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WO2016027990A1 - Composition pharmaceutique comprenant dusp5 en tant que substance active pour prévenir ou traiter des maladies métaboliques osseuses - Google Patents

Composition pharmaceutique comprenant dusp5 en tant que substance active pour prévenir ou traiter des maladies métaboliques osseuses Download PDF

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WO2016027990A1
WO2016027990A1 PCT/KR2015/007384 KR2015007384W WO2016027990A1 WO 2016027990 A1 WO2016027990 A1 WO 2016027990A1 KR 2015007384 W KR2015007384 W KR 2015007384W WO 2016027990 A1 WO2016027990 A1 WO 2016027990A1
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dusp5
stat3
activity
cells
bone
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Korean (ko)
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조미라
문수진
박진실
문영미
임미애
김은경
변재경
김성민
서현범
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Industry Academic Cooperation Foundation of Catholic University of Korea
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Industry Academic Cooperation Foundation of Catholic University of Korea
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

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  • the present invention relates to a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including DUSP5 as an active ingredient, a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, and a method for inhibiting the activity of STAT3 using DUSP5.
  • Bone is bone cells (osteocytes), osteoclasts (osteoclasts), osteoblasts (osteoblasts), hydroxyapatite crystals, collagenous fibers, glycosaminoglycans It consists of spaces such as bone matrix and bone marrow cavity, vascular canals, canaliculi and lacunae (Stavros CM, Endocrine Reveiws, 21). 2), 115-137 (2000)). Bones serve to mechanically support the body, protect vital organs, provide the microenvironment needed for hemopoiesis, and store calcium and other minerals.
  • Bone growth, development, and maintenance occur continuously throughout life. Aged bone is destroyed and new bone is regenerated instead. This turnover occurs mainly in basic multicellular units (BMUs), which are composed of osteoclasts and osteoblasts, which restore the bone's microscopic damage caused by growth and stress and maintain bone function. The destruction or absorption of aged bone is performed by osteoclasts. On the other hand, osteoblasts are responsible for the formation of new bone. Osteoclasts are attached to the surface of the bone to secrete acids and degrading enzymes to remove bone matrix such as apatite crystals and collagen that make up the bone and destroy the bone. Osteoblasts synthesize and release the bone matrix Skeletal formation is achieved by controlling the concentration of and phosphorus (Stavros CM, Endocrine Reviews, 21 (2), 115-137 (2000)).
  • Bone metabolic disease results from the disruption of the balance between osteoclasts and osteoblasts in vivo.
  • Representative examples of bone metabolic diseases include osteoporosis.
  • Osteoporosis refers to a condition in which total bone mass decreases due to an increase in osteoclast activity compared to osteoblasts. When osteoporosis occurs, the width of the cortical bone is reduced, the cavity of the bone marrow is enlarged, the reticulum is lowered, and the bone continues to be porous. As osteoporosis progresses, the bone's physical strength decreases, causing low back pain and joint pain, and the bone easily breaks down even with a slight impact.
  • Bone metabolic diseases include, but are not limited to, bone metastasis lesions in which tumors such as breast cancer and prostate cancer have metastasized to bone, primary tumors (such as multiple myeloma) in bone, rheumatoid or degenerative arthritis, and bacteria that cause periodontal disease.
  • Periodontal disease caused by destruction of alveolar bone, inflammatory alveolar bone resorption disease after implantation of dental implants, inflammatory bone resorption disease caused by implants implanted to fix bone in orthopedic areas, and various genetic Paget's disease caused by predisposition.
  • myeloma is a disease in which bone is easily fractured with severe pain, and is caused by tumor cells promoting osteoclast activity.
  • Breast and prostate cancers easily metastasize to bones, which also promote osteoclast activity and destroy bones.
  • tumor necrosis factor (TNF), interleukin-1, and interleukin-6 produced by immune response enhance the activity of osteoclasts in the joint cavity by Local bone breakdown occurs.
  • TNF tumor necrosis factor
  • interleukin-1 interleukin-1
  • interleukin-6 interleukin-6
  • osteoclast inhibitors As osteoclast inhibitors, estrogen, calcitonin, vitamin D and its analogues (Vitamin analogues), bisphosphonates and the like are known (Jardine et al., Annual Reports in Medicinal Chemistry, 31, 211 (1996). )).
  • an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.
  • Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, including both DUSP5 as an active ingredient.
  • another object of the present invention includes the step of transforming the cells with an expression vector comprising a promoter and a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked to the cell in vitro, to express the DUSP5 gene, in vitro It is to provide a method for reducing the activity or expression of STAT3 within.
  • the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.
  • the DUSP5 may be composed of the peptide of SEQ ID NO: 1.
  • the peptide of DUSP5 may be encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the DUSP5 may inhibit osteoclast differentiation by inhibiting the expression or activity of RANKL, RANK, NFATc1 and TRAP.
  • the bone metabolic disease is osteoprosis (osteoprosis), Paget disease (paget disease), metastatic cancer (metastatic cancer) or rheumatoid arthiritis (rheumatoid arthiritis), bone metastatic cancer (bone metastatic cancer) , Solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease and inflammatory bone resorption disease It may be selected.
  • the DUSP5 may be to inhibit or reduce the activity of Th17, and to promote or increase the activity of Regulatory T cells (Treg).
  • the DUSP5 may have an activity of inhibiting phosphorylation of STAT3.
  • the DUSP5 may be contained in the composition in the form of an expression vector comprising a promoter and the DUSP5 base sequence of SEQ ID NO: 2 operably linked thereto.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of STAT3-mediated diseases, including both DUSP5 as an active ingredient.
  • the STAT3-mediated disease is selected from the group consisting of arthritis, peritonitis, multiple sclerosis, psoriasis, asthma, edema, bronchial spasms, allergies, inflammatory diseases, autoimmune diseases, infectious diseases and necrotic diseases It may be.
  • the DUSP5 may have an activity of inhibiting phosphorylation of STAT3.
  • the DUSP5 may be composed of the peptide of SEQ ID NO: 1.
  • the peptide of DUSP5 may be encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the DUSP5 may be to inhibit or reduce the activity of Th17, and to promote or increase the activity of Regulatory T cells (Treg).
  • the present invention includes the step of transforming the cells with an expression vector comprising a promoter and a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto in vitro, expressing the DUSP5 gene, STAT3 in vitro It provides a method for reducing the activity or expression of.
  • the present invention relates to a pharmaceutical composition for preventing or treating bone metabolic diseases, including DUSP5 as an active ingredient, a pharmaceutical composition for preventing or treating STAT3 mediated diseases, and a method for inhibiting STAT3 activity using DUSP5.
  • DUSP5 can effectively inhibit the expression or activity of RANKL, RANK, NFATc1 and TRAP associated with osteoclast differentiation, inhibit or reduce the activity of Th17 and promote or increase the activity of Regulatory T cells (Tregs). It is possible to simultaneously act and inhibit the STAT3 activity by inhibiting the phosphorylation of STAT3, and thus will be useful as a new therapeutic agent for treating bone metabolic diseases and STAT3-mediated diseases.
  • Figure 1 confirms the effect of arthritis treatment by DUSP5, the results of analyzing the arthritis index and the incidence of arthritis by DUSP5 administration in the mouse group induced arthritis with collagen (Fig. The result confirmed by the crude staining method (Fig. 1b) and the result of analyzing the degree of reduction of immunoglobulin in the mouse serum is shown (Fig. 1c).
  • Figure 2 shows the results of analyzing the anti-inflammatory activity by DUSP5, after killing the arthritis-induced mouse group, and shows the result of observing the expression level of inflammatory factors in the joint tissue by immunostaining method (Fig. 2a), Fig. 2b is a blood vessel The result of observing the expression level of the formation factor by immunostaining method is shown.
  • FIG. 3 is a result of analyzing the effects on the differentiation of Th17 cells and Treg cells by DUSP5, and shows the results of analysis of Th17 cells and Treg cell differentiation according to pcDNA-DUSP5 and MOCK vector injection by flow cytometry (FIG. 3A). ), FIG. 3b shows the results of immunofluorescence microscopy using antibodies to IL-17, Foxp3, CD4, and CD25, and FIG. 3c shows the results of analyzing the expression level of each factor by RT-PCR. It is shown.
  • FIG. 4 is a result of analyzing the regulatory activity of ERK, Th17 and Tregs by DUSP5, 4a transduced CD4 + T cells with pcDNA-DUSP5 and MOCK vectors, respectively, DUSP5, IL-17, Gene expression levels of ERK and Foxp3 were analyzed by RT-PCR
  • FIG. 4B is a result of analysis of the production amount of IL-17, IL-21 and TNF-a in cell culture by ELISA.
  • 4C shows the results of RT-PCR analysis of the expression level of DUSP5, IL-17, ERK and Foxp3 in cells after DUSP5 siRNA treatment
  • FIG. 4D shows IL-17, IL-21 and TNF-a. The result of analyzing the amount of production is shown.
  • 5 is a result of analyzing the effect on the activity of regulating STAT3 and STAT5 by DUSP5, 5a obtained spleen from mice immunized with CII and immunized the degree of phosphorylation of STAT3 and STAT5 by DUSP5 treatment in spleen cells 5 shows the results of observing the degree of phosphorylation through Western blot, and FIG. 5 c shows the result of analyzing the phosphorylation of ERK by immunofluorescence staining.
  • 6 is a result of analyzing the osteoclast differentiation inhibitory activity by DUSP5
  • 6a is a result showing the expression of RANKL, RANK, NFATc1 in the tissue of the disease model through tissue staining
  • 6b is a disease mouse model After induction of osteoclast differentiation from bone marrow, the decrease of TRAP-positive cells by DUSP5 treatment was analyzed
  • 6c shows the expression of factors related to osteoclast differentiation in cells upon overexpression and suppression of DUSP5. The results of the analysis on RT-PCR are shown.
  • the present invention is characterized in that it provides a pharmaceutical composition for the prevention or treatment of bone metabolic diseases, including both DUSP5 as an active ingredient.
  • Dual-specificity phosphatase 5 (DUSP5) is known to act as an inhibitor of MAPKs, and unlike DUSP1 and DUSP4, it acts to inhibit the activity of ERK, while JNK and p38 MAPK do not inhibit activity.
  • DUSP5 can be used as a marker for diagnosing cancer (KR 20110078453A), and only discloses that it can be used for diagnosing obesity (KR 20090031400A). It has not been reported that metabolic diseases can be treated and STAT3-mediated diseases including autoimmune diseases can be prevented and treated.
  • the present invention is characterized by identifying a new use in terms of the medical use of DUSP5, in particular, the pharmaceutical use of DUSP5 identified in the present invention can be used for the prevention or treatment of bone metabolic diseases, STAT3 It can be used as a pharmaceutical composition for the prevention or treatment of mediated diseases, it was confirmed that it can be used to reduce the activity or expression of STAT3.
  • DUSP5 according to the present invention is excellent in inhibiting the differentiation of osteoclasts, in particular of the factors involved in osteoclast differentiation RANKL, RANK, NFATc1 and TRAP It was confirmed that there is an action that inhibits expression or activity.
  • Osteoclasts are known to be differentiated by various differentiation-inducing factors in macrophage family progenitor cells.
  • RANKL receptor activator of nuclear factor kappa B ligand
  • RANK receptor activator of nuclear factor kappa B
  • Differentiated osteoclasts were activated by NF-kB, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt MITF activation, etc. It is known to promote the expression of osteoclast specific proteins such as cathepsin K and calcitonin receptor.
  • DUSP5 of the present invention has an effect of effectively inhibiting the expression of osteoclast specific proteins and osteoclast differentiation related factors, thereby preventing or treating osteo metabolic diseases caused by osteoclast differentiation.
  • the bone metabolic disease of the present invention is not limited thereto, but osteoporosis (osteoprosis), Paget disease, (metastatic cancer) or rheumatoid arthiritis (rheumatoid arthiritis), bone metastatic cancer (bone metastatic cancer) , Solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease and inflammatory bone resorption disease Can be selected.
  • DUSP5 is characterized by inhibiting or reducing the activity of Th17 and simultaneously inducing an action of promoting or increasing the activity of Regulatory T cells (Treg).
  • T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body and undergo a series of differentiation processes to differentiate into T cells with unique characteristics.T cells, which have completed differentiation, are largely divided into type 1 helper cells (Th1) and type 2 according to their function. It is divided into helper cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.
  • Tregs immunoregulatory T cells
  • Th17 cells are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF- ⁇ in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF- ⁇ in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.
  • Th17 cells Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of autoimmune diseases which are not controlled by Treg cells among autoimmune diseases, development of therapeutic agents for autoimmune diseases that target the inhibition of Th17 cell activity has been highlighted.
  • STAT Signal transducers and activators of transcription
  • cytokines cytokines, hormones, growth factors, etc.
  • phosphorylation of tyrosine residues dimers by the interaction of the SH2 domain A dimer is formed that enters the nucleus and binds to a specific promoter.
  • the signaling system of these STAT proteins can be inhibited by dephosphorylation and protein degradation.
  • STAT3 is not only a hematological cancer such as leukemia, but also breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and prostate cancer. It is active in various solid cancers and has become an important anticancer target (Hua Yu and Richard Jove, Nature Review Cancer., 2004, 8, 945).
  • STAT3 has been known to inhibit apoptosis, induce angiogenesis, and induce immune evasion (Wang T. et al., Nature Medicine., 2004, 10, 48). Inhibition of STAT3 activity is a complex anticancer mechanism that can control tumors, and since STAT3 protein is involved in various intracellular functions as well as tumors, its inhibitor discovery can be developed as an immunosuppressive agent.
  • the immune system controls specific immune responses to autoantigens in a normal state, and also suppresses immune responses to external antigens. For example, a pregnant woman's response to an unborn baby and a chronically infected microorganism Reaction. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen specific immunotolerance can be induced.
  • Treg immunoregulatory T cells
  • Investigating some patients who accidentally acquired immunotolerance against transplanted antigens or experimentally induced animal models showed that all three of these mechanisms are involved in immunological tolerance. Is attracting attention as an important cell involved in controlling almost all immune responses of living body such as autoimmune, tumoral immunity, infectious immune response as well as transplantation immune response.
  • immunoregulatory T cells ie immunoregulatory T lymphocytes (Tregs), whose presence has recently been identified, can be largely divided into natural and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, are cells. Is newly immunized from the thymus and is present at 5-10% of the peripheral CD4 + T lymphocytes in normal individuals. The mechanism of immunosuppression of this cell is not yet known, but it has recently been discovered that the expression control factor of the gene, Foxp3, plays an important role in the differentiation and activity of the cell.
  • Tregs immunoregulatory T lymphocytes
  • peripheral natural T cells can be differentiated into cells that exhibit immunosuppressive effects upon stimulation of autologous or external antigens under certain circumstances, which are called adaptive or inducible Tregs and secrete IL-10. These include Tr1, Th3 and CD8 Ts that secrete TGF- ⁇ .
  • Th17 cells are differentiated into Th17 cells through differentiation in addition to Treg cells.
  • Th17 cells are formed in the presence of TGF- ⁇ in common with Treg cells, but Treg cells do not require IL-6, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF- ⁇ and secreting IL-17.
  • Th17 cells are characterized by having cytotoxicity that maximizes the signal of the inflammatory response and accelerates disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the ways to treat immune diseases.
  • DUSP5 of the present invention can prevent and treat not only bone metabolic diseases, but also immune diseases, and can also prevent and treat diseases caused by STAT3 mediation.
  • STAT3-mediated disease refers to a disease caused by activation of the STAT3 pathway, which is known in the art to indicate that overexpression, hypersecretion or overactivity of interleukin-1 ⁇ and / or interleukin-6 induces activation of the STAT3 pathway. It is known that interleukin-1 ⁇ and / or interleukin-6 are known to increase in expression, secretion or activity by inflammatory diseases, autoimmune diseases, destructive bone diseases, infectious diseases, degenerative diseases and necrotic diseases, etc. It is known to be due to STAT3 pathway activation induced by interleukin-1 ⁇ and / or interleukin-6. Therefore, in the development of a therapeutic agent for the disease, it is obvious that the substance that inhibits the activation of STAT3 is one of the methods for treating these diseases.
  • STAT3-mediated diseases that can be prevented or treated by the compositions of the present invention include arthritis, peritonitis, multiple sclerosis, psoriasis, asthma, edema, rheumatoid arthritis, degenerative arthritis, bronchial spasms, allergies, inflammatory diseases, Autoimmune diseases, destructive bone diseases, infectious diseases, degenerative diseases, necrotic diseases and inflammatory periodontal diseases.
  • the present invention provides an expression vector comprising a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto to a cell in vitro.
  • an expression vector comprising a DUSP5 nucleotide sequence of SEQ ID NO: 2 operably linked thereto to a cell in vitro.
  • DUSP5 an active ingredient contained in the composition provided in the present invention, may be a functional equivalent to a polypeptide having an amino acid sequence represented by SEQ ID NO: 1.
  • the term 'functional equivalent' refers to a sequence homology with at least 60%, preferably 70%, more preferably 80% or more of the amino acid sequence represented by SEQ ID NO. 1 as a result of the addition, substitution or deletion of amino acids. It refers to a polypeptide that exhibits substantially homogeneous activity with DUSP5 of the present invention.
  • substantially homogeneous activity refers to the activity of DUSP5 described above.
  • Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted or added.
  • Substitution of amino acids may preferably be conservative substitutions, examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids may preferably be located at portions not directly involved in the activity of DUSP5 of the invention.
  • the functional equivalent may also include polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of DUSP5 and its physiological activity.
  • polypeptide derivatives in which some chemical structures of the polypeptide are modified while maintaining the basic backbone of DUSP5 and its physiological activity.
  • fusion proteins made by fusion with other proteins while maintaining structural changes and physiological activities for altering the stability, storage, volatility or solubility of the polypeptide of the present invention may be included.
  • DUSP5 protein can also be obtained using genetic recombination techniques.
  • a polynucleotide encoding a DUSP5 protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured so that the DUSP5 protein is expressed, and the DUSP5 protein is recovered from the host cell. It can be obtained by the process.
  • Proteins are expressed in selected host cells and then subjected to conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, molecular sieve chromatography for isolation and purification. (Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In general, these are used in combination to separate proteins of high purity.
  • the present invention can provide a pharmaceutical composition
  • a pharmaceutical composition comprising a vector comprising a promoter and a polynucleotide encoding DUSP5 operably linked thereto.
  • the polynucleotide encoding the DUSP5 may be isolated or artificially modified in nature, the base sequence encoding the DUSP5 protein may be modified by substitution, deletion or insertion of one or more nucleic acid bases, such The protein expressed by the modification should not contain significant changes in its biological functionality. Such modifications include modifications to heterologous homologous genes.
  • the polynucleotide encoding the DUSP5 protein is preferably characterized by being represented by the nucleotide sequence of SEQ ID NO: 2, but not limited to the above polynucleotide, that is, the nucleic acid sequence and 70% or more, preferably 80% or more More preferably, it is represented by the base sequence which has 90% or more homology.
  • the polynucleotide encoding the DUSP5 protein of the present invention may be provided in the form of a recombinant expression vector operably linked to the vector expressing it.
  • Expression vectors comprising DUSP5 polynucleotides include, but are not limited to, plasmids, phages, cosmids, viral vectors or other mediators known in the art. Vectors can self replicate or integrate into host DNA.
  • Polynucleotides encoding the DUSP5 protein can be combined with expression control sequences such as promoter / enhancer sequences and other sequences required for transcription, translation or processing. Regulatory sequences include tissue-specific regulatory and / or inducible sequences as well as direct constitutive expression of nucleotides.
  • the design of the expression vector can be determined by factors such as the host cell to be transfected, the level of expression desired, and the like.
  • a non-viral vector or a viral vector may be used as the expression vector expressing the DUSP5 protein included in the composition of the present invention.
  • Viral vectors include, for example, replication defective retroviruses, adenoviruses, adenovirus-associated viruses, and the like. Viral vectors must meet the following criteria: (1) be able to infect the cells of interest, and thus a viral vector with an appropriate host range must be selected, and (2) the delivered genes will be preserved in the cells for an appropriate period of time. Must be able to be expressed and expressed, and (3) the vector must be safe for the host.
  • MLV murine leukemia virus
  • JC JC
  • SV40 polyoma
  • Epstein-Barr virus papilloma virus vaccinia
  • vaccinia Epstein-Barr virus papilloma virus
  • vaccinia Epstein-Barr virus papilloma virus
  • vaccinia Epstein-Barr virus papilloma virus
  • vaccinia Epstein-Barr virus papilloma virus
  • vaccinia Epstein-Barr virus papilloma virus
  • vaccinia vaccinia
  • poliovirus herpes virus
  • Sindbis virus lenti Viruse
  • the expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells Can be introduced into cells by the method of (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988). .
  • the meaning of including the polynucleotide encoding the DUSP5 protein as an active ingredient includes the polynucleotide itself encoding the DUSP5 protein as an active ingredient, and the expression vector into which the polynucleotide encoding the DUSP5 protein is inserted. Means to include as an active ingredient.
  • the term 'treatment' reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition.
  • the term 'treatment' refers to the act of treating when 'treating' is defined as above.
  • 'treatment' or 'therapy' of an immune disease in a mammal may include one or more of the following:
  • composition according to the invention may comprise a pharmaceutically effective amount of DUSP5 protein alone or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of an immune disease.
  • the pharmaceutically effective amount of DUSP5 protein according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight.
  • the pharmaceutically effective amount may be appropriately changed according to the degree of symptoms of immune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.
  • the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans.
  • carriers, 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.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • composition 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 determined by the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on several factors, and the composition according to the present invention may be administered in parallel with known compounds having the effect of preventing, ameliorating or treating the symptoms of the disease.
  • the present inventors prepared a collagen-induced arthritis mouse model to determine whether Dusp5 has a therapeutic effect on rheumatoid arthritis and performed the following experiment. At this time, the weight (weight) of each mouse used in the experiment was used a mouse 300g.
  • test animals were 6-week-old male DBA / 1J mice, and to prepare an arthritis animal model, type 2 collagen (Cll) was dissolved in 0.1 N acetic acid solution to 4 mg / ml, followed by dialysis buffer. , 50mM Tris, 0.2N Nacl) and mixed in equal amounts with complete Freund's adjuvant (CFA, Chondrex) containing M. tuberculosis and subcutaneously injected at the base of the tail of the mouse to obtain 100 ⁇ l of immunogen per animal. (Ie 100 ⁇ l / 100 ⁇ g).
  • CFA complete Freund's adjuvant
  • Arthritis was induced by intradermal injection of collagen type 2 into DBA1 / J mice, and 100 days after the induction of arthritis, 100 ⁇ g of pcDNA-DUSP5 containing the DUSP5 gene and a MOCK vector were injected intravenously with a control group. . After the induction of arthritis, the arthritis index was evaluated for 18 weeks, and the arthritis index was evaluated from 1 to 4 points.
  • the best arthritis index per head is 4, so the best disease index per mouse is 16.
  • DUSP5 cDNA clone GenBank No .: NM_001085390.1
  • restriction enzymes HindIII and Xho 1 inserted into pcDNA3.1 + (Invtrogen) to insert DUSP5 cDNA.
  • Generated recombinant vector pcDNA-DUSP5
  • transformed E. coli with the recombinant vector transformed E. coli with the recombinant vector
  • isolate plasmid DNA with plasmid extraction kit Qiagan
  • the correct recombinant plasmid vector was selected and then cultured to obtain a large amount of plasmid vector DNA.
  • Paraffin containing tissues were cooled to low temperature to a solid state and then trimmed to a suitable size.
  • Joint sections (5 ⁇ m) were prepared and placed on a glass slide treated with adhesive, and paraffin was removed with an organic solvent such as xylene. Since most dyes are aqueous solutions, the tissue-adhered slides were hydrated from a high concentration of alcohol to a low concentration of alcohol solution, and then stained first with an aqueous solution of hematoxylin, followed by secondary dyeing with eosin. The dyes were stained deeply and then subjected to a differentiation process to suitably match the degree of staining of samples overly stained with alcohol solution. In addition, histological examination was performed by toluidine blue and safranin O staining to confirm the degree of cartilage destruction.
  • immunohistochemical staining was performed to analyze the effects of VEGF and MMP9 on inflammatory cells such as IL-1 ⁇ , IL-6, and TNF- ⁇ and neovascularization factors in arthritis animal models. Histochemical staining was performed using the Vectastain ABC kit. That is, the primary antibody is reacted at 12 ° C. or more at 12 ° C., washed with PBS, reacted with biotin-conjugated secondary antibody (antirabbit IgG antibody) for 20 minutes, washed with PBS, and then reacted with streptatadine solution containing peroxidase. After reacting for 20 minutes, it was washed with water and developed using DAB chromogen.
  • the cells were washed with distilled water and embedded with an aqueous adhesive, and the expression levels of IL-1 ⁇ , IL-6, TNF- ⁇ , VEGF and MMP9 were observed by light microscopy.
  • a group reacted with PBS instead of the primary antibody was used.
  • mRNA expression levels were analyzed.
  • the cDNAs were isolated from the masons used in the above experiments, and the expression levels of each gene described below were measured. Using the primers, the mRNA expression level of each gene was measured by realtime PCR.
  • Primer sequence Primer Name Primer sequence (5'-3 ') IL-1 ⁇ forward GGA TGA GGA CAT GAG CAC ATT C IL-1 ⁇ reverse GGA AGA CAG GCT TGT GCT CTG A IL-6 forward ATG CTC CCT GAA TGA TCA CC IL-6 reverse TTC TTT GCA AAC AGC ACA GC TNF- ⁇ forward ATG AGC ACA GAA AGC ATG ATC TNF- ⁇ reverse TAC AGG CTT GTC ACT CGA ATT VEGF forward TCT TCA AGC CGT CCT GTG TG VEGF reverse AGG ACC ATT TAC ACG TCT GC MMP9 forward CTG TCC AGA CCA AGG GTA CAG CCT MMP9 reverse GAG GTA TAG TGG GAC ACA TAG TGG ⁇ -actin forward GAA ATC GTG CGT GAC ATC AAA G ⁇ -actin reverse TGT AGT TTC ATG GAT GCC ACA G
  • DUSP5 inhibits the production of inflammatory autoantibodies in the arthritis biological model
  • the blood was collected from each mouse and centrifuged. Serum was separated by using a total IgG and CII specific IgG and IgG2a antibody was performed by enzyme-linked immunoassay (ELISA).
  • ELISA enzyme-linked immunoassay
  • the arthritis index in the arthritis mouse group injected with the recombinant vector overexpressing DUSP5 is MOCK.
  • Arthritis onset analysis results showed that the group of mice injected with the recombinant vector overexpressing DUSP5 significantly inhibited the arthritis onset index compared to the control group injected with the MOCK vector. The results showed that arthritis is rarely developed.
  • IL-1 ⁇ , IL-6, TNF- ⁇ and VEGF and MMP9 all showed a significant inhibitory effect in the group of mice injected with DUSP5 compared to the control group (see FIGS. 2A and 2B). This inhibition was found to result from inhibition of gene expression (see FIG. 2C).
  • the present inventors can not only suppress the occurrence of arthritis by DUSP5 of the present invention, but also inhibit the destruction of joints, the infiltration of inflammatory cells in joints, the expression of angiogenesis-related factors and the production of inflammatory cytokines.
  • Inflammatory antibodies and inflammatory autoantibodies specific to the CII antigen were also found to have a selective and effective inhibitory activity.
  • the cells were isolated from the mice used in the above experiments. After the first immunization with CII and the mice were killed 56 days later, the cells and tissues of the spleen and the lymph node were separated. At this time, the experimental group was used as a mouse group injected with a vector expressing DUSP5, the control group was used as a group injected with a MOCK vector.
  • an optimal cutting temperature compound (OCT compound) was embedded using the spleen and lymph nodes of the mouse obtained by the above method, and then tissue was rapidly liquefied in liquefied nitrogen. Cool and attach to slides 7 ⁇ m thick using a frozen slicer. Sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes.
  • the primary antibody FITC-labeled anti-mFoxp3 Ab, PE-labeled anti-mCD4 Ab, APC-labeled anti-CD25 Ab diluted 1: 100 in PBS (pH7.5) and FITC for Th17 cell analysis. Reaction with -labeled anti-mCD4 Ab, PE-labeled anti-mIL-17 at 4 ° C. overnight, washed with PBS solution the next day, and stained tissues were analyzed by confocal microscopy.
  • RT-PCR In order to analyze the simultaneous regulation of T h 17 and Tregs according to DUSP5 overexpression at the gene expression level, gene expression levels of factors related to TH17 cell differentiation and factors related to Treg cell differentiation were analyzed by RT-PCR. Each cell for analysis was using the cell group used for the flow cytometry, and after obtaining the total RNA from these cell groups, RT-PCR was performed using the primers in the table below.
  • Primer sequence Primer Name Primer sequence (5'-3 ') IL-17 forward CCT CAA AGC TCA GCG TGT CC IL-17 reverse GAG CTC ACT TTT GCG CCA AG IL-21 forward CCC TTG TCT GTC TGG TAG TCA TC IL-21 reverse ATC ACA GGA AGG GCA TTT AGC IRF4 forward GCA GCT CAC TTT GGA TGA CA IRF4 reverse CCA AAC GTC ACA GGA CAT TG AHR Forward AGC AGC TGT GTC AGA TGG TG AHR reverse CTG AGC AGT CCC CTG TAA GC RoRrt forward TGT CCT GGG CTA CCC TAC TG RoRrt reverse GTG CAG GAG TAG GCC ACA TT Foxp3 forward GGC CCT TCT CCA GGA CAG A Foxp3 reverse GCT GAT CAT GGC TGG GTT GT
  • the TH17 cell group of IL-17 + was more than doubled in the experimental group injected with DUSP5 compared to the control group, and Foxp3-expressing Treg cell group was 1.6 times. It was found to increase by a factor of 2.
  • DUSP5 of the present invention has an action of inhibiting the differentiation and activity of TH17 cells and at the same time promoting the differentiation and activity of Treg cells, and this action is TH17 / Treg cell differentiation. It was found that it is through the regulation of the gene associated with.
  • Example 2 the effect of THD / Treg cell differentiation in the mouse was injected by injecting the DUSP5 expression vector into the disease mouse model.
  • the activity was reproved through in vitro experiments.
  • CD4 + T cell separation from spleen and lymph node cells was first performed by spleen and lymph node cells with CD4 coated microbeads at 4 ° C. for 15 minutes. After the reaction, washing with MACs buffer, CD4 + T cells were isolated. Isolated CD4 + T cells were washed with PBS and cell cultures containing 10% fetal calf serum, penicillin (100 U / mL) and streptomycin (100 g / mL) inactivated at 55 ° C. for 30 minutes (RPMI1640 medium). , Gibco BRL, USA).
  • the cells were seeded in a 24-well plate coated with 1 ⁇ g / mL of anti-CD3 antibody to 1 ⁇ 10 6 cells, and Th17 cells were seeded.
  • anti-CD28 antibody 1 ⁇ g / mL TGF- ⁇ 2ng / ml, IL-6 20ng / ml, anti-IL-4 10ng / ml, anti-IFNr 10ng / ml Cells were cultured for 3 days to induce differentiation into Th17 cells.
  • Cells for in vitro experiments were isolated from CD4 T cells from spleens of 35-day-old mice after the first immunization with the CII, and the cells were transduced with pcDNA-DUSP5 or MOCK vector and then 4 hours later.
  • the cells were treated with 1 ⁇ g / mL of anti-CD28 antibody, 2 ⁇ g of TGF- ⁇ , 20 ng / ml of IL-6, 10 ⁇ g / ml of anti-IL-4, and 10 ⁇ g / ml of anti-IFNr. After incubation, cells were collected and subjected to the RT-PCR method described above to analyze mRNA levels of IL-17, Foxp3 and ERK2 following DUSP5 overexpression under Th17 differentiation conditions.
  • the present inventors collected the culture supernatant of the cells used in the above ⁇ 3-1> and confirmed that the supernatant was monoclonal anti-IL-17, anti-inhibition to determine whether the production of inflammatory cytokines is inhibited by DUSP5 in vitro -TNF-a and anti-IL-21 were reacted at 4 ° C. overnight at 2 ⁇ g / mL, respectively, followed by blocking nonspecific binding with blocking solution (1% BSA / PBST).
  • biotinylated anti-IL-17, anti-TNF-a and anti-IL-21 were reacted at room temperature for 2 hours, washed four times, and then diluted with an ExtraAvidin-Alkaline Phosphatase conjugate and added for 2 hours at room temperature. Reacted. Thereafter, PNPP / DEA solution was added, followed by color development, and absorbance at 405 nm was measured.
  • siRNA Dharmacon, DUSP5
  • CD4 T cells isolated from spleen in ⁇ 3-1>.
  • siGENOME SMARTpool M-057231-01-0010
  • 19 and siGENOME non-targeting siRNA pool were treated to inhibit the expression of DUSP5 in cells.
  • the cells were incubated for 20 hours under the Th17 differentiation condition of ⁇ 3-1>, and the cells were collected and subjected to the RT-PCR method described above to perform IL-17, Foxp3 and MRNA levels of ERK2 were analyzed.
  • Example 1 the group was first immunized with CII and the mice injected with the DUSP5 expression vector and the mice injected with the MOCK vector were killed, and the spleen was separated, and then the degree of phosphorylation of STAT3 and STAT5 by DUSP5 in the tissues was determined. Observation was made through confocal microscopy and Western blot.
  • the mouse joints are separated and prepared as an Optimal Cutting Temperature compound (OCT compound), and then embedded in the tissue, and then the tissue is removed from the liquid nitrogen. Rapid cooling and attached to slides 7 ⁇ m thick using a frozen slicer. Sections were then fixed with acetone and 10% normal goat serum was applied to block nonspecific reactions for 30 minutes. And PE-labeled anti-STAT3 Tyr 705 (pSTAT3 Tyr 705 ), PE-labeled anti-STAT3 Ser 727 (pSTAT3 Ser 727 ) and anti-STAT5 Tyr 694 (pSTAT3 Tyr ) to detect phosphorylated STAT3 and STAT5.
  • OCT compound Optimal Cutting Temperature compound
  • DUSP5 of the present invention plays a role of reducing CD4 + ERK + cell number and also phosphorylation of ERK.
  • DUSP5 also influences the activity of STAT3 and STAT5, and that DUSP5 affects the differentiation of TH17 and Treg through the regulation of STAT3 and STAT5 activity, in particular DUSP5. It was found that the activity of STAT3 and STAT5 by the opposite results.
  • DUSP5 can simultaneously regulate TH17 / Treg by regulating the activity of ERK and transcription factors that regulate TH17 / Treg cells.
  • Example 1 primary immunization with CII and the injection of DUSP5 expression vector were performed 56 days later, and mice were killed to obtain bone tissue and bone marrow cells of the mice.
  • a group of MOCK vector injected instead of DUSP5 expression vector was used.
  • the obtained joint tissues were measured by immunohistochemical staining to confirm the expression level of RANKL, RANK, and NFATc1, which induce osteoclasts.
  • the Vectastain ABC kit was used, and primary antibodies (antibodies against RANKL, RANK, and NFATc1) were reacted at 4 ° C.
  • the joints of the collagen-induced arthritis disease animal model showed increased expression of RANKL, RANK, and NFATc1 markers of osteoclasts, whereas in the joints of mice injected with DUSP5 The expression of cell markers was shown to be markedly reduced.
  • the present inventors obtained bone marrow cells from the mouse group used in the experiment, and obtained monocytes from the bone marrow cells, and then, bone marrow of the animal model injected with the DUSP5 vector was treated with RANKL (50ng / ml) and M-CSF (10ng / ml) After stimulation, TRAP-positive cells, osteoclast factors, were observed by TRAP staining using a commercial kit.
  • the DUSP5-treated group showed a decrease in the number of TRAP positive marker cells and the expression level of TRAP compared to the non-treated group.
  • DUSP5 of the present invention has an activity for inhibiting osteoclast differentiation through the experiment of ⁇ 5-1>.
  • In vitro experiments confirmed the inhibitory activity of osteoclast differentiation as follows, ie for this purpose, the cells were stimulated with RANKL (50ng / ml) in RAW264.7 cell line, and then pcDNA- DUSP5 vector and MOCK vector (control) were injected, and the stimulated cells were treated with siRNA and scrambled siRNA (control) for DUSP5, respectively, and then cultured these cell lines for 72 hours. After that, gene expression levels of DUSP5, Cathepsin K, integrin beta3, RANK, and TRAP were analyzed by RT-PCR.
  • Primer sequence Primer Name Primer sequence (5'-3 ') Cathepsin K Forward CAG CAG AGG TGT GTA CTA TG Cathepsin K Reverse GCG TTG TTC TTA CGA GC Integrin- ⁇ forward CTG TGG GCT TTA AGG ACA GC Integrin- ⁇ reverse GAG GGT CGG TAA TCC TCC TC RANK forward CGA GGA AGA TTC CCA CAG AG RANK reverse CAG TGA AGT CAC AGC CCT CA TRAP forward TCC TGG CTC AAA AAG CAG TT TRAP reverse ACA TAG CCC ACA CCG TTC TC TC
  • the gene expression of the osteoclast factors Cathepsin K, integrin beta3, RANK, and TRAP was increased when the siRNA for DUSP5 was inhibited in the cells.
  • all of these osteoclast factors were significantly inhibited in expression.
  • DUSP5 of the present invention can effectively inhibit the differentiation of osteoclasts and thus can be usefully used for the treatment of bone diseases caused by osteoclast differentiation.

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Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies métaboliques osseuses et une composition pharmaceutique pour la prévention ou le traitement de maladies médiées par STAT3, qui contiennent DUSP5 en tant que substance active, et un procédé pour inhiber l'activité STAT3 au moyen de DUSP5. La présente invention peut être avantageusement utilisée en tant que nouvel agent thérapeutique capable de traiter des maladies métaboliques osseuses et des maladies médiées par STAT3 étant donné que DUSP5 peut inhiber efficacement l'expression de l'activité de RANKL, RANK, NFATc1, et TRAP, qui sont associés à la différenciation des ostéoclastes ; peut simultanément inhiber ou réduire l'activité de Th17 et favoriser ou augmenter l'activité de lymphocytes T régulateurs (Treg) ; et peut inhiber l'activité de STAT3 par l'intermédiaire de l'inhibition de la phosphorylation de STAT3.
PCT/KR2015/007384 2014-08-19 2015-07-16 Composition pharmaceutique comprenant dusp5 en tant que substance active pour prévenir ou traiter des maladies métaboliques osseuses Ceased WO2016027990A1 (fr)

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CN113332436A (zh) * 2021-06-25 2021-09-03 北京大学口腔医学院 Dusp5在制备预防、缓解和/或治疗骨质疏松药物中的应用
CN113616631A (zh) * 2021-08-26 2021-11-09 中国人民解放军陆军军医大学第二附属医院 Dusp6抑制剂bci在制备骨质疏松药物中的应用
CN114259564A (zh) * 2021-11-30 2022-04-01 清华大学 Hsp90抑制剂阻碍stat3线粒体转运和治疗哮喘的新应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113332436A (zh) * 2021-06-25 2021-09-03 北京大学口腔医学院 Dusp5在制备预防、缓解和/或治疗骨质疏松药物中的应用
CN113616631A (zh) * 2021-08-26 2021-11-09 中国人民解放军陆军军医大学第二附属医院 Dusp6抑制剂bci在制备骨质疏松药物中的应用
CN113616631B (zh) * 2021-08-26 2022-03-08 中国人民解放军陆军军医大学第二附属医院 Dusp6抑制剂bci在制备骨质疏松药物中的应用
CN114259564A (zh) * 2021-11-30 2022-04-01 清华大学 Hsp90抑制剂阻碍stat3线粒体转运和治疗哮喘的新应用
CN114259564B (zh) * 2021-11-30 2023-03-14 清华大学 Hsp90抑制剂阻碍stat3线粒体转运和治疗哮喘的新应用

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