WO2005039570A1

WO2005039570A1 - TGF-β INFORMATION TRANSFER PATHWAY INHIBITOR

Info

Publication number: WO2005039570A1
Application number: PCT/JP2004/016370
Authority: WO
Inventors: Soichi Kojima; Wakako Kondo; Hideaki Kakeya; Hiroyuki Osada; Yasuharu Sakamoto; Tadashi Nakata
Original assignee: Riken
Priority date: 2003-10-28
Filing date: 2004-10-28
Publication date: 2005-05-06
Also published as: JPWO2005039570A1; JP4688680B2

Abstract

A TGF-β information transfer pathway inhibitor comprising a low-molecular compound capable of inhibiting TGF-β information transfer pathway. There is provided a TGF-β information transfer pathway inhibitor comprising as an active ingredient a compound of the formula: (1) wherein R1 is hydroxymethyl, etc. and R2 is p-methoxyphenyl, etc.

Description

Specification

TGF-β signaling pathway inhibitor

The present invention relates to a TGF- 3 signaling pathway inhibitor containing cytoxazone or a derivative thereof as an active ingredient, and a therapeutic and / or prophylactic agent for a TGF-β-related disease. Background art

TGF- / 3 is a common etiologic agent in liver diseases such as liver fibrosis, cirrhosis, hepatitis, or hepatic regeneration failure, and attempts to treat or prevent the disease by suppressing its activity have been made. Has been done. For example, antibody therapy using neutralizing antibodies against TGF-j3, or gene therapy using dominant negative TGF-receptor gene or soluble TGF-β receptor gene, can stop the action of TGF-J3 at the entrance. Reported reports (Schuppan et al. Digestion 59: 385-390, 1998; Qi et al. Proc Natl Acad Sci USA 96: 2345-2349, 1999; and Ueno et al. Hum Gene Ther 11: 33-42, 2000). On the other hand, the present inventors have focused on the fact that TGF-j3 is produced as a latent inactive molecule and is converted to an active molecule by protease (TGF- / 3 activation reaction). Although animal models have shown the possibility of inhibiting disease by inhibiting the TGF j3 activation reaction using a synthetic protease inhibitor or an antibody (Akitaetal. Gastroenterology 123: 352-364, 2002), It has been revealed that the activation reaction is specific to a disease state, a fibrous tissue, and an isoform, and that the adaptation symptoms are limited. On the other hand, for cytoxazone (CXZ), its purification method, the activity of regulating the cytokin production of immune cells, and its synthesis method have been reported (Kakeya H et al., J Antibio 1998; 51: 1126-1128; Kakeya H et al.). , J Org Chem 1999; 64: 1052-1053; JP-A-11-209355; and JP-A-2000-86639. Power and other biological activities were unknown. Disclosure of the invention

TGF-β strongly promotes the production of extracellular matrix by mesenchymal cells and suppresses the proliferation of epithelial cells, resulting in hepatic fibrosis, cirrhosis, atherosclerosis, pulmonary fibrosis, dermal fibrosis, renal failure, It is a homodimer with a molecular weight of 25 kD and has a variety of biological activities, including the formation of pathological conditions of sclerosing diseases such as uterine fibroids and suppression of the function of immunocompetent cells. As described above, studies in animal models using neutralizing antibodies to TGF- / 3 have revealed that sclerosing diseases can be prevented and treated by suppressing the action of TGF-β. Furthermore, development of gene therapy using mutant TGF-receptors has been attempted. However, in antibody therapy and gene therapy, problems remain in terms of the required amount and administration method, and clinical application is not immediately possible, and the development of small molecule inhibitors based on the mechanism of action of TGF-) 3 has been developed. Is desired. That is, an object of the present invention is to provide a TGF-β signaling pathway inhibitor comprising a low-molecular compound that can inhibit the TGF-j3 signaling pathway.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, cytoxazone represented by the formula (1) or a derivative thereof (hereinafter, also abbreviated as CXZ) as defined in the present specification is used for transmitting TGF- The present invention has been completed by finding for the first time inhibition of the pathway.

That is, according to the present invention, the following formula (1):

(Wherein R ¹ is a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl group, a hydroxypropyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- or para-hydroxyphenyl group An ortho-, meta- or para-nitrophenyl group, an ortho-, meta- or para-aminophenyl group, a linear or branched alkyl group having 1-20 carbon atoms, 1-20 carbon atoms And R ² represents a hydroxymethyl group, a hydroxyxyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-alkyl group. , Meta- or para-hydroxyphenyl, ortho-, meta- or para-nitrophenyl, ortho-, meta- or para-aminophenyl, benzyl, ortho-, meta- or para-methoxybenzyl, ortho-, meta- Or para-hydroxybenzyl group, ortho-, meta- or para-nitrobenzyl group, ortho-, meta- or para-aminobenzyl group, linear or branched alkyl group having 1 to 20 carbon atoms, carbon number 1 to 20 linear or branched alkoxy groups. A TGF-signalling pathway inhibitor comprising a compound represented by the formula (1) as an active ingredient.

Preferably, the TGF-i3 signaling pathway inhibitor of the present invention inhibits the TGF-i3 signaling pathway by suppressing the phosphorylation of Smad2.

According to another aspect of the invention, the following formula (1):

(In the formula, R ¹ is a hydroxymethyl group, a hydroxyshethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- or para-hydroxy group. Droxyphenyl group, ortho-, meta- or para-nitrophenyl group, ortho-, meta- or para-aminophenyl group, linear or branched alkyl group having 1 to 20 carbon atoms, linear chain having 1 to 20 carbon atoms R ² represents a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an o / to, a meta- or para-methoxyphenyl group, an ortho-, Meta- or para-hydroxyphenyl group, ortho-, meta- or para-nitrophenyl group Ortho-, meta- or para-aminophenyl, benzyl, ortho-, meta- or para-methoxybenzyl, ortho-, meta- or para-hydroxybenzyl, ortho-, meta- or para-nitrobenzyl , Ortho, Meta

— Or a para-aminobenzyl group, a linear or branched alkyl group having 1 to 20 carbon atoms, or a linear or branched alkoxy group having 1 to 20 carbon atoms. A therapeutic and / or prophylactic agent for a TGF-β-related disease, comprising a compound represented by the formula (1) as an active ingredient.

Preferably, the TGF-] 3-related disease is liver fibrosis-cirrhosis, hepatitis, liver regeneration failure, arteriosclerosis, pulmonary fibrosis, dermal fibrosis, renal failure, or uterine fibroids.

Preferably, R ¹ is a hydroxymethyl group and R ² is a para-methoxyphenyl group. Particularly preferably, the compound used in the present invention is a compound represented by the following formula (2).

Brief Description of Drawings

FIG. 1 shows the TGF-signaling pathway.

A: Factors and factors that increase the synthesis and secretion of TGF-

T G F-3, vitamin A, antiestrogen, bleomycin,

Dexamethasone, virus infection, lymphocyte activation, fracture, liver fibrosis, myocardial infarction, liver damage

B: Factor that releases TGF—] 3 from extracellular matrix

Elastase, chymase, plasmin, thrombin

C: factor that activates TGF-

Four

Replacement form (Rule 26) Acid, alkali, heat, denaturant, reactive oxygen species (ROS),

Endoglycosidase, thrombospondin, serine protease, integrin, matrix meta-oral protease

D: Factors that cause TGF-activation in cells

Mixed culture, vitamin Α, vitamin D, antiestrogen, bleomycin, dexamethasone, lipopolysaccharide, IgG, interferon, carcinogenesis

E: Factors that increase the expression of TGF_j3 receptor

Vitamin A, liver fibrosis

F: factors that reduce the expression of TGF-j3 receptor

Carcinogenesis, extracellular matrix

FIG. 2 shows inhibition of liver pathogenesis by inhibition of TGF- / 3 activation using protease inhibitors.

FIG. 3 shows the structure of cytoxazone. R 1 represents a hydroxymethyl group, and R 2 represents a p-methoxyphenyl group.

FIG. 4 shows suppression of hepatic stellate cell activation by cytoxazone through suppression of TGF-J3 activity. A) morphological changes, B) suppression of target gene expression, C) suppression of TGF-] 3-dependent transcriptional activity

FIG. 5 shows that the amount of Smad2 and the amount of phosphorylated Smad2 are reduced by cytoxazone treatment.

FIG. 6 shows the reduction of the amount of Smad-target gene complex by cytoxazone treatment. FIG. 7 shows suppression of TGF-β-dependent transcription activation activity by cytoxazone treatment in hepatic stellate cells and mink lung epithelial cells.

FIG. 8 shows suppression of signaling downstream of the TGF-] 3 receptor by cytoxazone treatment.

FIG. 9 shows suppression of Smad activity by cytoxazone treatment.

FIG. 10 shows the effects of cytoxazone treatment on Smad mRNA expression and protein content. FIG. 11 shows the effect of cytoxazone treatment on nuclear localization of Smad protein. FIG. 12 shows suppression of transcriptional activation activity of procollagen gene promoter by cytoxazone treatment.

FIG. 13 shows the recovery of liver regeneration by cytoxazone in mice.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

Transforming Growth Factor (TGF)-is responsible for the pathogenesis of sclerosing diseases and suppresses the function of immunocompetent cells, while suppressing the overproduction of proteases to prevent the breakdown of lung tissue and emphysema. It is a 25 kD molecular weight homodimeric multifunctional cytokine that has a variety of biological activities, such as preventing or inhibiting the growth of cancer cells. TGF- / 3 is produced as an inactive latent form with a molecular weight of about 300 kD that cannot bind to the receptor, and is activated on and around the target cell surface to become an active form that can bind to the receptor and exerts its action (Figure 1 ).

The action of TGF-β in target cells is transmitted through a series of protein phosphorylation pathways that are responsible for signaling Smads. First, when activated TGF- binds to type II TGF-β receptor on the surface of target cells, two types of type II receptor and type I TGF-β receptor

A receptor complex consisting of two molecules is formed, and the type II receptor phosphorylates the type I receptor. Next, when the phosphorylated type I receptor phosphorylates Smad2 or Smad3, phosphorylated Smad2 or Smad3 forms a complex with Smad4 and translocates to the nucleus, where CAGA located in the target gene promoter region is present. It binds to a target sequence called box and induces the transcriptional expression of the target gene together with coactivator (Fig. ι). The present inventors believe that latent TGF- / 3 is activated by protease cleavage in the liver, and exhibits potent fibrogenesis-inducing ability and hepatocyte proliferation-inhibiting ability. It was found that it causes regenerative failure, and it was reported that inhibition of TGF- activation by using a protease inhibitor can suppress the formation of pathology (Fig. 2) (Okuno M et al., Gastroenterology 2001; 120: 1784-1800). ; Akita K Et al., Gastroenterology 2002; 123: 352-364). Based on these findings, we have reported the development of a therapeutic method using an antibody against protease, and the production of an antibody recognizing a specific activation reaction in disease state, tissue, and isoform (Japanese Patent Application No. 2002-057253). , Japanese Patent Application No. 2003-313014).

The present inventors subsequently hoped that a combined use of a TGF-signaling inhibitor and a protease inhibitor that suppresses the activation of TGF-β would produce a synergistic inhibitory effect on disease state formation. Screening of TGF- / 3 signaling inhibitor was performed. As a result, a cytoxazone having an oxazolidinone ring (FIG. 3; abbreviated as cytoxazone: CXZ) was found. R1 is a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- or para-hydroxyphenyl group, an ortho-, meta- or A paranitrophenyl group, an ortho-, meta-, or para-aminophenyl group, a linear or branched alkyl group having 1 to 20 carbon atoms, and a linear or branched alkoxy group having 1 to 20 carbon atoms. R2 represents a hydroxymethyl group, a hydroxyxyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho, a meta- or para-methoxyphenyl group, an ortho-, meta-, or para-hydroxyphenyl group , Ortho-, meta- or para-nitrophenyl group, ortho , Meta- or para-aminophenyl, benzyl, ortho-, meta- or para-methoxybenzyl, ortho-, meta- or para-hydroxybenzyl, ortho-, meta- or para-nitrobenzyl, ortho-, meta- A mono- or para-aminobenzyl group, a linear or branched alkyl group having 1 to 20 carbon atoms, or a linear or branched alkoxy group having 1 to 20 carbon atoms.

Cytoxazone is an antibiotic produced and secreted by Streptomyces and is known to suppress the production of cytokines IL4 and IL10 from immunocompetent Th2 cells (Kakeya H et al., J Antibio 1998; 51 Kakeya H et al., J Org Chem 1999; 64: 1052-1053). The discovery and purification of cytoxazone or a derivative thereof is described in JP-A-11-209355, and its organic synthesis is described in JP-A-2000-86639, And Tetrahedron Lett 1999; 40: 4203-4206. As described above, the compound represented by the formula (1) used in the present invention is a known compound, and can be obtained or synthesized according to the description in the above literature.

The present invention relates to a TGF-] 3 signaling pathway inhibitor, and a therapeutic and / or prophylactic agent for a TGF-β-related disease, comprising a compound represented by the formula (1) defined herein as an active ingredient. Things. Hereinafter, these may be collectively referred to as the drug of the present invention.

As the drug of the present invention, the compound represented by the formula (1) may be used alone as it is, but it is usually supplied in the form of a pharmaceutical composition using a pharmaceutically acceptable pharmaceutical additive. preferable. The medicament of the present invention can be administered orally or parenterally. Pharmaceutically acceptable pharmaceutical additives referred to herein include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners , Thickeners, flavoring agents, solubilizers or other additives.

Pharmaceutical compositions suitable for oral administration include, for example, tablets, granules, capsules, powders, solutions, suspensions, and syrups. Pharmaceutical compositions suitable for parenteral administration include: For example, injections, drops, suppositories, transdermal absorbents, etc. can be mentioned, but the form of the drug of the present invention is not limited to these. Examples of the form of parenteral administration include various administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection. Injectables may be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions or emulsions. You can also. Such injections can be sterilized by filtration sterilization through a pateria-retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of ready-to-use preparations. That is, a sterile solid composition can be obtained by freeze-drying or the like and dissolved in sterile distilled water for injection or another solvent before use.

The agent of the present invention can be administered to mammals including humans. The drug of the present invention Dosage should be adjusted according to the patient's age, sex, weight, symptoms, route of administration and other conditions.Generally, 0.001 mg Zk to 1000 mg / kg per adult per day , Preferably in the range of 0.01 mg / kg to 100 mg Z kg, particularly preferably in the range of lm g Z kg to 50 mg / kg, for example, 20 mg. Z kg. The above dose of the drug may be administered daily or at intervals of several days, for example, every 1 to 4 days.

The agent of the present invention can be used as a TGF-3 signaling pathway inhibitor, and can be used, for example, as a therapeutic and / or prophylactic agent for TGF-related diseases. Examples of the TGF-] 3-related disease include a disease in which the pathogenesis factor is TGF-] 3 (for example, a liver disease in which the pathogenesis factor is TGF-β). Specific examples of TGF-β-related diseases include hepatic fibrosis, cirrhosis, hepatitis, hepatic regeneration failure, arteriosclerosis, pulmonary fibrosis, dermal fibrosis, renal failure, or uterine fibroids. In addition, a therapeutic and / or prophylactic agent for a TGF-] 3-related disease is to be interpreted in a broad sense including suppressing or preventing the onset and / or progression of the disease.

The therapeutic effect of the agent of the present invention on the symptoms of a TGF-] 3-related disease can be tested and examined by administering it to a disease model animal according to a conventional method. For example, for liver diseases such as hepatitis (for example, viral hepatitis (type I, type II, type C, type III, etc.)), cirrhosis, or drug-induced liver injury, the model animals already reported can be used. (For example, see “Tests for the preparation of disease-specific model animals and development of new drugs · Experimental methods”, pp. 119-129 and p. 349-358, 1993, Technical Information Association, etc.).

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example

Example 1: Inhibition of hepatic stellate cell activation by CXZ

According to a previously reported method (Okuno M et al., Gastroenterology 2001; 120: 1784-1800), hepatic stellate cells were isolated from the liver of male Wistar rats (weight 350-400 g; Japan Clea). Away, 10 cm culture plastic dishes or-out the ³ · 5 ^Χ 10 ⁶ cells per sheet, 10% © Shi calf serum (FCS; Invitrogen Corporation) Dulbecco's modified Eagle's culture areas, including (DMEM; Invitrogen Corp. ) The cells were cultured. Compounds having the following structure from the next day

(Hereinafter abbreviated as CXZ), changed to a serum-free medium, cultured for another 6 days, and cultured on a plastic dish to observe the effect of the drug on hepatic stellate cell activation induced by microscopy. Was observed.

As shown in Fig. 4A, CXZ suppressed activation of stellate cells (morphological change accompanied by disappearance of oil droplets containing vitamin A). The effect is dose-dependent manner, the optimum value is one fc at a final concentration of 150 μ g / ml 7 This ₀

Next, the expression levels of several genes [TGF-] 31, type I procollagen α2, and α-smooth muscle actin] whose expression is known to be induced by stellate cell activation The changes in DNA were examined by the reverse transcription polymerase chain reaction (RT-PCR) method. Changes in the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were measured as an internal standard. Extract and purify RNA from cells that have been observed under a microscope using QIAGEN's REASY RNA Purification Kit, and use M-MLV reverse transcriptase (manufactured by Invitrogen) according to the manufacturer's instructions. stained with Echijiumu Puromaido by passing a (1 mu _beta) reverse transcribed into cDNA, each cDNA fragment using the following sense and antisense primers to the cDNA of this as铸型to then amplified by PCR to 2% Agarosugeru, The relative amounts were compared and observed under UV light.

Rat TGF-β 1

5'-GGACTCTCCACCTGCAAGAC-3 '(SEQ ID NO: 1)

Ten

Replacement form (Rule 26) 5, -CTCTGCAGGCGCAGCTCTG-3 '(SEQ ID NO: 2)

Rat type I procollagen α 2

5'-CCTGGTCCTCATGGTTCTGT-3 '(SEQ ID NO: 3)

5'-CTGGTCAGCCCTGTAGAAGC-3 '(SEQ ID NO: 4)

Rat _alpha smooth muscle actin

5, -ACTGGGACGACATGGAAAAG-3 '(SEQ ID NO: 5)

5, -GTCCAGAGCGACATAGCACA-3 '(SEQ ID NO: 6)

Rat glyceraldehyde-3-phosphate dehydroza "Ize (GAPDH)

5'-GCTGAGAATGGGAAGCTGGT-3 '(SEQ ID NO: 7)

5'-CCTTGGCAGCACCAGTGGAT-3 '(SEQ ID NO: 8)

As shown in Fig. 4 C, CGF-treated cells (CXΖ) showed higher levels of TGF-β1 and type I procollagen a 2 (procollagen) than control cells (treated with 0.5% DMS0 as a solvent). ), The amount of α-smooth muscle actin (a SMA) gene was decreased. At this time, there was no change in the amount of the internal standard dalyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

Since the activation of hepatic stellate cells depends on TGF-β produced by the stellate cells themselves, it was determined whether the inhibitory effect of CXZ on stellate cell activation was due to the suppression of TGF-β activity. The activity of TGF-β was examined by a reporter assay using mink lung epithelial (MLE) cells, which are most commonly used, and rat hepatic stellate cells.

Seed 1.0 x 10 ⁵ MLE cells per 3.5 cm culture plastic dish, and cultured in DMEM (Invitrogen) containing 10% FCS (Invitrogen). -Luciferase in the promoter of pPAI-1 which is a typical target gene of

(Puc) reporter-fused pPAI-1—Luc with Lipofectamine Plus reagent

(Invitrogen) was used to perform 500 ng transfection per dish. After 2 hours of transfection, add 1 ng / ml human recombinant TGF-β1

(R & D), 150 μg / ml CXZ, or a serum-free medium containing these combinations, and pre-cultured for 2 days to prepare a cell extract, and then luciferase activity in the extract. Was measured with a luminometer (manufactured by Packard) using the Dual Lucif erase Assay Kit from Promega. In order to detect the efficiency of the transfection, transfection was carried out simultaneously with Mycobacterium luciferase, and the measurement results were corrected with the value of Mycobacterium luciferase and the amount of protein in the extract, and then the value of TGF- / 31 alone was corrected. The relative value was set to 100% (FIG. 4C, lanes 1 to 3). The amount of protein was determined using PIERCE BCA Assay Kit and standard serum albumin. After isolation of stellate cells from rat liver, seed 4.0 × 10 ⁵ cells per 3.5 cm plastic dish, and replace with serum-free medium containing CXZ the next day after culturing in DMEM containing 10% FCS. After culturing for another 4 days, 500 ng of pPAI-1-Luc per dish was transfected using the Lipofectamine Plus reagent in the same manner as for MLE cells. An extract was prepared, and the luciferase activity in the extract was measured. The results are expressed as relative values with the value of CXZ-untreated cells (stimulated with endogenous TGF-β) as 100%, after correcting for the value of Mycobacterium luciferase and the amount of protein in the extract. (Figure 4C, lanes 4 and 5). As shown in FIG. 4C, CXZ showed remarkable suppression of Luc activity induced by TGF-j3 (lane 2 vs. lane 3 (MLE cells); lane 4 vs. lane 5 (star cells)). This result indicates that CXZ suppresses the activity of TGF- / 3, strongly suggesting that CXZ has an activity of inhibiting the signaling pathway of TGF-J3. Example 2: Inhibition of TGF-β signaling pathway by CXZ

As described in Example 1, the effect of TGF-β is on the phosphorylation of Smad by the receptor, the translocation of the phosphorylated Smad complex to the nucleus, and the transcription of the phosphorylated Smad complex by binding to the target gene promoter. Is transmitted by the route of promotion. Therefore, to determine whether CXZ inhibits the TGF- signaling pathway, the total amount of Smad2, the amount of phosphorylated Smad2, and the amount of phosphorylated Smad complex bound to the target gene promoter were analyzed by Western plot analysis and gel shift analysis. It was examined from the measurement results of Sey.

First, the western blot analysis was used to determine the total amount of Smad2 and the amount of phosphorylated Smad2. The result is explained. Cell extracts were prepared from rat hepatic stellate cells isolated and cultured as in the experiment in Figure 4A and treated with cytoxazone at a final concentration of 100 / zg / ml and 200 ^ g / ml using 1XSDS sample buffer. After electrophoresis on a 10% polyacrylamide gel, it was electrophoretically transferred to a PVDF (polybutene difluoride) membrane. Estane blot analysis of proteins on the membrane was performed using Smad2-recognized mouse monoclonal antibody (final concentration 1 μg / ml; Pharmingen) and phosphorylated Smad2-recognized ゥ sagi polyclonal antibody (final concentration 2 μg / ml; Upstate Biotechnology) and a combination of horseradish luoxidase-conjugated goat anti-mouse or ephedra IgG antibody (final 1: 750 dilution) according to a previously reported method (Okuno M et al., Gastroenterology 2001; 120: 1784-1800). ). Bands were detected on the Amersham (Buckinghamshire, UK) ECL system.

As a result, as shown in Fig. 5, the CXZ-treated cells (lanes 2 and 3) showed higher CXZ levels than the control cells (cells treated with 0.5% DMS0 as the solvent: lane 1). The total amount of Smad2 and the amount of phosphorylated Smad2 decreased depending on the concentration. At this time, the internal standard GAPDH amount had not changed.

Next, the results of measurement of the amount of phosphorylated Smad complex bound to the target gene promoter by gel shift assay will be described. A nuclear extract was prepared from rat hepatic stellate cells isolated, cultured, and treated with drugs as described in the experiment in Fig. 4A according to the method described above (Shimada J et al., Mol Endocrinol 2002; 15: 1677-1692). Radiolabeled CAGA box sequence (binding sequence of phosphorylated Smad complex present in PAI-1 promoter) After incubating with oligonucletide containing ice on ice, phosphorylation on 4¾ polyacrylamide gel After separating the Smad-CAGA box complex and the free CAGA box, the amount of the phosphorylated Smad-CAGA box complex was measured by autoradiography.

As a result, as shown in Fig. 6, the amount of phosphorylated Smad-CAGA box complex in CXZ-treated cells (lane 3) was lower than that in control cells (cells treated with 0.5% DMS0 as a solvent: lane 2). Had decreased. Lane 1 contains the unlabeled CAGA box sequence. This is the result when gonurequotide was added to the nuclear extract derived from the control cells, indicating that the obtained band was a specific band.

Furthermore, when the change in the amount of TGF-β receptor was measured by the RT-PCR method, it was found that the amount of TGF-receptor did not change in the CXZ-treated cells. In addition, CXZ did not affect the activation of latent TGF-β.

From the above results, it was found that CXZ suppressed the signaling pathway of TGF-) 3 by suppressing the phosphorylation of Smad2. Example 3: Inhibition of TGF-β-dependent transcriptional activation activity by CXZ in hepatic stellate cells and mink lung epithelial cells

Twenty-four hours after isolation, hepatic stellate cells cultured in a 35-mm dish were treated with or without 150 g / ml of CXZ for 3 days, and pRL-CMV (Renilla luciferase, 50 ng / dish). ) Together with ρΡΑΙ-1-Luc (500 ng / dish), a PAI-1 promoter luciferase expression vector, or 9CAGA-Luc (500 ng / dish), a vector containing 9 copies of CAGA sequence (See the left graph in Fig. 7). The transfected cells were treated for another 24 hours under the same conditions. 3 5 mm dishes on 2 X 1 0 ⁵ mink lung epithelial cells (MELC) 2 4 hours after plating, cells were treated for 3 days in the presence or absence of CXZ of 0.99 mu g / ml Then, transfection was performed using a combination of 9CAGA-Luc (500 ng / dish) and pRL-CMV (50 ng / dish) (the right graph in FIG. 7). The day after transfection, cells were treated with 5 ng / ml recombinant human TGF-1 for 16 hours in the presence or absence of 150 μg / ml CXZ. Cell lysates were prepared from both cells, luciferase activity in each lysate was measured, changes in firefly luciferase activity were calculated, normalized to the luciferase activity of CXZ-untreated control cells, and plotted. Fig. 7 shows the results. As can be seen from the results in Fig. 7, the luciferase reporter transmission driven by nine CAGA boxes, the target motif to which the Smad2 / Smad4 complex or Smad3 / Smad4 complex binds, which is a signaling factor for TGF- It was demonstrated that CXZ suppressed the TGF-J3-dependent transcriptional activation activity using the offspring. Example 4: Inhibition of signaling downstream of the TGF- / 3 receptor

3 5 mm dishes on 2 X 1 0 ⁵ mink lung epithelial cells (MELC) 2 4 hours after plating, was treated in the presence or absence of cells of 150 g / ml CXZ 3 days, Roroarufaiota Transfections were performed using a combination of -1-Luc (500 ng / dish) and pRL-CMV (50 ng / dish) and a vector that constitutively expresses the active TGF-jS receptor I. The transfected cells were further treated with soil CXZ under the same conditions for another 24 hours. Thereafter, luciferase activity was measured in the same manner as in Example 3.

Fig. 8 shows the results. The results of FIG. 8, which compares the effects of cytoxazone under the presence of the TGF- / 3 receptor, demonstrate that cytoxazone exerts an effect on signaling downstream of the TGF-) 3 receptor. Example 5: Smad activity suppression

Twenty-four hours after isolation, 35 mm dishes of hepatic stellate cells were treated in the presence or absence of 150 g / ml CXZ for 3 days, and 9CAGA-Luc (500 ng / dish) and pRL-CMV ( A combination of 50 ng / dish) and several combinations of Smads 2, 3, and 4 (200 ng each Zdish) were transfection. The transfected cells were treated for another 24 hours under the same conditions. Cell lysates were prepared, luciferase activity was measured in each lysate, changes in firefly luciferase activity were calculated, plotted after normalization to Renilla luciferase activity of CXZ-untreated control cells. The results are shown in FIG. When TGF- signaling is activated (phosphorylated), Smad2 or Smad3 present in the cytoplasm is phosphorylated to form a complex with Smad4, and phosphorylated Smad2 / Smad4 complex or phosphorylated Smad3 The mechanism by which the / Smad4 complex translocates to the nucleus and binds to the target sequence CAGA box, activates basic transcription factors together with other transcription factors and transcription mediators, and triggers transcription of the target gene (Fig. 1). The results in Figure 9 show the effect of CXZ on hepatic stellate cells that forcibly expressed Smad2, Smad3, and Smad4. CXZ suppresses the expression of Smad3 as shown in Example 6, which is rescued by forced expression of Smad3. In contrast, the expression of Smad2 does not change, but the activity of forced Smad2 is suppressed in Smad2 / Smad4 + Smad3 / Smad4 and Smad2 / Smad4 cells. (Including phosphorylation of Smad2). Example 6: Effect of Smad on mRNA expression and protein content

After treating primary hepatic stellate cells in the presence or absence of 150 / zg / ml CXZ for 4 days, cell lysate was prepared, mRNA was isolated, and the change in mRNA amount of Smad shown in RT- It was evaluated by PCR (left figure in FIG. 10).

Rad Smad2

5'-GCCCCAACTGTAACCAGAGA-3 '(SEQ ID NO: 9)

5'-CGCTCTGGGTTTTGACTAGC-3 '(SEQ ID NO: 10)

Rat Smad3

5'-CTGTGAGTTCGCCTTCAACA-3 '(SEQ ID NO: 11)

5'-TGTGAAGCGTGGAATGTCTC-3 '(SEQ ID NO: 12)

Rat Smad4

5'-AGTCCCTTCAAGCTGTCCTATTGTA-3 '(SEQ ID NO: 13)

5'-CGGGTAGATCTTGTGGACGGCGTCA-3 '(SEQ ID NO: 14)

Rat Smad7

5'-CCAACTGCAGACTGTCCAGA-3 '(SEQ ID NO: 15)

5'-AACCAGGGAACACTTTGTGC-3 '(SEQ ID NO: 16)

After treating HSC-T6 cells in the presence or absence of 150 μg / ml CXZ for 4 days, cell lysates were prepared, and total protein and phosphorylated protein of Smad 2 and Z or 3 were prepared. Western blot using anti-Smad 2/3 antibody and anti-phospho Smad 2/3 antibody (Figure 10 right).

As shown in FIG. 10, a significant difference was seen in Smad3 at the mRNA level, and a difference was seen in the Smad2 / 3 complex protein in the protein. Example 7: Effect of Smad protein on nuclear localization

After treating primary hepatic stellate cells in the presence or absence of 150 μg / ml CXZ for 4 days, the cells were fixed, stained with anti-Smad2 / 3 antibody, and observed with a confocal laser microscope. The results are shown in FIG. Smad2 / 3 was more concentrated in the nucleus in CXZ-untreated cells (control), while CXZ-treated cells were observed to spread throughout the cells. Example 8: Suppression of transcriptional activation activity of procollagen gene promoter

Primary liver stellate cells were treated for 3 days in the presence or absence of CXZ of _{150 μ § / πι1, pCollagen-} Luc (500 ng / dish)及Pi pRL - CMV Trang combinations (50 ng / dish) Sufuwekushiyon The treatment was continued for another 24 hours under the same conditions. Cell lysates were prepared, luciferase activity in each lysate was measured, changes in firefly luciferase activity were calculated, and plotted after normalization to Renilla luciferase activity of CXZ-untreated control cells.

The results are shown in FIG. The results in FIG. 12 indicated that CXZ suppressed the transcriptional activation activity of the procollagen gene promoter. Example 9: Restoration of liver regeneration by cytoxazone in mice

Mice pre-treated with LPS and partially hepatectomized were treated with CXZ (15 mg / kg body weight / 0; ip) (no control). 48 hours after the start of the partial hepatectomy, the liver was isolated, a section was prepared, stained with an anti-PCNA antibody, and observed with a light microscope. The results are shown in FIG. In Fig. 3, the nucleus stained brown is the regenerated cell nucleus, and the PCNA labeling index, which is the result of counting the number of nuclei, is 28.5 ± 3.74 in the control. In contrast, the value was 80.8 ± 3.89 in the CXZ-administered case. Cytosoxazone was shown to restore liver regeneration.

(Summary of Examples)

As described above, CXZ has the effect of blocking the TGF-β signaling pathway by decreasing the phosphorylated Smad responsible for TGF-β signaling, thereby inhibiting the action of TGF-β. β-related diseases, such as liver stellate cells, which cause liver cirrhosis. did. By using CXZ or its derivatives, it is possible to develop preventive and therapeutic drugs for TGF-related diseases such as various sclerotic diseases based on blockade of TGF- / 3 signaling pathway, which was difficult with conventional technology Can be expected. Industrial applicability

In the present invention, based on the finding that cytoxazone suppresses TGF-β-dependent activation of hepatic stellate cells, the present inventors have found inhibition of the TGF-signalling pathway as a new biological activity of cytoxazone or a derivative thereof. The use of a TGF-signalling pathway inhibitor containing cytoxazone or a derivative thereof as an active ingredient according to the present invention makes it possible to establish a method for treating and preventing TGF-related diseases.

Claims

The scope of the claims

1. The following equation (1):

(Wherein R ¹ is a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl 'group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- or para-hydroxyphenyl group) Group, ortho-, meta- or para-nitrophenyl group, ortho-, meta- or para-aminophenyl group, linear or branched alkyl group having 1 to 20 carbon atoms, straight-chained alkyl group having 1 to 20 carbon atoms Represents a linear or branched alkoxy group, and R ² is a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- Mono- or para-hydroxyphenyl, ortho-, meta- or para-nitrophenyl Ortho-, meta- or para-aminophenyl, benzyl, ortho-, meta- or para-methoxybenzyl, ortho-, meta- or para-hydroxybenzyl, ortho-, meta- or para-nitrobenzyl, It represents an ortho-, meta- or para-aminobenzyl group, a linear or branched alkyl group having 1 to 20 carbon atoms, and a linear or branched alkoxy group having 1 to 20 carbon atoms. A TGF-] 3 signaling pathway inhibitor comprising a compound represented by the formula (1) as an active ingredient:

2. The TGF—] 3 signaling pathway inhibitor according to claim 1, which inhibits the TGF— signaling pathway by suppressing phosphorylation of Smad2.

3. The following equation (1):

(Wherein R ¹ is a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- or para-hydroxyphenyl group, an ortho-hydroxyphenyl group, 1, meta- or para-nitrophenyl group, ortho-, meta- or para-aminophenyl group, linear or branched alkyl group having 1 to 20 carbon atoms, linear or branched chain having 1 to 20 carbon atoms R ² is a hydroxymethyl group, a hydroxyxethyl group, a hydroxypropyl group, a hydroxybutyl group, a phenyl group, an ortho-, meta- or para-methoxyphenyl group, an ortho-, meta- Or para-hydroxyphenyl, ortho-, meta- or para-nitrophenyl , Ortho-, meta- or para-aminophenyl, benzyl, ortho-, meta- or para-methoxybenzyl, ortho-, meta- or para-hydroxybenzyl, ortho-, meta- or para-nitrobenzyl Groups, ortho-, meta- or para-aminobenzyl groups, linear or branched alkyl groups having 1 to 20 carbon atoms, and linear or branched alkoxy groups having 1 to 20 carbon atoms. ) A therapeutic or Z- or prophylactic agent for a TGF-related disease, comprising a compound represented by the formula

4. The drug according to claim 3, wherein the TGF- / 3-related disease is liver fibrosis, cirrhosis, hepatitis, hepatic regeneration failure, arteriosclerosis, lung fibrosis, dermal fibrosis, renal failure, or uterine fibroids.

5. The drug according to any one of claims 1 to 4, which is an R ¹ force hydroxymethyl group and an R ² force paramethoxyphenyl group.

6. The drug according to any one of claims 1 to 5, wherein the compound represented by the formula (1) is a compound represented by the following formula (2).

21 Replacement form (Rule 26)