KR20090028880A - Pharmaceutical compositions for preventing or treating fibrosis - Google Patents

Abstract

A pharmaceutical composition including a transglutaminase(TGase 2) inhibitor or N-acetylcysteine(NAC) is provided to prevent or cure fibrosis and to specifically show the molecular mechanism about the fibrosis. A pharmaceutical composition for preventing and treating fibrosis comprises (a) pharmaceutically effective dose of a transglutaminase(TGase 2) inhibitor or N-acetylcysteine(NAC); and (b)a pharmaceutically allowable carrier. A screening method of a material for preventing and treating fibrosis comprises the steps of: (i) contacting a testing material with a transglutaminase(TGase 2) protein; and (ii) measuring the activity of the TGase 2. The transglutaminase(TGase 2) inhibitor is the cystamine or the cysteamine.

Description

Pharmaceutical Compositions for Preventing or Treating Fibrosis

The present invention relates to a novel pharmaceutical composition for preventing or treating fibrosis and a method for screening a fibrosis therapeutic agent.

The tissue includes a well-ordered cell population bound to the extracellular matrix and surrounded by a vascular network. Fibrosis or Fibrosis is an abnormal accumulation of collagen matrix following injury or inflammation that changes the structure and function of various tissues. Regardless of the location of the invention of fibrosis, most etiology of fibrosis involves excessive accumulation of collagen matrix to replace normal tissue. Progressive fibrosis in the kidneys, liver, lungs, heart, bone or bone marrow, and skin is a major cause of death or pain (Border, et al., New Engl . J. Med . 331: 1286 (1994). Meanwhile, the development of fibrosis is associated with excessive expression and excessive production of transforming growth factor β (TGFβ) in tissues.

TGFβ, on the other hand, affects growth, differentiation and gene expression in various cells. In mammals it has three types of isoforms: TGFβ1, TGFβ2 and TGFβ3 (de Martin, et al. EMBO J. 6: 3673-3677 (1987); and Derynck, et al. EMBO J. 7: 3737-3743 (1988)).

Activating TGFβ binds to Type III receptors on cell membranes, and Type III receptors deliver TGFβ to Type I and Type II receptors. The TGFβ-coupled receptor then phosphorylates Smad2 or Smad3, and phosphorylated Smad2 or 3 binds to Smad4 and migrates into the cell nucleus, thereby regulating the expression of many genes.

TGFβ increases the expression of fibronectin, laminin and collagen, which are important components of the extracellular matrix (ECM), reduces the expression of the metalloprotease (MMP), a matrix degrading enzyme, and inhibits MMP. By increasing the expression of MMP), which in turn acts to increase the ECM.

In addition, TGFβ is known to be involved in the pathogenesis of several diseases. Decreased TGFβ causes atherosclerosis, whereas increased TGFβ is the cause of several types of fibrosis.

Although the cause-effect relationship of TGFβ in the pathogenesis of fibrotic disease, ie fibrosis, is well established, little has been reported about the mechanism by which TGFβ causes fibrotic disease. In addition, fibrotic diseases are commonly associated with oxidative stress, but the mechanism is also unknown.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made efforts to develop molecular mechanisms for the development of fibrosis, to discover new targets of fibrosis therapeutics and to develop novel fibrosis therapeutics. As a result, the activation of intracellular TGase 2 mediated by TGFβ2 (transforming growth factor β2) signaling has been investigated. The present invention has been completed by discovering a novel fibrosis therapeutic agent that is directly involved in the development of fibrosis and capable of effectively blocking the TGFβ2-TGase 2 pathway.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis.

Another object of the present invention is to provide a method for screening a material for preventing or treating fibrosis.

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

According to one aspect of the invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a transglutaminase 2 inhibitor or N-acetylcysteine (NAC); And (b) provides a pharmaceutical composition for preventing or treating fibrosis comprising a pharmaceutically acceptable carrier.

The present inventors have made efforts to develop molecular mechanisms for the development of fibrosis, to discover new targets of fibrosis therapeutics and to develop novel fibrosis therapeutics. As a result, the activation of intracellular TGase 2 mediated by TGFβ2 (transforming growth factor β2) signaling has been investigated. New fibrosis therapies have been found that are directly linked to the development of fibrosis and can effectively block the TGFβ2-TGase 2 pathway.

The pharmaceutical composition of the present invention contains a TGase 2 inhibitor or NAC as an active ingredient.

According to a preferred embodiment of the present invention, the TGase 2 inhibitor is cystamine, cysteamine, monodansil cardaberine, putriscine, histamine, methyl amine, iodoacetamide, 8-phenyl propionyl thiocholine, mono Amine, diamine, gamma-amino benzoic acid, 1- (5-aminopentyl) -3-phenylthiourea, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl Ester, CuSO ₄ and tolbutamide.

According to a more preferred embodiment of the present invention, the TGase 2 inhibitor is cystamine or cystiamine.

Fibrosis or fibrotic disorders prevented or treated by the present invention can be specified as acute or chronic and exhibit common characteristics such as excessive collagen accumulation and loss of function following replacement of normal tissue by fibrous tissue. Acute fibrosis includes reactions to trauma, infections, surgery, burns, radiation and chemotherapy. Chronic fibrosis is caused by viral infections, diabetes, obesity, fatty liver, high blood pressure, scleroderma and other chronic diseases that cause fibrosis.

Thus, fibrosis prevented or treated by the present invention may include fibrosis caused by a pathological condition or disease, fibrosis caused by radiation damage, and chemotherapeutic agents (e.g., bleomycin, chlorambucil, cyclophosphamide, methotroxate, All fibrotic diseases, including fibrosis caused by mustin or procarbazine).

Fibrosis that is prevented or treated by the pharmaceutical composition of the present invention may be located in various parts of the body. Fibrosis can be located in the kidneys, and glomerulonephritis (Yoshioka et al., Lab Invest 68: 154-63 (1993)), diabetic nephropathy (Yamamoto et al., Proc Natl Acad Sci USA 90: 1814-8 (1993)), transplant rejection (Shihab et al., J Am Soc Nephrol 4: 671 (1993)), HIV nephropathy (Border et al., J Am) Soc Nephrol 4: 675 (1993)), fibrosis is observed in IgA nephropathy and lupus nephropathy; For liver, cirrhosis (Castilla et al., N Engl J Med 324: 933-940 (1991) and Nagy et al., Hepatology 14: 269-73 (1991)), venous-obstructive disease (Anscher et al., N Engl J Med 328: 1592-8 (1993)), fibrosis is observed in hepatitis C virus infection, alcohol induced liver fibrosis and autoimmune liver fibrosis; For lungs, idiopathic fibrosis (Anscher et al., N Engl J Med 328: 1592-8 (1993) and Brockelmann et al., Proc Natl Acad Sci USA 1991; 88: 6642-6), autoimmune fibrosis (Deguchi, Ann Rheum Dis 1992; 51: 362-5), and fibrosis is observed in bleomycin-induced fibrosis and the like; For skin, systemic sclerosis (Kulozik et al., J Clin Invest 1990; 86: 917-22), keloids (Peltonen et al., J Invest Dermatol 1991; 97: 240-8), Wounds (Ghahary et al., J Lab Clin Med 1993; 122: 465-73) and eosinophilia-pain syndrome (Varga et al., Ann Intern Med Fibrosis is observed in 1992; 116: 140-7); In the central nervous system, intraocular fibrosis (Conner et al., J Clin Invest 1989; 83: 1661-6) of fibrosis; In the cardiovascular system, vascular restenosis (Nikol et al., J Clin Invest 1992; 90: 1582-92); For nasal, fibrosis is observed in nasal polyposis (Ohno et al., J Clin Invest 1992; 89: 1662-8); Fibrosis is also observed in bone or bone marrow (Reith, JD et al., Am J Srg Pathol , 1996 20 (11): 1368-1377); Fibrosis is also observed in endocrine organs (Endocrinology, 3rd Edition, Edited by Leslie J. DeGroot, Vol. 1, pp. 165-177 and pp. 747-751); And gastrointestinal relations (Tahara, E., J. Cancer Res . Clin . Oncol . , 1990, 116 (2), 121-131).

Fibrosis prevented or treated by the pharmaceutical composition of the present invention includes fibrosis occurring at the various positions described above.

Preferably, the fibrosis prevented or treated by the pharmaceutical composition of the present invention is kidney, liver or pulmonary fibrosis.

As active ingredients in the compositions of the present invention, the above-mentioned TGase 2 inhibitor or NAC, as well as pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof are included.

The term “pharmaceutically acceptable salts” refers to salts of the aforementioned TGase 2 inhibitors or NACs with the desired pharmacological effect. Such salts include inorganic acids such as hydrochloride, hydrobromide and hydroiodide, acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, p-toluenesulfonates, bisulfates, sulfamate, sulfates, Naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, hep Formed with organic acids such as tanoate, hexanoate, 2-hydroxyethanesulfate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate do.

The term “pharmaceutically acceptable hydrate” refers to a hydrate of the aforementioned TGase 2 inhibitor or NAC with the desired pharmacological effect. The term “pharmaceutically acceptable solvate” refers to a solvate of the aforementioned TGase 2 inhibitor or NAC with the desired pharmacological effect. The hydrates and solvates may also be prepared using the acids described above.

The term “pharmaceutically acceptable prodrug” refers to a TGase 2 inhibitor or a derivative of NAC that must be bioconverted before exerting the pharmacological effects of the TGase 2 inhibitor or NAC described above. Such prodrugs are prepared for prolongation of duration of action and reduction of side effects in order to improve chemical stability, patient solubility, bioavailability, organ selectivity or convenience of preparation. The preparation of the prodrugs of the present invention is carried out in conventional methods in the art using TGase 2 inhibitors or NAC (e.g. Burger's Medicinal Chemistry and Drug Chemistry, 5th ed., 1: 172-178 and 949-982 (1995)). It can be easily prepared accordingly.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

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

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Can be. On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.0001-100 mg / kg (body weight) per day.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention effectively blocks the TGFβ2-TGase 2 pathway, a mechanism that causes fibrosis, to prevent or treat fibrosis.

According to another aspect of the invention, the invention provides a method for screening a material for the prevention or treatment of fibrosis comprising the following steps:

(a) contacting a test substance with a transglutaminase 2 protein; And

(b) measuring the activity of the TGase 2.

The screening method of the present invention is based on the discovery that TGase 2 is involved in fibrosis.

According to the first step in the screening method of the present invention, the test substance to be analyzed is contacted with TGase 2. The TGase 2 employed is one that exists intracellularly or extracellularly. Extracellular TGase 2, ie purified recombinant TGase 2, can be obtained through various recombinant DNA techniques known in the art. In addition, as the extracellular TGase 2, a peptide of TGase 2 having an intrinsic activity of TGase 2 can also be used.

The test substance screened by the method of the invention is a single compound or a mixture of compounds (eg, a natural extract or cell or tissue culture). Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA).

Finally, the activity of TGase 2 treated with the test substance is measured. If the TGase 2 activity to which the test substance is treated is lower than the TGase 2 activity to which the test substance is not treated, the test substance is selected as a fibrosis treatment agent.

The measurement of TGase 2 activity can be carried out through various methods known in the art, preferably according to (i) in vitro transamidation assay or (ii) in situ transamidation assay.

In vitro transamidation assay measures in vitro TGase 2 activity by measuring incorporation of [ ₁₄ C] putrycin into N, N'-dimethylcasein. More specifically, TGase 2 treated with the test substance or cells treated with the test substance were crushed to obtain a cell lysate, which contained a substrate solution ([ ₁₄ C] putrycin and N, N'-dimethylcasein). ) And react. Then, the reaction is stopped and TGase 2 activity is measured by measuring radioactivity bound to N, N'-dimethylcasein.

According to the in situ transamidation assay, an amine compound in which an affinity material is bound is first treated with cells that are not crushed. Most preferred as an affinity material is biotin, preferred as an amine compound is polyamine, most preferably pentylamine. Biotinylated pentylamine (BP) is treated and treated in cells. Proteins bound to BP in cells can be measured by various methods. For example, the BP bound protein can be analyzed by the solid-state microtiter plate method. BP-treated cells were crushed to obtain lysate, and then the microtiter plate was coated with the lysate. Subsequently, the plate is treated with the signal generating-substance conjugated with streptadibin (or avidin). The signal generating agent is an enzyme (eg alkaline phosphatase, β-galactosidase, horseradish peroxidase, β-glucosidase and cytochrome P ₄₅₀ ), radioactive substance (eg C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET).

When a radioisotope is used as a signal generating substance, the BP-binding protein formed in the final process of the present invention can be detected by measuring radioactivity.

When an enzyme catalyzing a color reaction is used as a signal generating substance, a BP-binding protein can be detected by adding a substrate used for the enzymatic reaction to measure the reaction product. For example, when alkaline phosphatase is used as a signal-generating substance, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-) as substrates Colorimetric substrates such as AS-B1-phosphate) and enhanced chemifluorescence (ECF) can be used, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucifer Genine (bis-N-methylacridinium nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5 Substrates such as -tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

By measuring the signal coming from the signal generating substance, the intracellular BP-binding protein can be quantified.

In addition to the solid-state microtiter plate method described above, BP-bound proteins can be analyzed by western blotting. Western blotting method can be used conventionally performed methods (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). The Western blotting method preferably comprises the steps of (i) pulverizing the cells treated with the test substance; (Ii) SDS-PAGE the cell lysate; (Iii) transferring the protein on the gel complete with SDS-PAGE to the NC membrane; And (iii) treating the NC membrane with a signal generating-substance conjugated with streptadibin (or avidin).

In addition, through the cell staining method, it is possible to analyze BP-bound intracellular proteins. For example, cells cultured and treated in glass coverslips placed on plates are labeled with BP and then immobilized, followed by permeabilization of the cell membranes with a suitable surfactant (eg, Triton X-100). The cells are then treated with streptavidin (or avidin) to which the fluorescent material is bound, and the cells are then observed with a confocal microscope. Examples of the fluorescent material are fluorescein (fluorescein), fluoresein isothiocyanate (FITC), rhodamine 6G (rhodamine 6G), rhodamine B (rhodamine B), TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole) and Coumarin.

According to a preferred embodiment of the present invention, the screening method of the present invention is in situ It is carried out according to the transamidation analysis protocol. Because direct measurement of TGase 2 activity in cells is a measure of true in vivo TGase 2 activity , in situ rather than in vitro transamidation assay Transamidation assay protocols are preferred.

According to a more preferred embodiment of the present invention, the screening method of the present invention comprises the following steps.

(a) treating a test substance to a cell comprising TGase 2;

(b-1) treating the cells with a biotinylated polyamine, preferably biotinylated pentylamine (BP);

(b-2) crushing the cells treated with the polyamine to obtain a cell lysate;

(b-3) coating the cell lysate on a microtiter plate;

(b-4) treating the plate generating signal-conjugated streptavidin (or avidin) to the plate; And

(b-5) measuring the signal from the signal generator-substance.

If the signal from step (b-5) is smaller than the signal from the control, i.e., cells not treated with the test substance, it is selected as a fibrosis therapeutic agent.

The present invention clearly shows the molecular mechanisms for the development of fibrosis and provides new molecular targets for the treatment of fibrosis. In addition, the fibrosis therapeutic agent provided in the present invention can very efficiently inhibit the onset of fibrosis or treat fibrosis, and has high safety in vivo.

As described in detail above, the present invention provides a pharmaceutical composition for preventing or treating fibrosis. The present invention also provides a method for screening a substance for preventing or treating fibrosis. The present invention clearly shows the molecular mechanism for fibrosis and provides new molecular targets for the treatment of fibrosis. In addition, the fibrosis therapeutic agent provided in the present invention can very efficiently inhibit the onset of fibrosis or treat fibrosis, and has high safety in vivo.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Example 1 Analysis of Involvement of TGase 2 in Fibrosis

20-week-old C57BL / 6 (TGase 2 ^{+ / +} , Charles River Laboratories) and TGase 2-deficient (TGase 2 ^-/- ) mice (De Laurenzi, V. & Melino, G .. Gene disruption of tissue transglutaminase.Mol . Cell Biol. 21: 148-155 (2001)) was used to investigate whether TGase 2 is involved in fibrosis, particularly pulmonary fibrosis. Wild type C57BL / 6 and TGase 2 ^{− / −} mice were injected intranasally with phosphate buffered saline (PBS) or bleomycin (2.0 mg / kg, Dong-A) as a negative control. One month later, the mice were sacrificed to collect lung tissue, fixed with formalin, and then subjected to Mason trichrome staining. Images of the lung tissue were obtained by magnification 400 times.

As can be seen in Figure 1, pulmonary fibrosis was induced by bleomycin in normal C57BL / 6 mice. In contrast, bleomycin-induced pulmonary fibrosis was not observed in TGase 2 ^{− / −} mice. These experimental results indicate that TGase 2 activity plays a critical role in the development of fibrosis.

Example 2: Analysis of the inhibitory effect of fibrosis development of NAC or cystiamine

Infusion of bleomycin (1.5 or 2.0 mg / kg) into 20-week-old C57BL / 6 mice immediately following intraperitoneal injection of PBS, NAC (60 mg / kg, Sigma), or cystiamine (50 mg / kg, Sigma) It was. One month later, the mice were sacrificed to collect lung tissue, fixed in formalin, then subjected to Mason trichrome staining, and images of the lung tissue were magnified 400 times.

As can be seen in FIG. 2, NAC partially inhibited the development of pulmonary fibrosis caused by bleomycin. Cystiamine, which directly inhibits the activity of TGase 2, showed a much better inhibitory effect on lung fibrosis than NAC.

Example 3: Intracellular TGase Activity Measurement

Intracellular TGase2 activity was measured by determining the biotinylated pentylamine (BP, Pierce) which bind to cellular proteins (Shin, DM et al. Cell-type specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation. J. Biol . Chem . 279: 15032-15039 (2004). The TGase2 activity value was normalized by subtracting the value representing the endogenous biotin-conjugated protein obtained in the absence of BP, and describing how many times the increase was compared with the control group without the oxidative stress inducing factor. TGase2 activity was analyzed as band strength (probed with streptavidin-HRP (SA)) of cellular proteins cross-linked with BP.

Example 4: Cell Culture Experiment

Human primary lung fibroblasts, IMR90 cells (ATCC) supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (100 U / ml), streptomycin sulfate (100 μg / ml) and glutamine (2 mM) It was maintained in DMEM (Dulbecco's modified Eagle's medium, GIBCO). IMR90 cells were incubated at 37 ° C. for 12 hours in DMEM containing 2% FBS and then exposed to media containing TGFβ1 (R & D system). Cystamine (1 mM, Sigma) and NAC (2.5 mM, Sigma) were used to inhibit TGase2 and TGFβ, respectively.

Example 5: Western Blotting Assay

Cells were sonicated in buffer (50 mM Tris-Cl, pH 6.8, 6 M urea, 2% SDS, 40 mM dithiothreitol and protease inhibitor cocktails) to obtain total cell extracts. Total cell extracts were centrifuged (12,000 × g, 10 min, 4 ° C.) and then protein concentrations were determined by BCA method. Cell extracts (30 μg) were isolated in 8% or 12% SDS-PAGE. Monoclonal Antibodies to Actin (Sigma), Monoclonal Antibodies to α-Smooth Muscle Actin (DAKO Corporation), Monoclonal Antibodies to Fibronectin (Santa Cruz), Monoclonal Antibodies to TGase2 (Shin, DM et al. J.) ... Biol Chem 279: 15032-15039 (2004)), PARP (poly (ADP-ribose) single polyclonal antibody for the monoclonal antibody (Cell signaling), and phosphorylated Smad3 to the polymerase) (Wilkes, MC et al , Mol. Cell. Biol. 23: 88788889 (2003)) was used to analyze the expression of proteins.

3A-3B are Western blotting results showing that TGFβ1 but not TGFβ2 increases expression of FN (pirbonectin) in a dose dependent manner. As shown in FIG. 3A, human lung fibroblasts (IMR90) were treated with TGFβ1 or TGFβ2 and the expression of FN or α-smooth muscle actin (α-SMA), a molecular marker of fibrosis, was examined. Treatment with TGFβ1 for 24 hours resulted in concentration-dependent increases in FN and α-SMA. In contrast, when treated with TGFβ2, α-SMA was increased, but FN expression was not changed. The expression of TGase was little changed by 24 hours of treatment with both TGFβs. As can be seen in Figure 3b, the intracellular activity of TGase was increased by 48 hours of treatment with TGFβ1. These results show that TGFβ1 is a major isoform that induces FN in lung fibroblasts.

Example  6: Extracellular  Substrate analysis

To determine the amount of fibronectin (FN) deposited on the extracellular matrix (ECM), human primary lung fibroblasts, IMR90 cells, were cultured in 10-cm tissue culture dishes, 0.1% (w / v) Soluble in 1 ml of sodium dioxysulfate-2 mM EDTA (DOC-EDTA). DOC-EDTA-soluble fractions were stored for protein analysis (BCA analysis), and insoluble residues remaining on the plate, consisting mainly of ECM, were used for further experiments. For immunoassay, DOC-EDTA-insoluble protein was blocked with milk-Tween-PBS, followed by 0.2 μg / ml of monoclonal anti-TGase antibody CUB7042 or rabbit anti-probronectin (clone IST-1, Sigma) The reaction was reacted with 1: 1000 dilution overnight at 4 ° C. The deposited amount of each protein was determined by reaction with O-phenylenediamine dihydrochloride (Sigma). Absorbance was measured at 490 nm using a microplate spectrophotometer (Molecular Devices) to quantify.

4A-4F show that TGFβ1-induced activation of TGase2 promotes the deposition of FN in ECM. In FIG. 4A, TGase2 intracellular activity was induced by 48 hours (not 24 hours) treatment of TGFβ1. The increase in TGase2 activity was extinguished by treatment with cystamine (TGase inhibitor, Sigma) or N-acetylcysteine (NAC) (2.5 mM).

In FIG. 4B, cystamine and NAC inhibited TGFβ1-mediated induction of FN and α-SMA, which demonstrates that activation of TGase2 influences the expression of fibrous components. 1. NT (not treated); 2. TGFβ1 treatment; 3. TGFβ1 and cystamine treatment; 4. TGFβ1 and NAC treatment.

On the other hand, TGase2 is known to crosslink ECM molecules such as collagen I, III and IV, and fibronectin, resulting in aggregation of proteins resistant to proteolytic enzymes. To investigate whether activated TGase2 induced the deposition of ECM molecules, the amount of fibronectin in the detergent non-soluble matrix was measured. As can be seen in FIG. 4C, treatment with 1 ng / ml of TGFβ1 increased the amount of FN bound to the detergent insoluble matrix.

4D shows the deposition of FN by treatment with TGFβ1 at 4 ng / ml.

This deposition of FN is inhibited by cystamine or NAC (FIGS. 4C and 4D), and the results indicate that the transamidation activity of TGase2 crosslinks ECM molecules.

4E demonstrates the role of TGase2 by measuring the amount of BP-binding protein, and the amount of this BP-binding protein was reduced by cystamine or NAC.

As can be seen in FIG. 4F, the binding of TGase 2 itself to ECM was not observed.

The experimental results described above demonstrate that TGFβ1-induced activation of TGase 2 leads to fibrous deposition of ECM molecules via a transamidation reaction.

Example  7: cell proliferation and Apoptosis  analysis

The increase in cell number was analyzed according to the manufacturer's protocol using the MTT assay kit (Roche), and expressed as an increased fold compared to samples not treated with TGFβ1. Cell proliferation assay was performed according to the manufacturer's protocol using Brd-U binding kit (Roche). For apoptosis analysis, we measured the sub-G1 fraction or measured caspase activity by FACS analysis. Caspase activity was determined using a chromogenic substrate, Ac-DEVD-pNA for caspase 3, and Ac-LEVD-pNA for caspase 9 (A.G. Scientific, Inc.). Cell extracts were prepared by freeze-thaw in Lysis buffer (100 mM HEPES, pH 7.5, 0.1% CHAPS, 0.1% Triton X-100, 100 mM EDTA), followed by centrifugation (12,000 xg, 10 minutes, 4 ° C). It was. Cell extracts (30 μg) were added to assay buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT) containing chromogenic substrate (200 μM) and reacted at 37 ° C. for 4 hours. . Caspase activity was quantified by measuring absorbance at 490 nm and expressed as relative activity compared to untreated cells (negative control).

5A-5B show that cystamine and NAC inhibit TGFβ-induced proliferation of lung fibroblasts. Increased proliferation of fibroblasts after injury is a major cause of fibrosis. As can be seen in Figure 5a, the cell number of IMR90 was increased by the treatment of TGFβ1. The effect of TGFβ1 on cell proliferation could be observed more clearly at 48 hours after treatment. The number of fibroblasts was not affected by the treatment of TGFβ2.

In FIG. 5B, IMR90 cells were treated with TGFβ1 in the presence or absence of cystamine or NAC. Both cystamine and NAC strongly inhibited the proliferation of lung fibroblasts by TGFβ1.

The experimental results demonstrate that TGase2 contributes to inducing fibrosis by promoting the proliferation of lung fibroblasts.

6A-6B show that cystamine or NAC does not affect apoptosis of TGFβ-treated cells. Apoptosis of TGFβ-treated cells in the presence or absence of cystamine or NAC was investigated by measuring PARP cleavage (FIG. 6A) or sub-G1 fraction (FIG. 6B). In TGFβ-treated cells, TGFβ and cystamine treated cells, and TGFβ and NAC treated cells, there were no significant differences in PARP cleavage or sub-G1 fractions.

Thus, it can be seen that cystamine and NAC do not adversely affect cells.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is an image showing the results of Mason trichrome staining analysis of the involvement of TGase 2 in fibrosis. B6, C57BL / 6 wild type mice; TGase2 ^null , TGase 2-deficient (TGase 2 ^-/- ) mice; BLM, bleomycin.

Figure 2 is an image showing the results of Mason trichrome staining analysis of the effect of inhibiting fibrosis development of N-acetylcysteine (NAC) or cystiamine. BLM 1.5 and BLM 2.2 represent bleomycin doses of 1.5 mg / kg and 2.0 mg / kg. NAC, N-acetylcysteine; CYST, cystiamine.

3A-3B show that TGFβ1 but not TGFβ2 increases the expression of FN (pirbonectin) in a quantity dependent manner. In FIG. 3A, human lung fibroblasts (IMR90) were treated with TGFβ1 or TGFβ2 for 24 hours and the expression of FN or α-smooth muscle actin (α-SMA), a molecular marker of fibrosis, was examined. 3B shows the results of 48 hours of treatment with TGFβ1. Intracellular activity of TGase was increased by 48 hours of treatment with TGFβ1. The numbers above the gel photograph are the throughput of TGFβ1 or TGFβ2.

4A-4F show that TGFβ1-induced activation of TGase2 promotes the deposition of FN in ECM. NT, not treated; Cyst, cystamine. In FIG. 4A, TGFβ1 was treated for 24 hours or 48 hours in the presence or absence of cystamine or NAC, and TGase2 intracellular activity was measured. 4B analyzes the effect of cystamine or NAC on the induction of TGFβ1-mediated expression of FN and α-SMA. 1. NT (not treated); 2. TGF β1 treatment; 3. TGFβ1 and cystamine treatment; 4. TGFβ1 and NAC treatment. 4C-4D show the results of analyzing the extent of FN invasion by treatment with TGFβ1 in the presence or absence of cystamine or NAC. 4E elucidates the role of TGase2 by measuring the amount of BP-binding protein. Figure 4f is the result of analyzing the deposition of TGase 2 itself.

5A-5B show that cystamine and NAC inhibit TGFβ-induced proliferation of lung fibroblasts. The amount of cystamine and NAC treated was 1 mM (Cystamine) and 2.5 mM (NAC), respectively.

6A-6B show that cystamine or NAC does not affect apoptosis of TGFβ1-treated cells. Apoptosis of TGFβ1-treated cells in the presence or absence of cystamine or NAC was investigated by measuring PARP cleavage (FIG. 6A) or sub-G1 fraction (FIG. 6B). The throughput of TGFβ1 is 1 ng / ml and the amounts of cystamine and NAC treated are 1 mM (Cystamine) and 2.5 mM (NAC), respectively.

Claims (5)

(a) a pharmaceutically effective amount of a transglutaminase 2 inhibitor or N-acetylcysteine (NAC); And (b) a pharmaceutically acceptable carrier. The method of claim 1, wherein the TGase 2 inhibitor is cystamine, cystiamine, monodansil cardaberine, putricin, histamine, methyl amine, iodoacetamide, 8-phenyl propionyl thiocholine, monoamine, diamine, gamma- Amino benzoic acid, 1- (5-aminopentyl) -3-phenylthiourea, N-benzyloxy carbonyl, 5-deazo-4-oxonorvaline, p-nitrophenylester, glycine methyl ester, CuSO ₄ and tol Composition selected from the group consisting of butamides. The composition of claim 2, wherein the TGase 2 inhibitor is cystamine or cystiamine. Screening methods for the prevention or treatment of fibrosis comprising the following steps: (a) contacting a test substance with a transglutaminase 2 protein; And (b) measuring the activity of the TGase 2. The method of claim 4, wherein the TGase 2 is present in the cell or extracellularly.

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