-SMA As long as it has the properties of binding specifically recognizing a protein expressed from a gene, it includes functional fragments of antibody molecules, as well as a complete form with the full length of two heavy and two light chains. The functional fragment of the molecule of the antibody means a fragment having at least an antigen-binding function, and includes, but is not limited to, Fab, F (ab'), F (ab')2, and Fv.

For the purpose of the present invention, the protein expression level measurement includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Surface Enhanced Laser Desorption/ Ionization Time of Flight Mass Spectrometry), radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry ( liquid chromatography-Mass Spectrometry (LCMS), liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS), western blot, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.

The term "measurement of mRNA expression level" used in the present invention is a process of determining the expression level of the genes of α-SMA affecting collagen accumulation in systemic sclerosis and measuring the amount of mRNA. Analysis methods for this include reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), real-time reverse transcriptase reaction (Real-time RTPCR), and RNase protection assay (RPA). , Northern blotting, DNA chips, etc., but are not limited thereto.

The agent for measuring the mRNA level of the gene may specifically include a primer pair, a probe or an antisense nucleotide that specifically binds to the α-SMA gene, and the nucleic acid information of the genes is known to GeneBank, etc. Based on this, primers or antisense nucleotides that specifically amplify specific regions of these genes can be designed.

Another aspect of the present invention relates to a composition for preventing, improving or treating systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, "prophylaxis" refers to all actions that inhibit or delay the onset of the administration of the composition.

In the present invention, "improvement" or "treatment" refers to all actions in which symptoms of the disease are improved or beneficially changed by administration of the composition.

In a specific embodiment of the present invention, an attempt was made to discover a therapeutic agent for systemic sclerosis using a newly established systemic sclerosis skin model in the present invention. First, 800 drugs approved by the FDA were identified in iPSC-F to confirm that collagen accumulation was reduced. After treatment, the drug for reducing cell proliferation was first screened, and it was further confirmed that the expression of total collagen and the expression of α-SMA were reduced by using the first selected drug (FIG. 6 ). Through this, it was found that dactinomycin and raloxifene, which inhibit α-SMA expression, inhibit collagen accumulation very well, and thus have a prophylactic or therapeutic effect on fibrosis disease.

In addition, in order to verify the effect of Raloxifen on the treatment of systemic sclerosis, cell proliferation decreases when treated with fibroblasts having increased cell proliferation by TGF-β1, the thickness of the 3D fibroblast layer decreases, and α-SMA It was confirmed that expression and collagen expression decreased (FIG. 7 ). Furthermore, it was confirmed that the expression of the fibrosis factor markers COL1A1, COL3A1, and ACTA2 was reduced by treatment with raloxifene in the Bleomycin model, which is an animal model of systemic sclerosis, and the increased thickness of skin tissue was reduced and lung fibrosis was reduced (FIG. 8 ).

Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, the active ingredient of the composition for the treatment of systemic sclerosis of the present invention, raloxifene (raloxifene) is used as a concept containing a pharmaceutically acceptable salt of raloxifene, it may be a compound represented by the following formula (1).

[Formula 1]

Each of R1 and R2 may independently form a nitrogen-containing heterocycle group with a nitrogen atom adjacent to an alkyl group having 1 to 5 carbon atoms.

The raloxifene of the present invention may include hydrates of raloxifene, raloxifene derivatives, and the like within a range having the same efficacy as raloxifene, and may also include solvent compounds or stereoisomers thereof.

The method for obtaining the raloxifene is not particularly limited, and may be used that is isolated from nature or manufactured or commercially available by a chemical synthesis method known in the art.

In the present invention, the term “pharmaceutically acceptable salt” or “salt thereof” may be an acid addition salt formed by free acid. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. In addition, the same molar amount of the compound and the acid or alcohol in water (eg, glycol monomethyl ether) can be heated and then the mixture is evaporated to dryness or the precipitated salt can be suction filtered.

As the free acid, an inorganic acid or an organic acid may be used. As a non-limiting example of the inorganic acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid may be used, and these may be used alone or by mixing two or more.

The salt of the raloxifene may include all salts of acidic or basic groups that may be present in the compound of the raloxifene, unless otherwise indicated. For example, the salt of the raloxifene may include sodium, calcium and potassium salts of the hydroxy group, and the salt of the amino group may include hard bromide, sulfuric acid, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, Succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and the like, and may be prepared through a method of preparing a salt known in the art. have.

In addition, the composition for preventing or treating systemic sclerosis according to the present invention may contain a pharmaceutically effective amount of raloxifene or a salt thereof alone, or may include one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount in the above refers to an amount sufficient to prevent, improve and treat symptoms of systemic sclerosis.

The pharmaceutically effective amount of raloxifene or a salt thereof according to the present invention is generally 0.001 to 1,000 mg/day, and preferably 0.01 to 500 mg/day, based on an adult patient weighing 60 kg. However, the pharmaceutically effective amount may be appropriately changed according to the degree of symptoms of systemic sclerosis, the patient's age, weight, health status, sex, administration route, and treatment period.

In addition, “pharmaceutically acceptable” in the above refers to a composition that is physiologically acceptable and does not generally cause an allergic reaction such as gastrointestinal disorder, dizziness or similar reaction when administered to a human. Examples of the carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc., and these formulations include diluents (e.g. lactose, dext, in addition to active ingredients). Rose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycols). Tablets may also contain a binder such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally, such as starch, agar, alginic acid or its sodium salt. Disintegrants or boiling mixtures and/or absorbents, colorants, flavors, and sweeteners.

The pharmaceutical composition containing the raloxifene of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and parenteral administration is a method of injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. Depends on At this time, in order to formulate a formulation for parenteral administration, a pharmaceutical composition containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and this is an ampoule or vial. It can be prepared in unit dosage form. The composition may be sterile and/or contain preservatives, stabilizers, hydrating or emulsifying accelerators, adjuvants such as salts and/or buffers for osmotic pressure control, and other therapeutically useful substances, conventional methods of mixing, granulation It can be formulated according to the chemical or coating method.

The composition for the prevention or treatment of systemic sclerosis according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is the route of administration, age, sex, weight of the patient and It may be appropriately selected according to various factors such as the severity of the patient, and the composition for preventing or treating systemic sclerosis according to the present invention may be administered in combination with other compounds having an effect of preventing, improving or treating symptoms of systemic sclerosis. have.

In one embodiment of the present invention, it is confirmed that Dactinomycin decreases the expression of α-SMA, inhibits collagen production, and treats Tactinomycin to reduce the proliferation of cells increased by TGF-β1. (Fig. 6).

Therefore, the composition for preventing or treating systemic sclerosis of the present invention may further include dactinomycin as an active ingredient.

In another aspect, the present invention also provides a health functional food for preventing or improving systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

The specific contents of the raloxifene are as described above.

In the present invention, the term "health functional food" refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills, etc. using ingredients or ingredients having useful functionality for the human body. Here, the term'functional' refers to obtaining a useful effect for health use such as adjusting nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the art, and may be prepared by adding raw materials and ingredients conventionally added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food.

In the health functional food for preventing or improving systemic sclerosis according to the present invention, when the raloxifene is used as an additive of the health functional food, it can be added as it is or used with other foods or food ingredients, and is suitable according to a conventional method Can be used. The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health, or treatment.

The formulation of the dietary supplement can be in the form of powders, granules, pills, tablets, capsules, as well as in the form of regular food or beverages.

The type of the food is not particularly limited, and examples of foods to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. , Various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and may include all foods in the ordinary sense.

The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). In general, the amount of the compound in the health food can be added to 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Among the functional foods according to the present invention, the beverage may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a conventional beverage. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural sweeteners such as Martin and Stevia extracts, synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g per 100 mL of the beverage according to the present invention, preferably about 0.02 to 0.03 g.

In addition to the above, the health functional food for preventing or improving systemic sclerosis according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, and pH adjusting agents , Stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages. In addition, the health functional food composition for preventing or improving systemic sclerosis of the present invention may contain natural fruit juice, fruit juice beverage and fruit for the production of vegetable beverages. These ingredients can be used independently or in combination. The ratio of these additives is not limited, but is generally selected from 0.01 to 0.1 parts by weight compared to 100 parts by weight of the functional food of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Preparation and characterization of induced pluripotent stem cells (iPSCs) specific to systemic sclerosis (SSc) patients

<1-1> Recruitment of patients and isolation of induced pluripotent stem cells

Blood from patients with systemic sclerosis was obtained from Rheumatology Internal Medicine at St. Mary's Hospital in Seoul. Whole blood was diluted with phosphate buffered saline (PBS) and centrifuged at 850 xg for 30 minutes through a Ficoll gradient. Peripheral Blood Cells (PBMCs) were collected and frozen. PBMCs were thawed prior to use in the experiment and resuspended in StemSpan culture (STEM CELL Technological, Vacouver, British Colubia, Canada) supplemented with CC110 cytokine cocktail (STEMCELL). Cells were maintained at 5% CO ₂ , 37° C. for 5 days prior to reprogramming. The study was approved by The Institutional Review Board (IRB) of the Catholic University of Korea, St. Mary's Hospital in Seoul.

<1-2> Reprogramming of induced pluripotent stem cells from systemic sclerosis patients

Induced pluripotent stem cells (iPSCs) isolated Peripheral Blood Cells (PBMCs) from the blood of patients with systemic sclerosis and treated with Sendai virus containing Yamanaka effector to obtain iPSCs. In general, induced pluripotent stem cell reprogramming refers to a technique for inducing pluripotent stem cells by artificially overexpressing factors required for reprogramming in somatic cells. Methods for overexpressing the gene include viruses, plasmid vectors, mRNA, and proteins. 3×10 ⁵ PBMCs were plated on 24-well plates. Reprogramming was induced using the CytoTune-iPSC Sendai reprogramming kit. Virus transduction was performed with a multiplex infection of 7.5 per 3×10 ⁵ cells. After treating the virus, the cells were centrifuged at 1,160 xg and 37°C for 30 minutes and then cultured at 5% CO ₂ and 37°C. The next day, the cells were transferred to a 24-well plate coated with Vitronectin (Life Technologies), centrifuged at 1,160 xg, 37°C for 10 minutes, and then cultured at 5% CO ₂ , 37°C. Essential 8 (Life Technologies) was added to the culture solution in a 1:1 ratio. Reprogrammed cells were maintained and expanded in Essential 8 culture by daily culture exchange.

<1-3> Cell morphology confirmation

Reprogrammed cells were maintained and expanded in Essential 8 culture by daily culture exchange. To obtain a colony suitable for cell morphology, 5x10 ³ cells were plated on a 6-well plate coated with Vitronectin and maintained for 5 days to confirm iPSC morphology using a Leica DMi8 microscope (FIG. 1B).

<1-4> AP staining

IPSC generated and maintained through Example 1-2 above for AP staining (Alkaline Phosphatase staining) was spread on 5x10 ³ cells in Vitronectin coated 6-well plate and expanded for 5 days. Staining of undifferentiated iPSC colonies was performed using an alkaline phosphatase detection kit (Millipore, Billerica, MA, USA). The culture was removed, and fixed with 4% formaldehyde for 1-2 minutes. The remaining fixative was washed with PBS containing 0.05% Tween-20. Fast Red Violet, naphthol AS-BI phosphate solution and water were mixed at a ratio of 2:1:1 and stained for 15 minutes. After washing with PBS containing 0.05% Tween-20, PBS was added to prevent drying. Stained colonies were measured using a Leica DMi8 microscope. This experiment confirmed the iPSC starch differentiation ability (FIG. 1C).

<1-5> Cell immune staining

For immunofluorescence staining, iPSCs generated and maintained through Example 1-2 above were spread on 5x10 ³ cells on a Vitronectin coated 6-well plate and expanded for 5 days. After expansion, washed with PBS and fixed with 4% formaldehyde for 30 minutes. After removing the remaining fixative with an ammonium chloride solution, 0.1% Triton X-100 (BIOSESANG) was treated to increase cell permeability. After blocking the cells for 30 minutes at room temperature with PBS containing 2% bovine serum albumin (BSA), the primary antibody was diluted in PBA at the following dilution ratio: OCT4 (1/100; Santa Cruz, CA, USA) , KLF4(1/250; Abcam, Cambridge, UK), SOX2(1/100; BioLegend, San Diego, CA, USA), TRA-1-60(1/100; Millipore), TRA-1-81(1 /100; Millipore) and SSEA4 (1/200; Millipore). Primary antibodies were incubated for 2 hours at room temperature. DAPI was used as a nuclear stain. After staining, the cells were washed and sealed using ProLong Antifade reagent. Colonies stained with a Carl Zeiss immunofluorescence microscope were detected.

As a result, expression of iPSC markers SSEA4, OCT4, TRA-1-60, SOX2, TRA-1-81, and KLF4 was confirmed by IFA (FIG. 1D).

<1-6> Perform qRT-PCR

For qRT-PCR, total RNA was extracted using Trizol, and cDNA was synthesized using the Revert Aid TM First Strand cDNA Synthesis kit. QRT-PCR was performed on the synthesized cDNA using LightCycle 480 SYBR Green. The primers used are shown in [Table 1]. All experiments were repeated three times, and the threshold of the average cycle was used to calculate gene expression for averaging GAPDH as an internal control.

Primer sequence for identification of starch potential marker gene Target gene direction Base sequence (5'->3') Sequence number size OCT4 Forward ACCCCTGGTGCCGTGAA One 190 Reverse GGCTGAATACCTTCCCAAATA 2 SOX2 Forward CAGCGCATGGACAGTTAC 3 321 Reverse GGAGTGGGAGGAAGAGGT 4 NANOG Forward AAAGGCAAACAACCCACT 5 270 Reverse GCTATTCTTCGGCCAGTT 6 LIN28 Forward GTTCGGCTTCCTGTCCAT 7 122 Reverse CTGCCTCACCCTCCTTCA 8 GAPDH Forward ACCCACTCCTCCACCTTTGA 9 101 Reverse CTGTTGCTGTAGCCAAATTCGT 10

As a result, gene expression of the iPSC markers OCT4, SOX2, NANOG, and LIN28 was confirmed (FIG. 1E).

<1-7> teratoma formation observation

For observation of teratoma formation, immunodeficient mice such as SCID mice and Nude mice are generally used to induce tumors, and SCID mice are used in the experiment. 1x10 ⁶ iPSCs generated and maintained through Example 1-2 were mixed with Matrigel (BD Biosciences) in a ratio of 1:1. Cells were injected into the testis of SCID mice using an insulin syringe, and the tumors generated after 8-12 weeks were extracted to perform hematoxylin and eosin staining.

As a result, it was confirmed that dedifferentiated stem cells having stem cell functions capable of differentiating into endoderm, mesoderm, and ectoderm were generated (FIG. 1F).

Differentiation of systemic sclerosis specific iPSCs into keratinocytes

<2-1> keratinocyte differentiation protocol

The iPSCs generated and maintained through Example 1 were resuspended in Aggrewell culture (STEMCELL) and cultured (EB; Embryonic Body) using a Hanging drop culture method. Cells were incubated at 5% CO ₂ at 37° C. for one day. The formed EB was harvested and maintained by adding 1 ng/ml BMP4 to the Essential 8 culture. The next day, EBs were harvested and attached to collagen IV coated plates. During the differentiation period, the culture was changed once every 2 days. The culture was exchanged for keratin differentiation culture 1 (DMEM/F12 3:1, 2% fetal bovine serum (FBS), 0.3 mmol/L L-ascorbic acid, 5 μg/mL insulin, and 24 μg/mL adenine), and 3 It was maintained for 7 days by adding μM RA (Retinoic acid), 25 ng/mL BMP4, and 20 ng/mL EGF. After 7 days (Day8), the culture was exchanged for keratin differentiation culture 2 (defined keratinocyte serum-free medium, 0.3 mmol/l L-ascorbic acid, 5 μg/ml insulin, 10 μg/ml adenine), and 3 μM RA ( Retinoic acid), 25 ng/mL BMP4, and 20 ng/mL EGF were added and maintained for 4 days. After 4 days (Day12), the culture was exchanged for keratin differentiation culture 3 (defined keratinocyte serum-free medium and keratinocyte serum-free medium (1:1)), and 10 ng/mL BMP4 and 20 ng/mL EGF were added. Differentiated keratinocytes were maintained and expanded (FIG. 2A ).

<2-2> Confirmation of keratinocyte morphology and marker

The keratinocytes formed in Example 2-1 were harvested on the 21st day after the start of differentiation to confirm the morphology using a Leica DMi8 microscope. After washing the harvested cells with PBS and fixing the cells with 4% formaldehyde for 30 minutes for immunofluorescence staining, the remaining fixative is removed with ammonium chloride solution, and then treated with 0.1% Triton X-100 (BIOSESANG). Permeability was increased. After blocking the cells for 30 minutes at room temperature with PBS containing 2% bovine serum albumin (BSA), the primary antibody was diluted in PBA at the following dilution ratio: p63 (1/100; Abcam, Cambridge, UK), KRT14 (1/100; Abcam, Cambridge, UK). Primary antibodies were incubated for 2 hours at room temperature. DAPI was used as a nuclear stain. After staining, the cells were washed and sealed using ProLong Antifade reagent. Keratin cells stained with a Carl Zeiss immunofluorescence microscope were detected.

As a result, expression of p63 and KRT14 markers of keratinocytes was confirmed by IFA (FIG. 2B ).

<2-3> Confirmation of iPSC and Neuroectodermal Marker Expression Reduction

The keratinocytes formed in Example 2-1 were harvested on the 21st day after the start of differentiation, and total RNA was extracted using Trizol for qRT-PCR, and cDNA was synthesized using Revert Aid TM First Strand cDNA Synthesis kit. . QRT-PCR was performed on the synthesized cDNA using LightCycle 480 SYBR Green. The primers used are shown in [Table 2]. All experiments were repeated three times, and the threshold of the average cycle was used to calculate gene expression for averaging GAPDH as an internal control.

As a result, it was confirmed that the expression of the iPSC markers OCT4 and the neuroectoderm markers PAX6 and SOX1 is reduced (FIG. 2C ).

<2-4> Confirmation of expression of keratinocyte markers using qRT-PCR

The keratinocytes formed in Example 2-1 were harvested on the 21st day after the start of differentiation, and total RNA was extracted using trizol for qRT-PCR, and cDNA was synthesized using Revert Aid TM First Strand cDNA Synthesis kit. Did. QRT-PCR was performed on the synthesized cDNA using LightCycle 480 SYBR Green. The primers used are shown in Table 2. All experiments were repeated three times, and the threshold of the average cycle was used to calculate gene expression for averaging GAPDH as an internal control.

Primer sequence for identifying keratinocyte differentiation markers Target gene direction Base sequence (5'->3') Sequence number size hOCT4 Forward ACCCCTGGTGCCGTGAA 11 190 Reverse GGCTGAATACCTTCCCAAATA 12 hPAX6 Forward GTCCATCTTTGCTTGGGAAA 13 110 Reverse TAGCCAGGTTGCGAAGAACT 14 hSOX1 Forward CACAACTCGGAGATCAGCAA 15 133 Reverse GGTACTTGTAATCCGGGTGC 16 hNp63 Forward GGAAAACAATGCCCAGACTC 17 294 Reverse GTGGAATACGTCCAGGTGGC 18 hKRT5 Forward ACCGTTCCTGGGTAACAGAGCCAC 19 198 Reverse GCGGGAGACAGACGGGGTGATG 20 hKRT14 Forward GCAGTCATCCAGAGATGTGACC 21 181 Reverse GGGATCTTCCAGTGGGATCT 22 hGAPDH Forward ACCCACTCCTCCACCTTTGA 23 110 Reverse CTGTTGCTGTAGCCAAATTCGT 24

As a result, it was confirmed that the expression of the markers of keratinocytes Np63, KRT5 and KRT14 increased (FIG. 2D ).

Differentiation of systemic sclerosis specific iPSCs into fibroblasts

<3-1> Fibroblast differentiation protocol

The iPSCs generated and maintained through Example 1 were resuspended in Aggrewell culture (STEMCELL) and cultured (EB; Embryonic Body) using a Hanging drop culture method. Cells were incubated at 5% CO ₂ at 37° C. for one day. The formed EB was harvested and kept in Essential 8 culture. The next day, EB was harvested and attached to a Matrigel coated plate. The culture was exchanged for fibroblast differentiation culture 1 (DMEM/F12 3:1, 5% fetal bovine serum (FBS), 5 μg/ml insulin, 0.18 mM adenine, and 10 ng/ml epidermal growth factor (EGF)). During the differentiation period, the culture was changed once every 2 days. After 3 days (Day4), 0.5 nM BMP4 was added to the fibroblast differentiation culture 1 and maintained for 3 days. Thereafter, the culture medium was exchanged with fibroblast differentiation culture medium 2 (DMEM/F12 1:1, 5% FBS, 1% nonessential amino acids) and maintained for 7 days. Seven days later (Day14), expanded cells were transferred to uncoated plates. At this time, the culture medium was replaced with fibroblast differentiation culture medium 1. Seven days later (Day 21), expanded cells were transferred to collagen 1 coated plates. The culture medium was expanded by maintaining the differentiated fibroblasts by changing the fibroblast differentiation culture medium once every 2 days (FIG. 3A ).

<3-2> Identification of fibroblast morphology and marker

The fibroblasts formed in Example 3-1 were harvested on the 28th day after the start of differentiation, and the morphology was confirmed using a Leica DMi8 microscope. After washing the harvested cells with PBS and fixing the cells with 4% formaldehyde for 30 minutes for immunofluorescence staining, the remaining fixative is removed with ammonium chloride solution, and then treated with 0.1% Triton X-100 (BIOSESANG). Permeability was increased. After blocking the cells for 30 minutes at room temperature with PBS containing 2% bovine serum albumin (BSA), the primary antibody was diluted in PBA at the following dilution ratio: Fibronectin (1/200; Abcam, Cambridge, UK), Vimentin (1/200; Abcam, Cambridge, UK). Primary antibodies were incubated for 2 hours at room temperature. DAPI was used as a nuclear stain. After staining, the cells were washed and sealed using ProLong Antifade reagent. Keratin cells stained with a Carl Zeiss immunofluorescence microscope were detected.

As a result, expression of fibronectin and vimentin, which are cell markers of fibroblasts, was confirmed by IFA (FIG. 3B).

<3-3> Expression of iPSC and fibroblast marker (ECM marker)

The fibroblasts formed in Example 3-1 were harvested on the 28th day after the start of differentiation, and total RNA was extracted using trizol for qRT-PCR, and cDNA was synthesized using Revert Aid TM First Strand cDNA Synthesis kit. Did. QRT-PCR was performed on the synthesized cDNA using LightCycle 480 SYBR Green. The primers used are presented in [Table 3]. All experiments were repeated three times, and the threshold of the average cycle was used to calculate gene expression for averaging GAPDH as an internal control.

Primer sequence for fibroblast differentiation marker and fibrosis factor expression confirmation Target gene direction Base sequence (5'->3') Sequence number size hOCT4 Forward ACCCCTGGTGCCGTGAA 11 190 Reverse GGCTGAATACCTTCCCAAATA 12 hCOL1A1 Forward CCCCTGGAAAGAATGGAGATG 25 148 Reverse TCCAAACCACTGAAACCTCTG 26 hCOL1A2 Forward GGATGAGGAGACTGGCAACC 27 77 Reverse TGCCCTCAGCAACAAGTTCA 28 hCOL3A1 Forward CGCCCTCCTAATGGTCAAGG 29 161 Reverse TTCTGAGGACCAGTAGGGCA 30 hACTA2 Forward AAAGCAAGTCCTCCAGCGTT 31 115 Reverse TTCACAGGATTCTGGGAGCG 32 hGAPDH Forward ACCCACTCCTCCACCTTTGA 33 110 Reverse CTGTTGCTGTAGCCAAATTCGT 34

As a result, it was confirmed that the expression of OCT4 expressed in iPSC decreased after differentiation, and the expression of fibroblast marker increased. At this time, it was confirmed that the expression of all fibroblast markers is increased in patients with systemic sclerosis compared to normal people (FIG. 3C ).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

Construction of 3D skin organoids and humanized mouse models

<4-1> 3D skin organoid production protocol

3D skin organoids were produced using keratinocytes and fibroblasts formed in Examples 2 and 3 at all times. 2x10 ⁵ fibroblasts after 28 days of differentiation were harvested and mixed with collagen 1 and plated on a 6-well size Transwell plate. Changed once every 2 days with fibroblast differentiation culture 1 for 5 days. When collagen 1 is gelated, 1×10 ⁶ keratinocytes after 21 days of differentiation are harvested and 50-100 μl of epithelial cell culture medium 1 (Epithelial Medium; EP1; DMEM/F12 3:1, 4 mM L-glutamine, 40 μM adenine, Resuspend in 10 μg/mL transferrin, 10 μg/mL insulin, and 0.1% FBS) and streak onto fibroblasts. After maintaining for 2 days (Day7), the culture was replaced with epithelial cell culture medium 2 (Epithelial Medium; EP2; prepared by adding 1.8 mM calcium chloride to the EP1 culture) and cultured for 2 days. After 2 days (Day 9), epithelial cell culture medium 3 (Epithelial Medium; EP3; DMEM/F12 1:1, 4 mM L-glutamine, 40 μM adenine, 10 μg/mL transferrin, 10 μg/mL insulin, 2% FBS , 1.8 mM calcium chloride). At this time, the culture medium was added only to the bottom of the insert of the transwell plate and maintained by an air-liquid interface culture method (Figure 4A).

<4-2> Schematic diagram of humanized mouse model and 3D skin transplantation protocol

The 3D skin organoids formed through Example 4-1 were implanted into mice using a suture fixed dressing method (FIG. 4B). The skin tissue of the anesthetized SCID mouse was cut to 1×2 cm (FIG. 4C), and then 3D skin organoids derived from iPSC were raised (FIG. 4D). Mouse skin and skin organoids to be implanted were closed 8-9 times (FIG. 4E ). After placing the gauze on the suture site, the suture was tied with the remaining suture (FIG. 4F-H). After dressing with a band, it was maintained while watching the progress of the mice (Fig. 4I-J).

Identification of systemic sclerosis disease modeling characteristics in vitro and in vivo

<5-1> Analysis of cell proliferation in vitro

For the analysis of the proliferation of iPSC and iPSC-derived fibroblasts of the systemic sclerosis patients formed in Examples 1 and 3, 5×10 ³ cells were plated in a 96-well plate. Cell proliferation was measured by using the Cell Counting Kit-8 (Dojindo) at 450 nm.

As a result, there was no difference in cell proliferation between normal iPSC and iPSC in systemic sclerosis patients (FIG. 5A), but it was confirmed that cell proliferation of iPSC-derived fibroblasts in systemic sclerosis patients was increased compared to normal iPSC-derived fibroblasts. (Fig. 5B).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

<5-2> In vitro fibrosis factor expression analysis

In order to confirm the expression of the fibrosis factor of iPSC-derived fibroblasts in patients with systemic sclerosis formed in Example 3, 1×10 ⁶ fibroblasts were plated on a 100 mm culture dish, maintained and expanded for 5 days. The protein was extracted using RIPA buffer (Sigma) to analyze the expression of fibrosis factors, and the protein concentration was quantified by Bradford assay. The same concentration of protein was loaded on 10% SDS-PAGE and the protein was transferred to polyvinylidene difluoride membranes. After blocking for 1 hour at room temperature with PBS containing 5% skim milk, the primary antibody was diluted in PBS containing 5% skim milk at the following dilution ratio: α-SMA (1/200; Abcam). Primary antibodies were incubated for 2 hours at room temperature. After washing with PBS containing 0.1% Tween-20, the peroxidase-linked IgG secondary antibody was cultured, and protein expression was measured using the ECL kit.

As a result, it was confirmed that the expression of fibrosis factor α-SMA is increased in iPSC-derived fibroblasts of patients with systemic sclerosis compared to normal iPSC-derived fibroblasts (FIG. 5C ). When this was quantified using the image J program, a statistically significant increase was confirmed (FIG. 5D).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

<5-3> Analysis of collagen expression in vitro

To confirm the collagen expression of iPSC-derived fibroblasts of the systemic sclerosis patient formed in Example 3, 1x10 ⁶ fibroblasts were plated on a 100 mm culture dish, maintained and expanded for 5 days. For collagen expression, a Hydroxyproline assay kit (Sigma) was used. The cells or cell cultures are harvested, mixed with hydrochloric acid in a ratio of 1:1, and incubated at 120°C for 3 hours. After incubation, 5 mg of activated charcoal was added, followed by centrifugation at 13,000 xg for 5 minutes to separate only the supernatant. The harvested supernatant was transferred to a 96-well plate and dried completely at 60°C. Add the Chloramine T/Oxidation Buffer mixture to the well and incubate for 5 minutes at room temperature. After adding the diluted DMAB Reagent and incubating at 60°C for 90 minutes, absorbance at 560 nm was measured.

As a result, it was confirmed that the expression of total collagen as a fibrosis factor is increased in iPSC-derived fibroblasts of systemic sclerosis patients compared to normal iPSC-derived fibroblasts (FIG. 5E).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

<5-4> Analysis of 3D fibroblast layer in vitro

A 3D fibroblast layer was produced using the fibroblasts formed in Example 3 at all times. 2x10 ⁵ fibroblasts after 28 days of differentiation were harvested and mixed with collagen 1 and plated on a 6-well size Transwell plate. Changed once every 2 days with fibroblast differentiation culture 1 for 5 days (FIG. 5F ).

As a result, it was confirmed that the thickness of the 3D fibroblast layer produced using iPSC-derived fibroblasts of patients with systemic sclerosis is increased compared to normal iPSC-derived fibroblasts (FIG. 5G).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

<5-5> In vitro fibrosis factor expression analysis

The fibroblasts formed in Example 3-1 were harvested on the 28th day after the start of differentiation, washed with PBS, and fixed for 30 minutes with 4% formaldehyde for immunofluorescence staining. After that, the remaining fixative was removed with ammonium chloride solution, and then treated with 0.1% Triton X-100 (BIOSESANG) to increase cell permeability. After blocking the cells for 30 minutes at room temperature with PBS containing 2% bovine serum albumin (BSA), the primary antibody was diluted in PBA at the following dilution ratio: α-SMA (1/200; Abcam). Primary antibodies were incubated for 2 hours at room temperature. DAPI was used as a nuclear stain. After staining, the cells were washed and sealed using ProLong Antifade reagent. The expression of α-SMA, a fibroblast fibroblast, was confirmed by IFA using a Carl Zeiss immunofluorescence microscope.

As a result, it was confirmed that the expression of fibrosis factor α-SMA is increased in iPSC-derived fibroblasts of patients with systemic sclerosis compared to normal iPSC-derived fibroblasts (FIG. 5H ).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and disease modeling in vitro of fibroblasts differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

<5-6> Systemic sclerosis modeling in vivo and histological analysis

3D skin organoids derived from iPSC, which were normal humans and systemic sclerosis patients formed in Example 4-1, were implanted into SCID mice. After 2 weeks of transplantation, skin tissue was harvested and paraffin blocks were prepared for staining analysis. The harvested skin tissue was fixed in 4% formaldehyde. The tissue was kept in the running water for one day to remove the remaining fixative. Dehydration was carried out sequentially while increasing the concentration of the ethanol solution. After dehydration, a transparent process was performed with xylene, followed by paraffin penetration. The next day, the tissue was fixed to a paraffin block and a tissue section was obtained using a microtome. The slide was dried at 60° C. for 1 hour. After the slides were deparaffinized with xylene, they were rehydrated while reducing the concentration of the ethanol solution, washed with tap water, and stained.

For hematoxylin and eosin (H&E) staining, sections were incubated in a Harris hematoxylin solution for 10 minutes. The slide was washed, decolorized in an ethanol solution with 1% HCl, and then neutralized in 0.2% ammonia water. Control staining was performed with an eosin solution for 1 minute 30 seconds. The slide was washed and dehydrated while increasing the concentration of ethanol. After removing the remaining ethanol with xylene, it was sealed using VectaMount Permanent Mounting Medium.

For Masson Trichrome staining, slides were buried overnight in Bouin solution. After washing the slides, nuclei were stained for 10 minutes with Weigert iron hematoxylin. The cytoplasm was stained for 5 minutes with biebrich scarlet-acid fuchsin. The solution was mixed with Phosphotungstic acid, Phosphomolybdic acid, and DW in a ratio of 1:1:2 for 10 minutes. The fiber was dyed by holding it in a 2% aniline blue solution for 5 minutes. The slide was washed and dehydrated while increasing the concentration of ethanol. The remaining ethanol with xylene was removed and sealed using VectaMount Permanent Mounting Medium.

The slides were kept for 1 hour in the Pyrosirius Red solution for Picrosirius Red staining. It was washed with acetic acid and dehydrated while increasing the concentration of ethanol. After removing the remaining ethanol with xylene, it was sealed using VectaMount Permanent Mounting Medium.

For immunochemical staining, endogenous peroxidase was blocked by incubation for 15 minutes in 3% hydrogen peroxide. After washing the slides, cells were blocked for 1 hour at room temperature with PBS containing 1% bovine serum albumin (BSA), and then the primary antibody was diluted in PBA at the following dilution ratio: collagen 3(1/200;Abcam ), α-SMA (1/200; Abcam). Primary antibodies were incubated at room temperature at 4°C for one day. The next day, washed with Tris buffered saline (TBS) containing 0.1% Tween-20 (TBST), the secondary antibody was maintained at room temperature for 10 minutes, and then cultured in ABC reagent for 10 minutes. The slides were washed with PBS and the DAB solution was maintained for 1 minute. Mayer's hematoxylin was applied for 1 minute as a nuclear stain. The slide was washed and dehydrated while increasing the concentration of ethanol. The remaining ethanol with xylene was removed and sealed using VectaMount Permanent Mounting Medium.

As a result, it was confirmed that the skin thickness of mice implanted with iPSC-derived 3D skin organoids in patients with systemic sclerosis was increased compared to mice implanted with normal 3D skin organoids derived from iPSC. In addition, it was confirmed that the expression of collagen 3 and α-SMA is increased in mice transplanted with iPSC-derived 3D skin organoids in patients with systemic sclerosis (FIG. 5I ).

Through the above results, the same trend as the clinical symptoms of systemic sclerosis was confirmed, and in vivo disease modeling of cells differentiated from induced pluripotent stem cells in patients with systemic sclerosis was verified.

Screening of antifibrotic agents using iPSC-derived systemic sclerosis model

<6-1> FDA Approved Drug Screening

In order to analyze the proliferation of iPSC-derived fibroblasts formed in Example 3, 5×10 ³ cells were plated in a 96-well plate. The smeared cells were treated with about 800 FDA-approved drugs to screen for drugs that reduce the proliferation (Fig. 6A). Cell proliferation was measured by using the Cell Counting Kit-8 (Dojindo) at 450 nm (Fig. 6B).

In order to detect a drug that reduces the total collagen expression using the selected drug, 1x10 ⁶ fibroblasts were plated on a 100mm culture dish as in Example 5-3 above, treated with the drug, and then maintained and expanded. . For collagen expression, a Hydroxyproline assay kit (Sigma) was used. The cells or cell cultures are harvested, mixed with hydrochloric acid in a ratio of 1:1, and incubated at 120°C for 3 hours. After incubation, 5 mg of activated charcoal was added, followed by centrifugation at 13,000 xg for 5 minutes to separate only the supernatant. The harvested supernatant was transferred to a 96-well plate and dried completely at 60°C. Add the Chloramine T/Oxidation Buffer mixture to the well and incubate for 5 minutes at room temperature. After adding the diluted DMAB Reagent and incubating at 60°C for 90 minutes, absorbance at 560 nm was measured.

As a result, it was confirmed that Dactinomycin and Raloxifene decreased the cell proliferation and increased total collagen expression (Fig. 6C-F).

<6-2> Analysis of fibrosis factor expression in vitro of target drug

In order to verify the antifibrotic effect of the drugs selected through the experiments, dactinomycin and raloxifene, 1x10 ⁶ fibroblasts were plated on a 100 mm culture dish, treated with the target drug, maintained and expanded. The protein was extracted using RIPA buffer (Sigma) to analyze the expression of fibrosis factors, and the protein concentration was quantified by Bradford assay. The same concentration of protein was loaded on 10% SDS-PAGE and the protein was transferred to polyvinylidene difluoride membranes. After blocking for 1 hour at room temperature with PBS containing 5% skim milk, the primary antibody was diluted in PBS containing 5% skim milk at the following dilution ratio: α-SMA (1/200; Abcam). Primary antibodies were incubated for 2 hours at room temperature. After washing with PBS containing 0.1% Tween-20, the peroxidase-linked IgG secondary antibody was cultured, and protein expression was measured using the ECL kit.

As a result, it was confirmed that raloxifene effectively decreased the expression of α-SMA than dactinomycin (FIG. 6G-I).

Validation of anti-fibrosis effect of raloxifene in vitro

<7-1> Confirmation of reduction in cell proliferation in vitro by treatment with raloxifene

Wound healing experiment was conducted to confirm the cell proliferation inhibitory effect of the drug selected in Example 6, raloxifene. Fibroblasts were plated in 6-well plates, maintained and expanded. When the cells filled the plate, the plate was scratched, and fibrosis induction with TGF-β1 and treatment with raloxifene drug were performed.

As a result, it was confirmed that the proliferation of cells increased by TGF-β1 was decreased by raloxifene (FIGS. 7A-D ). Through the above results, it was verified that raloxifene is effective in reducing cell proliferation.

<7-2> Confirmation of 3D Fibroblast Layer Thickness Reduction by Raloxifen Treatment

In order to confirm the effect of reducing the skin thickness of the drug selected in Example 6, raloxifene, the 3D fibroblast layer was induced with TGF-β1 and treated with raloxifene drug.

As a result, it was confirmed that the 3D fibroblast layer thickness increased by TGF-β1 was decreased by raloxifene (FIGS. 7E-G ). Through the above results, it was verified that raloxifene is effective in reducing the thickness of the 3D fibroblast layer.

<7-3> Confirmation of Reduction of Fibrosis Factor Expression by Raloxifen Treatment

In order to confirm the reduction effect of the expression of the fibrosis factor of the drug selected in Example 6, raloxifene, raloxifene was treated by concentration and the expression of α-SMA was confirmed. 1×10 ⁶ fibroblasts were plated on a 100 mm culture dish, treated with raloxifene, maintained and expanded. The protein was extracted using RIPA buffer (Sigma) to analyze the expression of fibrosis factors, and the protein concentration was quantified by Bradford assay. The same concentration of protein was loaded on 10% SDS-PAGE and the protein was transferred to polyvinylidene difluoride membranes. After blocking for 1 hour at room temperature with PBS containing 5% skim milk, the primary antibody was diluted in PBS containing 5% skim milk at the following dilution ratio: α-SMA (1/200; Abcam). Primary antibodies were incubated for 2 hours at room temperature. After washing with PBS containing 0.1% Tween-20, the peroxidase-linked IgG secondary antibody was cultured, and protein expression was measured using the ECL kit.

As a result, it was confirmed that the expression of α-SMA decreases as the treatment concentration of raloxifene increases (FIG. 7H-I ).

Next, raloxifene was treated by concentration and total collagen expression change was confirmed. In the same manner as in Example 5-3, 1×10 ⁶ fibroblasts were plated on a 100 mm culture dish, treated with drugs, and maintained and expanded. For collagen expression, a Hydroxyproline assay kit (Sigma) was used. The cells or cell cultures are harvested, mixed with hydrochloric acid in a ratio of 1:1, and incubated at 120°C for 3 hours. After incubation, 5 mg of activated charcoal was added, followed by centrifugation at 13,000 xg for 5 minutes to separate only the supernatant. The harvested supernatant was transferred to a 96-well plate and dried completely at 60°C. Add the Chloramine T/Oxidation Buffer mixture to the well and incubate for 5 minutes at room temperature. After adding the diluted DMAB Reagent and incubating at 60°C for 90 minutes, absorbance at 560 nm was measured.

As a result, it was confirmed that the expression of total collagen decreased as the treatment concentration of raloxifene increased (FIG. 7J ).

Validation of Raloxifene in Bleomycin, an animal model of systemic sclerosis

<8-1> Bleomycin model establishment protocol

A systemic sclerosis model was established by dissolving leucine (Dong-A ST, 15mg) in PBS and injecting it daily into the nape of the mouse. Raloxifen and bazedoxifene were injected subcutaneously for 21 days from 3 days after the injection of leucine (Fig. 8A).

<8-2> Analysis of fibrosis factor expression by treatment with raloxifene in bleomycin model

ECM gene expression was confirmed to verify the antifibrosis efficacy of raloxifene in the bleomycin model established in Example 8-1. After harvesting the skin tissue, total RNA was extracted using Trizol, and cDNA was synthesized using the Revert Aid TM First Strand cDNA Synthesis kit. QRT-PCR was performed on the synthesized cDNA using LightCycle 480 SYBR Green. The primers used are presented in [Table 4]. All experiments were repeated three times, and the threshold of the average cycle was used to calculate gene expression for averaging GAPDH as an internal control.

Primer sequence to confirm expression of fibrosis factor Target gene direction Base sequence (5'->3') Sequence number size mCOL1A1 Forward GCAACAGTCGCTTCACCTACA 35 138 Reverse CAATGTCCAAGGGAGCCACAT 36 mCOL3A1 Forward TGAGCGTGGCTATTCCTTCGT 37 76 Reverse GCCGTGGCCATCTCATTTTCAA 38 mACTA2 Forward GTTCTAGAGGATGGCTGTACTA 39 108 Reverse TTGCCTTGCGTGTTTGATATTC 40 mGAPDH Forward ACCCCAGCAAGGACACTGAGCAAG 41 92 Reverse TGGGGGTCTGGGATGGAAATTGTG 42

Through the above experiment, it was confirmed that the expression of the fibrosis factor markers COL1A1, COL3A1, and ACTA2 was decreased by treatment with raloxifene (FIGS. 8C-E ).

<8-3> Skin tissue thickness analysis by Raloxifen treatment in Bleomycin model

To verify the anti-fibrotic efficacy of raloxifene in the bleomycin model established in Example 8-1, the skin tissue was harvested and paraffin blocks were prepared. The harvested skin tissue was fixed in 4% formaldehyde. The tissue was kept in the running water for a day to remove the remaining fixative. Dehydration was carried out sequentially while increasing the concentration of the ethanol solution. After dehydration, a transparent process was performed with xylene, followed by paraffin penetration. The next day, the tissue was fixed to a paraffin block and a tissue section was obtained using a microtome. The slide was dried at 60° C. for 1 hour. After the slides were deparaffinized with xylene, they were rehydrated while reducing the concentration of the ethanol solution, washed with tap water, and stained.

For hematoxylin and eosin (H&E) staining, sections were incubated in a Harris hematoxylin solution for 10 minutes. The slide was washed, decolorized in an ethanol solution with 1% HCl, and then neutralized in 0.2% ammonia water. Control staining was performed with an eosin solution for 1 minute 30 seconds. The slide was washed and dehydrated while increasing the concentration of ethanol. After removing the remaining ethanol with xylene, it was sealed using VectaMount Permanent Mounting Medium.

For Masson Trichrome staining, slides were buried overnight in Bouin solution. After washing the slides, nuclei were stained for 10 minutes with Weigert iron hematoxylin. The cytoplasm was stained for 5 minutes with biebrich scarlet-acid fuchsin. The solution was mixed with Phosphotungstic acid, Phosphomolybdic acid, and DW in a ratio of 1:1:2 for 10 minutes. The fiber was dyed by holding it in a 2% aniline blue solution for 5 minutes. The slide was washed and dehydrated while increasing the concentration of ethanol. The remaining ethanol with xylene was removed and sealed using VectaMount Permanent Mounting Medium.

The slides were kept for 1 hour in the Pyrosirius Red solution for Picrosirius Red staining. It was washed with acetic acid and dehydrated while increasing the concentration of ethanol. After removing the remaining ethanol with xylene, it was sealed using VectaMount Permanent Mounting Medium.

For immunochemical staining, endogenous peroxidase was blocked by incubation for 15 minutes in 3% hydrogen peroxide. After washing the slides, cells were blocked for 1 hour at room temperature with PBS containing 1% bovine serum albumin (BSA), and then the primary antibody was diluted in PBA at the following dilution ratio: α-SMA (1/200; Abcam). Primary antibodies were incubated at room temperature at 4°C for one day. The next day, washed with Tris buffered saline (TBS) containing 0.1% Tween-20 (TBST), the secondary antibody was maintained at room temperature for 10 minutes, and then cultured in ABC reagent for 10 minutes. The slides were washed with PBS and the DAB solution was maintained for 1 minute. Mayer's hematoxylin was applied for 1 minute as a nuclear stain. The slide was washed and dehydrated while increasing the concentration of ethanol. The remaining ethanol with xylene was removed and sealed using VectaMount Permanent Mounting Medium.

As a result, it was confirmed that the thickness of the skin tissue increased in the bleomycin mouse model was decreased by raloxifen (FIG. 8B, F).

<8-4> Pulmonary Fibrosis Analysis by Raloxifen Treatment in Bleomycin Model

In order to confirm the effect of reducing pulmonary fibrosis by treatment with raloxifene in the bleomycin model established in Example 8-1, abrasion tricolor staining was performed. For Masson Trichrome staining, slides were buried overnight in Bouin solution. After washing the slides, nuclei were stained for 10 minutes with Weigert iron hematoxylin. The cytoplasm was stained for 5 minutes with biebrich scarlet-acid fuchsin. The solution was mixed with Phosphotungstic acid, Phosphomolybdic acid, and DW in a ratio of 1:1:2 for 10 minutes. The fiber was dyed by holding it in a 2% aniline blue solution for 5 minutes. The slide was washed and dehydrated while increasing the concentration of ethanol. The remaining ethanol with xylene was removed and sealed using VectaMount Permanent Mounting Medium.

As a result, it was confirmed that lung fibrosis, which was increased in the bleomycin mouse model, was decreased by raloxifene (FIG. 8G ).

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> PHAMACEUTICAL COMPOSITION COMPRISING RALOXIFENE FOR PREVENTING OR TREATING SYSTEMIC SCLEROSIS <130> 1064735 <160> 42 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OCT4 <400> 1 acccctggtg ccgtgaa 17 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OCT4 <400> 2 ggctgaatac cttcccaaat a 21 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SOX2 <400> 3 cagcgcatgg acagttac 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for SOX2 <400> 4 ggagtgggag gaagaggt 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NANOG <400> 5 aaaggcaaac aacccact 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for NANOG <400> 6 aaaggcaaac aacccact 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for LIN28 <400> 7 gttcggcttc ctgtccat 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for LIN28 <400> 8 ctgcctcacc ctccttca 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 9 acccactcct ccacctttga 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 10 ctgttgctgt agccaaattc gt 22 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hOCT4 <400> 11 acccctggtg ccgtgaa 17 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hOCT4 <400> 12 ggctgaatac cttcccaaat a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hPAX6 <400> 13 gtccatcttt gcttgggaaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hPAX6 <400> 14 tagccaggtt gcgaagaact 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hSOX1 <400> 15 cacaactcgg agatcagcaa 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hSOX1 <400> 16 ggtacttgta atccgggtgc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hNp63 <400> 17 ggaaaacaat gcccagactc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hNp63 <400> 18 gtggaatacg tccaggtggc 20 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hKRT5 <400> 19 accgttcctg ggtaacagag ccac 24 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hKRT5 <400> 20 gcgggagaca gacggggtga tg 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hKRT14 <400> 21 gcagtcatcc agagatgtga cc 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hKRT14 <400> 22 gggatcttcc agtgggatct 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hGAPDH <400> 23 acccactcct ccacctttga 20 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hGAPDH <400> 24 ctgttgctgt agccaaattc gt 22 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hCOL1A1 <400> 25 cccctggaaa gaatggagat g 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hCOL1A1 <400> 26 tccaaaccac tgaaacctct g 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hCOL1A2 <400> 27 ggatgaggag actggcaacc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hCOL1A2 <400> 28 tgccctcagc aacaagttca 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hCOL3A1 <400> 29 cgccctccta atggtcaagg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hCOL3A1 <400> 30 ttctgaggac cagtagggca 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hACTA2 <400> 31 aaagcaagtc ctccagcgtt 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hACTA2 <400> 32 ttcacaggat tctgggagcg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for hGAPDH <400> 33 acccactcct ccacctttga 20 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for hGAPDH <400> 34 ctgttgctgt agccaaattc gt 22 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mCOL1A1 <400> 35 gcaacagtcg cttcacctac a 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mCOL1A1 <400> 36 caatgtccaa gggagccaca t 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mCOL3A1 <400> 37 tgagcgtggc tattccttcg t 21 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mCOL3A1 <400> 38 gccgtggcca tctcattttc aa 22 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mACTA2 <400> 39 gttctagagg atggctgtac ta 22 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mACTA2 <400> 40 ttgccttgcg tgtttgatat tc 22 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mGAPDH <400> 41 accccagcaa ggacactgag caag 24 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mGAPDH <400> 42 tgggggtctg ggatggaaat tgtg 24

Claims (8)

Pharmaceutical composition for preventing or treating systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 1,
The composition is a pharmaceutical composition for preventing or treating systemic sclerosis, characterized in that it reduces the cell proliferation induced by TGF-β1 and reduces the thickness of the fibroblast layer or the thickness of skin tissue.
According to claim 1,
The composition is a pharmaceutical composition for preventing or treating systemic sclerosis, characterized by inhibiting or reducing the expression of the fibrosis factor marker induced by TGF-β1.
According to claim 3,
The fibrosis factor marker is COL1A1, COL3A1, ACTA2, characterized in that the pharmaceutical composition for preventing or treating systemic sclerosis.
According to claim 1,
The systemic sclerosis comprises systemic sclerosis of the lungs, liver, kidneys, cardiovascular system or skin, pharmaceutical composition for preventing or treating systemic sclerosis.
According to claim 1,
The systemic sclerosis is diffuse skin systemic sclerosis (dcSSc) or limited skin systemic sclerosis (IcSSc), a pharmaceutical composition for preventing or treating systemic sclerosis.
According to claim 1,
The composition is a pharmaceutical composition characterized in that it further comprises Dactinomycin (Dactinomycin) as an active ingredient.
Health functional food for preventing or improving systemic sclerosis (SSc) containing raloxifene or a pharmaceutically acceptable salt thereof as an active ingredient.

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