JP2017019741A - Inhibition of pulmonary fibrosis by inhibiting macrophage proliferation/differentiation or promoting macrophage aging - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for the inhibition of pulmonary fibrosis by inhibiting macrophage proliferation/differentiation or promoting macrophage aging.SOLUTION: The present invention provides a composition for promoting macrophage aging in fibrosis regions, comprising Sphingosine-1-phosphate receptor-2 (S1 P) antagonist as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a medicament that inhibits the proliferation and differentiation of macrophages involved in pulmonary fibrosis or promotes aging of macrophages.

  Pulmonary fibrosis (IPF: Idiopathic pulmonary fibrosis) is one of idiopathic interstitial pneumonia, and has the largest number of patients among idiopathic interstitial pneumonia and an extremely poor prognosis. Pulmonary fibrosis results from inflammation of the interstitium of the lungs, causing pulmonary fibrosis, reducing gas exchange in the lungs and causing respiratory failure.

  The cause of idiopathic pulmonary fibrosis is unknown, and secondary pulmonary fibrosis is known to develop due to collagen disease, radiation, drugs such as anticancer agents, poisoning, and the like.

  In Japan, the incidence is approximately 20 per 100,000 in the case of idiopathic.

  Treatment includes steroids, immunosuppressants, and virfenidone, but they are symptomatic and have limited effects. The prognosis for pulmonary fibrosis is very poor, and the survival time after diagnosis is only 2.5 to 3.5 years.

Sphingosine-1-phosphate (S1P) is a lipid mediator present in plasma at a concentration of several hundreds of nM. As a ligand for S1P receptors on the cell membrane, cell motility control, actin skeleton formation, cell proliferation, adhesion Causes various cellular responses such as bond formation. S1P ₁ (EDG-1), S1P ₂ (EDG-5), S1P ₃ (EDG-3), S1P ₄ (EDG-6), and S1P ₅ are S1P receptors, which are analogs of G protein-coupled receptors (EDG-8) has been identified so far.

It has been reported that antagonists of S1P receptors can be used to treat various diseases including fibrosis (see Patent Documents 1 and 2). However, only S1P ₁ (EDG-1) and S1P ₄ (EDG-6) have been identified as receptors associated with the disease.

International Publication No.WO01 / 03739 JP 2012-102124 A

  An object of the present invention is to provide a composition for inhibiting pulmonary fibrosis due to inhibition of macrophage proliferation / differentiation or promotion of aging of macrophages.

In order to study various cellular responses in vivo involving S1P ₂ which is a sphingosine-1-phosphate (S1P) receptor, the present inventors have produced mice knocked out of the S1P ₂ gene and conducted various studies. Went.

Among them, in order to investigate the relationship between pulmonary fibrosis and S1P ₂ , bleomycin administration, which is a method of creating a pulmonary fibrosis mouse model (bleomycin-induced mouse pulmonary fibrosis model) in wild type mice, was compared with wild type mice and S1P _2. It was performed on mice knocked out of the gene to examine whether the fibrosis of both mice was induced.

As a result, it was found that pulmonary fibrosis induced by bleomycin administration was mild in mice knocked out of the S1P ₂ gene.

The present inventors have further investigated the mechanism by which pulmonary fibrosis induced by bleomycin administration is mild in S1P ₂ gene knockout mice, and that S1P ₂ expressing cells are accumulated in the lung fibrosis region, and macrophages There is involved in fibrosis of the lung, aging macrophages advances in SlP ₂ gene knockout mice, further since the aging proceeds increased expression of collagen degrading enzymes lung fibroblasts are observed, SlP ₂ macrophages and lung It was found that lung fibrosis is promoted by suppressing fibroblast senescence.

  In addition, the present inventors have found that by inhibiting macrophage growth using a macrophage growth / differentiation inhibitor, lung fibrosis induced by bleomycin administration is suppressed.

Based on these findings, the present inventors have found that a substance that antagonizes the action of S1P ₂ suppresses pulmonary fibrosis by promoting cell senescence of macrophages and lung fibroblasts, and further inhibits proliferation and differentiation of macrophages. To suppress lung fibrosis, and found that a substance that antagonizes the action of S1P ₂ or a macrophage growth / differentiation inhibitor can be used as a suppressor of lung fibrosis, leading to the completion of the present invention. .

That is, the present invention is as follows.
[1] A composition for promoting aging of macrophages in a fibrosis region, comprising a sphingosine-1-phosphate type 2 receptor (S1P ₂ ) antagonist as an active ingredient.
[2] The composition according to [1], which further promotes the expression of collagenolytic enzyme in fibroblasts in the fibrosis region.
[3] The sphingosine-1-phosphate type 2 receptor (S1P ₂ ) antagonist is a pyrazolopyridine derivative represented by the following formula (1) or (2), or a pharmaceutically acceptable salt thereof: [1] or [2] composition:
(1) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(3,5- Dichloropyridin-4-yl) urea
Or (2) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(2,6) represented by the following formula -Dichloropyridin-4-yl) urea
[4] A lung fibrosis inhibitor comprising the composition according to any one of [1] to [3].
[5] A pulmonary fibrosis inhibitor comprising, as an active ingredient, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that inhibits macrophage proliferation and differentiation.
[6] Lung fibrosis inhibitor of [5], wherein the inhibitor of colony stimulating factor I receptor (CSF1R: Colony Stimulating Factor 1 Receptor) is any one of the following compounds (1) to (6):
(1)
BLZ945 (4- (2-((1R, 2R) -2-hydroxycyclohexylamino) benzo [d] thiazol-6-yloxy) -N-methylpicolinamide)
,
(2)
GW2580
(2,4-Pyrimidinediamine, 5-[[3-methoxy-4-[(4-methoxyphenyl) methoxy] phenyl] methyl])
,
(3)
Linigfanib (ABT-869)
(1- (4- (3-amino-1H-indazol-4-yl) phenyl) -3- (2-fluoro-5-methylphenyl) urea)
,
(4)
OSI-930
(3-[(4-quinolinylmethyl) amino] -N- [4- (trifluoromethoxy) phenyl] -2-thiophenecarboxamide)
,
(5)
CEP-32469
(Urea, N- [3-[(6,7-dimethoxy-4-quinazolinyl) oxy] phenyl] -N- [5- (2,2,2-trifluoro-1,1-dimethylethyl) -3-isoxazolyl] )
Or (6)
Pexidartinib (PLX3397)
(N- [5-[(5-chloro-1H-pyrrolo [2,3-b] pyridin-3-yl) methyl] -2-pyridinyl] -6- (trifluoromethyl) -3-pyridinemethanamine)
[7] contacting a cell with a candidate compound expressing SlP _2, screening methods fibrosis inhibitor, which comprises selecting the candidate compound that inhibits the activity or expression of SlP _2.
[8] The screening method according to [7], wherein a candidate compound is selected using an inhibitory activity of binding of radiolabeled S1P, protein kinase ERK activation, or intracellular signal transduction activation as an index.

The pulmonary fibrosis involves macrophages, and the macrophages proliferate and accumulate in the fibrotic region. The S1P ₂ antagonist of the present invention promotes aging of macrophages and fibroblasts, and the colony stimulating factor 1 receptor (CSF1R) inhibitor of the present invention suppresses macrophage proliferation and differentiation. This inhibits macrophage accumulation and suppresses pulmonary fibrosis.

FIG. 3 shows that S1P ₂ deletion suppresses pulmonary fibrosis induced by bleomycin. Sirius Red-stained images of the lungs of S1P ₂ gene-deficient (S1P ₂ -KO) mice (Fig. 1-1B) and wild-type mice (Fig. 1-1A) 33 days after intraperitoneal administration of bleomycin or saline (upper) And immunostained images of PDGFRα (bottom). The figure shows a representative stained image. FIG. 3 shows that S1P ₂ deletion suppresses pulmonary fibrosis induced by bleomycin. Lung fibrosis area (Fig. 1-2A) and pathological evaluation of lung fibrosis in S1P ₂ -KO mice (white circles) and wild type mice (black circles) 33 days after intraperitoneal administration of bleomycin or saline (Fig. 1) -2B). Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. FIG. 3 shows that S1P ₂ deletion suppresses pulmonary fibrosis induced by bleomycin. Fibronectin (Fig. 1-3A) and collagen Iα1 (Fig. 1-3B) in the lung tissue of S1P ₂ -KO mice (white bars) and wild-type mice (black bars) 33 days after intraperitoneal administration of bleomycin or saline ) And FSP1 (FIGS. 1-3C) mRNA expression. The data shows the relative amount as the mean ± standard error with the physiological saline-administered group of wild-type mice as 1. β-actin was used as an internal standard gene. The numbers in parentheses indicate the number of mice used in each group experiment. SlP ₂ deletion is a diagram showing the suppressing alveolitis disease caused by bleomycin administration. Soluble collagen content (Figure 2A) and total protein content (Figure 2B) in bronchoalveolar lavage fluid of S1P ₂ -KO mice (white circles) and wild-type mice (black circles) 33 days after intraperitoneal administration of bleomycin or saline , Total cell number (FIG. 2C), macrophage number (FIG. 2D). Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. It shows the results of expression analysis of SlP ₂ in lung tissue. The X-gal staining image of the lungs of S1P ₂ ^{LacZ / +} mice 33 days after intraperitoneal administration of bleomycin (FIG. 3-1B) or physiological saline (FIG. 3-1A) is shown. The figure shows a representative stained image. It shows the results of expression analysis of SlP ₂ in lung tissue. Double-stained images of X-gal staining and Mac-3 immunostaining of lungs of S1P ₂ ^{LacZ / +} mice 33 days after intraperitoneal administration of bleomycin (Fig. 3-2B) or physiological saline (Fig. 3-2A) Indicates. The figure shows a representative stained image. SlP ₂ expressed on bone marrow-derived cells is a diagram showing the involvement of the pulmonary fibrosis induced by bleomycin. FIG. 4A shows the lung fibrosis area of each bone marrow transplanted mouse 33 days after intraperitoneal administration of bleomycin. Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. 4B, C and D show the amount of soluble collagen in the bronchoalveolar lavage fluid of each bone marrow transplanted mouse 33 days after intraperitoneal administration of bleomycin (FIG. 4B), the total number of cells (FIG. 4C), the number of macrophages (FIG. 4D). Indicates. Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. It is a figure which shows that a macrophage growth and differentiation inhibitor (BLZ945) suppresses pulmonary fibrosis induced by bleomycin. Sirius Red of the lungs of each mouse in the BLZ945 administration group (FIG. 5-1C), the solvent administration group (FIG. 5-1B), and the non-administration group (FIG. 5-1A) 25 days after intraperitoneal administration of bleomycin or physiological saline A stained image is shown. The figure shows a representative stained image. It is a figure which shows that a macrophage growth inhibitor (BLZ945) suppresses the pulmonary fibrosis induced by bleomycin. Fibrosis region of the lungs of each mouse in the BLZ945 administration group, solvent administration group, and non-administration group 25 days after intraperitoneal administration of bleomycin (FIG. 5-2A). The amount of soluble collagen in bronchoalveolar lavage fluid (FIG. 5-2B), the total number of cells (FIG. 5-2C), and the number of macrophages (FIG. 5-2D) are shown. Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. The SlP ₂ -KO mice shows that advanced aging of macrophages. 3 shows SA-β-gal stained images of lungs of S1P ₂ -KO mice and wild-type mice 33 days after intraperitoneal administration of bleomycin or physiological saline. The figure shows a representative stained image. The SlP ₂ -KO mice shows that advanced aging of macrophages. The number of SA-β-gal positive cells per field of lung of S1P ₂ -KO mice (white circles) and wild type mice (black circles) 33 days after intraperitoneal administration of bleomycin or physiological saline is shown. Data are shown as mean ± standard error. The SlP ₂ -KO mice shows that advanced aging of macrophages. With cells of the F4 / 80 of SlP ₂ -KO mice and bronchoalveolar lavage fluid of wild type mice 33 days after intraperitoneal administration of bleomycin or saline exhibits fluorescence immunostaining images of p16 ^INK4a. The figure shows a representative stained image. The SlP ₂ -KO mice shows that advanced aging of macrophages. Number of p16 ^INK4a positive cells ^{compared to the} number of F4 / 80 positive cells in bronchoalveolar lavage fluid of S1P ₂ -KO mice (white circles) and wild type mice (black circles) 33 days after intraperitoneal administration of bleomycin or saline Indicates the percentage. Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. The SlP ₂ -KO mice shows that advanced aging of macrophages. The immunostained images of p16 ^INK4a in the lungs of S1P ₂ -KO mice (FIG. 6-5B) and wild type mice (FIG. 6-5A) 33 days after intraperitoneal administration of bleomycin or physiological saline are shown. The figure shows a representative stained image. FIG. 3 shows that the phenotype of S1P ₂ -KO lung fibroblasts is different from that of wild-type lung fibroblasts. The results of Western blot showing p16 ^INK4a protein expression in lung fibroblasts SlP ₂ -KO mice and wild-type mice 33 days after intraperitoneal administration of bleomycin or saline. FIG. 3 shows that the phenotype of S1P ₂ -KO lung fibroblasts is different from that of wild-type lung fibroblasts. P16 ^INK4a protein expression in lung fibroblasts of S1P ₂ -KO mice (white bars) and wild type mice (black bars) 33 days after intraperitoneal administration of bleomycin or physiological saline, p16 with GAPDH protein as internal standard The expression level of ^INK4a protein was corrected and calculated. The data shows the relative amount as the mean ± standard error with 1 as the lung fibroblasts isolated from the saline-administered group of wild-type mice. The numbers in parentheses indicate the number of mice used in each group experiment. FIG. 3 shows that the phenotype of S1P ₂ -KO lung fibroblasts is different from that of wild-type lung fibroblasts. 3 shows SA-β-gal stained images of lung fibroblasts of S1P ₂ -KO mice and wild-type mice 33 days after intraperitoneal administration of bleomycin or physiological saline. The figure shows a representative stained image. FIG. 3 shows that the phenotype of S1P ₂ -KO lung fibroblasts is different from that of wild-type lung fibroblasts. The ratio of the number of SA-β-gal positive cells to the total number of S1P ₂ -KO mice (white bars) and wild type mice (black bars) 33 days after intraperitoneal administration of bleomycin or physiological saline is shown. Data are shown as mean ± standard error. The numbers in parentheses indicate the number of mice used in each group experiment. FIG. 3 shows that the phenotype of S1P ₂ -KO lung fibroblasts is different from that of wild-type lung fibroblasts. Collagenase MMP1 (FIG. 7-5A), MMP2 (in the lung fibroblasts of S1P ₂ -KO mice (white bars) and wild-type mice (black bars) 33 days after intraperitoneal administration of bleomycin or physiological saline Fig. 7-5B) shows mRNA expression of MMP3 (Fig. 7-5C). The data are shown as relative + mean + standard error relative to lung fibroblasts isolated from the saline-administered group of wild type mice as 1. The numbers in parentheses indicate the number of mice used in each group experiment. S1P ₁ and S1P ₃ is a diagram showing that not involved in fibrosis-suppressing effect. In the figure, black circles indicate wild type mice (WT), and white circles indicate S1P ₁ ^+/− mice (FIG. 8A) or S1P ₃ -KO mice (FIG. 8B).

  Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for promoting aging of macrophages or fibroblasts, comprising an S1P ₂ antagonist (antagonist) as an active ingredient. The composition promotes senescence of macrophages or fibroblasts in a fibrotic region, thereby suppressing lung fibrosis and alveolar inflammation, and is used for prevention or treatment of pulmonary fibrosis be able to.

  Pulmonary fibrosis that can be prevented or treated by the composition of the present invention is idiopathic pulmonary fibrosis, which is one of idiopathic interstitial pneumonia (IIP).

Antagonists worked receptor molecule in vivo, it refers to a compound which blocks the signal transduction like, SlP ₂ antagonist has bound SlP ₂ antagonism in SlP _2.

Examples of the S1P ₂ antagonist include the pyrazolopyridine derivative represented by the following formula (1) or (2), or a pharmaceutically acceptable salt thereof.
(1) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(3,5- Dichloropyridin-4-yl) urea
(2) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(2,6- Dichloropyridin-4-yl) urea

Moreover, as an antagonist of SlP _2, including an antagonist antibody to SlP _2. Antibody, by binding to SlP _2, prevents the ligand binds to the SlP _2, inhibit signaling. Antibody, for example, an antibody that recognizes an epitope present in the site that binds the ligand SlP _2. The antibody includes both a monoclonal antibody and a polyclonal antibody, and is preferably a monoclonal antibody. Also included are humanized antibodies, human antibodies, and chimeric antibodies. An antagonist antibody against S1P ₂ can be prepared by a known technique, and may be selected based on the activity of blocking ligand binding to S1P ₂ .

  Examples of pharmaceutically acceptable salts include various inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, phosphate or nitrate; acetate, propionate, succinate, glycolate, Lactate, malate, oxalate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, tosylate or ascorbate Various organic acid addition salts such as aspartate or salts with various amino acids such as glutamate, but are not limited thereto. In some cases, it may be a hydrate, hydrate or solvate.

S1P ₂ is predicted to be involved in macrophage accumulation in fibrotic lesions by promoting monocyte extravasation or resident macrophage proliferation. At this time, by the action of SlP _2, aging macrophage is suppressed. Therefore, by using antagonists of SlP ₂ inhibiting the action of SlP _2, aging of macrophages is promoted, macrophage growth is inhibited. As a result, accumulation of macrophages at the fibrotic lesion site is suppressed, and fibrosis is suppressed.

In addition, macrophage S1P ₂ promotes mediator release and promotes senescence of fibroblasts in this fibrosis region through this action, and senescence-associated secretory phenotype (SASP) (J. Cell Biol., 2011, 192, 547-556) is predicted to change the expression of MMP (matrix metalloprotease), an enzyme that degrades collagen. Therefore, by using antagonists of SlP ₂ inhibiting the action of SlP _2, increased expression levels of MMP by fibroblasts, collagen degradation is accelerated, fibrosis is suppressed.

  Furthermore, the present invention includes a preventive or therapeutic agent for pulmonary fibrosis containing a macrophage growth inhibitor. The medicament is also a lung fibrosis inhibitor.

  Examples of the macrophage growth inhibitor include inhibitors of colony stimulating factor 1 receptor (CSF1R), which is a transmembrane receptor type tyrosine kinase. CSF1R (FMS, CD115) is a one-transmembrane tyrosine kinase that is highly expressed in cells such as macrophages, and CAF1R inhibitors can suppress lung fibrosis by inhibiting macrophage proliferation. .

  Examples of CSF1R inhibitors include the following compounds.

BLZ945 (4- (2-((1R, 2R) -2-hydroxycyclohexylamino) benzo [d] thiazol-6-yloxy) -N-methylpicolinamide)

GW2580
(2,4-Pyrimidinediamine, 5-[[3-methoxy-4-[(4-methoxyphenyl) methoxy] phenyl] methyl])

Linigfanib (ABT-869)
(1- (4- (3-amino-1H-indazol-4-yl) phenyl) -3- (2-fluoro-5-methylphenyl) urea)

OSI-930
(3-[(4-quinolinylmethyl) amino] -N- [4- (trifluoromethoxy) phenyl] -2-thiophenecarboxamide)

CEP-32469
(Urea, N- [3-[(6,7-dimethoxy-4-quinazolinyl) oxy] phenyl] -N- [5- (2,2,2-trifluoro-1,1-dimethylethyl) -3-isoxazolyl] )

Pexidartinib (PLX3397)
(N- [5-[(5-chloro-1H-pyrrolo [2,3-b] pyridin-3-yl) methyl] -2-pyridinyl] -6- (trifluoromethyl) -3-pyridinemethanamine)

  The medicament containing the composition of the present invention is a carrier, excipient, thickener, preservative, stabilizer, antioxidant, binder, disintegrant, wetting agent, lubricant, ordinarily used in formulation. Colorants, fragrances, flavoring agents, suspending agents, emulsifiers, solubilizers, buffering agents, tonicity agents, surfactants, soothing agents, sulfur-containing reducing agents, etc., improved absorption, solid stability Various other functional components can be blended as appropriate for the purpose of achieving the above. Examples of carriers and excipients are water-soluble or sparingly soluble, such as sugars, polysaccharides, dextrins, celluloses, synthetic or semi-synthetic polymers, amino acids, polyamino acids, proteins, phosphorus Examples thereof include lipids.

  Examples of the route of administration of the medicament containing the composition of the present invention include transdermal, intravenous, intramuscular, subcutaneous and oral. The dosage form of the therapeutic agent can be appropriately set depending on the administration method. Specific examples include solutions such as aqueous solutions, emulsions and suspensions, and tablets, granules and capsules. Usually, since oral administration is preferable, an aqueous solution or a tablet is preferable.

  The dose can be appropriately set depending on the administration method, the age, weight, and medical condition of the patient to be applied, but 0.005 to 5 mg / kg body weight is preferable at one time, and 0.005 to 1 mg / kg body weight is more preferable. It can be administered once a day or divided into several times every day.

The present invention encompasses a method of further inhibiting the activity or expression of SlP _2, screening for a compound having a fibrosis-suppressing effect. In the screening, for example, a cell such as a Chinese Hamster Ovary (CHO) cell overexpressing S1P ₂ is contacted with the candidate compound, and the activity of inhibiting the binding of radiolabeled S1P in the cell, Activation of intracellular signal transduction such as activation of protein kinase ERK and increase of intracellular free calcium concentration can be performed as an index. When a candidate compound has S1P ₂ antagonist activity, binding of radiolabeled S1P in the cell is inhibited, protein kinase ERK activity is activated, and intracellular signaling such as increased intracellular free calcium concentration is activated .

Have a relationship with the fibrosis inhibited is in S1P receptors in is only S1P _2, the S1P ₁ and S1P ₃ is not involved in inhibiting fibrosis is also shown in later comparative examples.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example Relationship between S1P ₂ and suppression of fibrosis (materials and methods)
The materials and methods used in the examples are as follows.

(mouse)
SlP ₂ gene defect (S1P ₂ -KO) mice (129Ola; C57BL / 6J mixed background ) was prepared by the method described in the literature (Cancer Res 2010, 16, 509-520 .). S1P ₂ -KO mice were backcrossed once with C57BL / 6J mice, and 8-10 week old male S1P ₂ -KO mice and littermate wild-type mice were used for the experiment. (S1P ₂ ^{LacZ / +} ) mice (129Ola; C57BL / 6J mixed background) in which β-galactosidase gene was inserted into the S1P ₂ locus were prepared by the method described in the literature (Cancer Res. 2010, 16, 509-520) did. S1P ₂ ^{LacZ / +} mice were backcrossed 3 times with C57BL / 6J mice, and 8-10 week old male S1P ₂ ^{LacZ / +} mice were used for the experiment. Mice were bred under conditions of 24 ° C, humidity of 60% or less, and 12-hour off-to-12-hour on cycle where food and water can be freely inoculated. Confirmation of mouse genotype was performed by PCR. Genomic DNA recovered from mouse tails by standard techniques was used as a template for PCR. In order to detect S1P ₂ gene-deficient allele and wild type allele, the following three kinds of primers were used.

Forward1; 5'-tggctacccgtgatattgctgaagagcttg-3 '(SEQ ID NO: 1)
Reverse1; 5'-tgagcagtgagttaagggtggcaaaggcaa-3 '(SEQ ID NO: 2)
Forward2; 5'-cagtgacaaaagctgccgaatgctgatgct-3 '(SEQ ID NO: 3)
In PCR, a cycle of 94 ° C. for 30 seconds, 59 ° C. for 45 seconds, and 2 ° C. for 60 seconds was repeated 40 times.

(Bleomycin-induced lung fibrosis model)
A bleomycin-induced mouse lung fibrosis model was prepared by intraperitoneally administering 0.035 mg / g body weight of bleomycin (Nippon Kayaku, Tokyo) to male mice (8-10 weeks old). A bleomycin solution (prepared to a concentration of 5 mg / ml with physiological saline) was intraperitoneally administered twice a week for 4 weeks for a total of 8 times, using a Terumo syringe equipped with a 27 gauge needle. In the control group, physiological saline (150 μl) was intraperitoneally administered. One week after the final administration (33 days after administration), the mice were euthanized and the following analysis was performed.

(Tissue staining)
Mice 33 days after intraperitoneal administration of bleomycin were euthanized by pentobarbital (Kyoritsu Pharmaceutical Co., Ltd., Tokyo) (50 mg / kg, ip) deep anesthesia and then exsanguinated by abdominal aortic amputation. After quickly opening the chest cavity, exposing the lungs and ligating the right hilar region, the right lung was removed and frozen in liquid nitrogen. The right lung was used for mRNA expression analysis. After making an incision in the left atrium, 4% paraformaldehyde solution (dissolved in 0.1 M sodium phosphate buffer (pH 7.4)) was injected into the right ventricle to fix the perfusion of the lung, and an 18-gauge needle was inserted from the upper part of the mouse trachea. Then, 0.5 mL of 4% paraformaldehyde solution containing 0.5% low melting point agarose (dissolved in 0.1 M sodium phosphate buffer (pH 7.4)) was injected into the trachea with a Terumo syringe. The trachea was tied as the 18 gauge needle was removed to keep the expanded lung fixed. After removing the lung, fix it in 4% paraformaldehyde solution for more than 24 hours, and then divide the left lung into three parts, hilar, central, and peripheral by sagittal section, embedded in paraffin, and sliced 5 μm paraffin sections were prepared.

  The increase in collagen fibers in the fibrosis region of the lung is confirmed by staining the left hilar, central and peripheral paraffin sections with Sirius red and then using the image analysis software Image-J software The ratio (% value) of the Sirius red positive area to the area was calculated. The fibrosis region of the entire lung was evaluated by the average of the fibrosis regions of the hilar region, central region, and peripheral region of the left lung. Infiltration and migration of fibroblasts in the fibrotic region of the lung were evaluated by immunostaining of paraffin sections using a goat polyclonal anti-PDGFRα antibody (AF1062, R & D systems). Immunostaining of paraffin sections was performed using the ABC method (VECTASTAIN). The secondary antibody used in the ABC method was colored by DAB (3,3′-Diaminobenzidine, tetrahydrochloride, Wako, Osaka). After completion of the reaction, the nucleus was stained with hematoxylin, dehydrated with ethanol, degreased with xylene, and encapsulated with Entellan New (Merck).

  The pathological evaluation of pulmonary fibrosis was performed based on the lung fibrosis score by Ashcroft. Under the microscope, the degree of lung fibrosis was graded from 0 to 8 from mild to severe, and scored by scoring all visual fields (more than 10 visual fields for each mouse). The degree of fibrosis of the whole lung was evaluated by the average of.

(Analysis of bronchoalveolar lavage fluid)
1. Collection of bronchoalveolar lavage fluid (BALF) After 33 days after intraperitoneal administration of bleomycin, mice were subjected to airway lavage and BALF was collected. First, pentobarbital (Kyoritsu Pharmaceutical Co., Ltd., Tokyo) (50 mg / kg, ip) was euthanized by deep anesthesia and then exsanguinated by abdominal aortic amputation. An 18-gauge needle was quickly inserted from the upper part of the mouse trachea, and 1 ml of phosphate-buffered saline (PBS) was injected into the trachea with a Terumo syringe and collected. This operation was repeated twice, and about 80% or more of BALF of the total injection volume (2 ml) was recovered. The recovered BALF was separated into a sediment (cell component) and a supernatant by centrifugation (450 × g, 4 ° C., 10 min).

2. Analysis of cell fraction of bronchoalveolar lavage fluid 1 ml of PBS was added to the sediment to suspend the cells, and the number of cells was counted under an optical microscope using a hemocytometer. The remaining sediment solution was adjusted so that the total number of cells was 100,000, and a smear was prepared. The smear was stained with Diff Quick (Sysmex International Reagents, Kobe). The number of macrophages was calculated by counting 150 to 200 cells per specimen.

3. Measurement of total protein in BALF
The total protein amount in BALF was measured by the Lowry method. A calibration curve was prepared using bovine serum albumin (BSA) (Wako, Osaka).

4). Measurement of soluble collagen in BALF
The amount of soluble collagen in BALF was measured by Sircol assay kit (Biocolor). A calibration curve was prepared using bovine collagen.

(MRNA expression analysis)
1. Extraction of total RNA 33 days after intraperitoneal administration of bleomycin, mice were euthanized by pentobarbital (Kyoritsu Pharmaceutical Co., Ltd., Tokyo) (50 mg / kg, ip) deep anesthesia and then rapidly exsanguinated by abdominal aortic amputation. Was extracted. Total RNA was extracted from isolated lung or cultured lung fibroblasts using TRIzol Reagent (Invitrogen).

2. Quantitative PCR reaction
A reverse transcription reaction was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) to synthesize cDNA from 2 μg of total RNA. A quantitative PCR reaction was performed using 1/5 of the product. A quantitative PCR reaction was performed using MESA Green qPCR MasterMix Plus (Eurogenetec) reagent and ABI PRISM 7300 sequence detection system (Applied Biosystems). In PCR, a cycle of 94 ° C. for 15 seconds and 60 ° C. for 60 seconds was repeated 40 times. In all experiments, dissociation curve analysis confirmed the dissociation temperature, and the PCR product was subjected to agarose gel electrophoresis to confirm amplification of the target product. For quantification of gene expression, β-actin was used as an internal standard gene, and analysis was performed by relative quantification using a comparative Ct method (ΔΔCT method).
The primers used are shown in Table 1.

(X-gal staining)
After euthanizing S1P ₂ ^{lacZ / +} mice 33 days after intraperitoneal administration of bleomycin with pentobarbital (Kyoritsu Pharmaceutical Co., Ltd., Tokyo) (50 mg / kg, ip) deep anesthesia, an incision was made in the left atrium 4% paraformaldehyde solution (dissolved in 0.1 M sodium phosphate buffer (pH 7.4)) was injected into the right ventricle to fix the lungs by perfusion. The tissue was embedded in a frozen tissue embedding agent OCT compound (Sakura Fine Chemical, Tokyo), sliced, and 10 μm frozen sections were prepared. For X-gal staining, treatment with 0.2% glutaraldehyde, followed by washing with PBS, X-gal staining solution (1 mg / ml X-gal (5-bromo-4-chloro-3-indolyi-β -D-galactopyranoside), 100 mM soudium phosphate, pH 7.4, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl ₂ ), and reacted overnight at 37 ° C to express the lacZ inserted into the S1P ₂ locus Analyzed. Nuclear fast Red (VECTOR) was used for counterstaining of nuclei. In addition, double staining of immunostaining using X-gal staining and rat monoclonal anti-Mac-3 antibody (553322, BD Biosciences) was performed after X-gal staining and immunostaining using ABC method. After completion of the reaction, it was dehydrated with ethanol, degreased with xylene and sealed with Entellan New.

(Bone marrow transplantation experiment)
The lethal dose of radiation 8 week old SlP ₂ -KO mice or wild type mice subjected to (4.8 Gy 2 times the irradiation of the 3 hour intervals), from the femur and tibia of SlP ₂ -KO mice or wild type mice The prepared bone marrow cells (1 × 10 ⁷ cells) were injected intravenously from the tail vein, and bone marrow transplantation was performed. Mice were allowed to recover for 6 weeks before analyzing bleomycin-induced lung fibrosis.

(BLZ945)
Male C57BL / 6J mice (8 weeks old) were orally administered 200 mg / kg body weight / day macrophage growth / differentiation inhibitor BLZ945 (Csf1 receptor kinase inhibitor) (Chemshuttle) daily from 3 days before the start of intraperitoneal administration of bleomycin. Administered. BLZ945 solution is 20% Captisol (Captisol To a concentration of 40 mg / ml. In the control group, 20% Captisol (100 μl) was orally administered.

(Isolation of lung fibroblasts)
The lungs removed from the mice were minced and incubated in DMEM medium containing Collagenase A (Roche). Thereafter, the cell suspension was centrifuged. The supernatant was removed, resuspended and seeded in a cell culture dish. After confirming cell adhesion, non-adherent cells (supernatant) were removed, and adherent cells were cultured and used in the experiment.

(Western blot)
After pulmonary fibroblasts were washed with PBS, the Laemmli SDS sample buffer with a double concentration was added and collected, and boiled for 5 minutes. The collected sample was separated by 15% SDS-PAGE. Then, after transferring to immobilon-P membrane (Merck), each band using rabbit polyclonal anti-p16 ^INK4a antibody (M-156, Santa Cruz Biotechnology) and rabbit monoclonal anti-GAPDH antibody (2118, Cell Signaling Technology) Was detected, and the intensity of the band was digitized using a densitometer. The expression level of p16 ^INK4a protein was corrected and calculated using GAPDH protein as an internal standard.

(SA-β-gal staining)
Cryosections of mouse lungs or cultured lung fibroblasts 33 days after intraperitoneal administration of bleomycin were treated with 0.2% glutaraldehyde, washed with PBS, and X-gal staining solution (1 mg / ml X-gal (5 -bromo-4-chloro-3-indolyi-β-D-galactopyranoside), 40 mM citrate-phosphate buffer, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl ₂ ), And the acid β-galactosidase activity observed in senescent cells widely known as aging markers was analyzed. Nuclear fast Red (VECTOR) was used for counterstaining of nuclei. In mouse lung, the number of SA-β-gal positive cells per visual field was determined, and in lung fibroblasts, the ratio of SA-β-gal positive cells to the total number of cells was determined.

(Immunofluorescent cell staining)
Cells from bronchoalveolar lavage fluid were seeded on culture chamber slides. Two hours later, cell adhesion was confirmed, non-adherent cells (supernatant) were removed, and the adherent cells were used in the experiment. The cells were washed with PBS, fixed with 4% paraformaldehyde solution, and then permeabilized with 1% Triton X-100 in PBS for 15 minutes. Cells were blocked with 5% normal goat serum (Wako, Osaka), incubated overnight with rat polyclonal anti-F4 / 80 antibody (Cl: A3-1 AbD Serotec) and rabbit polyclonal anti-p16 ^INK4a antibody, followed by goat The cells were incubated with AlexaFluor 488 or 584-conjugated secondary antibody (Molecular Probe) for 1 hour. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probe) for 5 minutes. The slides were encapsulated with an aqueous mounting medium fluoromount (Fluoromount ^™ , Diagnostic Biosystems) for fluorescent tissue staining, and observed with a confocal laser microscope (Zeiss Axiovert 200M with LSM5 Pascal). The ratio (%) of the number of p16 ^INK4a positive cells to the number of F4 / 80 positive cells was determined.

(Statistical processing)
All measured values were expressed as mean ± standard deviation. Two-way analysis of variance was used for the significant difference test, and Bonferroni method was used for the post hoc test, and a risk rate of less than 5% was determined to be statistically significant. In addition, FIG. 4 was analyzed using the Mann-Whitney method. Prism 6 software (GraphPad Software, San Diego, USA) was used for these statistical processing.

Result 1. In S1P ₂ gene disruption (knockout (KO)) mice, pulmonary fibrosis induced by bleomycin administration is mild.

Using a pulmonary fibrosis model induced by bleomycin administration, we examined how S1P ₂ affects pulmonary fibrosis.

A 0.035 mg / g body weight bleomycin solution was intraperitoneally administered twice a week for 4 weeks for a total of 8 times to wild type mice and S1P ₂ -KO mice. On day 33 after administration, lungs were removed from each mouse, and Sirius Red staining (collagen staining) and immunohistochemical staining of PDGFRα (lung fibroblast marker) were performed to examine the histopathological changes in the lung tissue (Fig. 1-1 and 1-2). In the lung tissue of wild-type mice, the Sirius Red positive region increased in the bleomycin administration group compared to the saline administration group, confirming the promotion of lung fibrosis (FIG. 1-1). In the fibrosis region, the PDGFRα positive region was strong. On the other hand, in the S1P ₂ -KO mice, pulmonary fibrosis induced by bleomycin administration was mild, and the PDGFRα positive region was also reduced (FIG. 1-1). After quantifying the fibrosis area and the degree of fibrosis using the Sirius Red stained image of the lung 33 days after administration, the S1P ₂ -KO mice showed a decrease in fibrosis area and the degree of fibrosis of the Ashcroft score. A decrease in numerical values was observed (FIGS. 1-2). Using quantitative PCR, mRNA expression of fibronectin, which is an extracellular matrix, and collagen type I α1, and FSP1, which is a marker of fibroblasts, were analyzed in lung tissue of bleomycin-treated mice (FIGS. 1-3). The administration of bleomycin increased the mRNA expression of fibronectin, collagen type I α1 and FSP1. In S1P ₂ -KO mice, mRNA expression of fibronectin, collagen type I α1 and FSP1 in the lung tissue was decreased as compared to wild-type mice (FIGS. 1-3).

From the above results, pulmonary fibrosis induced by bleomycin administration was mild in S1P ₂ -KO mice, and S1P ₂ was considered to be a factor promoting fibrosis.

2. In S1P ₂ -KO mice, alveolar inflammation by bleomycin administration is suppressed.
We confirmed how S1P ₂ affects the spread of inflammation to the alveolar space by bleomycin administration.

Bronchoalveolar lavage fluid of wild type mice and S1P ₂ -KO mice on day 33 after bleomycin administration was collected, and the amount of soluble collagen, total protein, total number of cells and macrophages in bronchoalveolar lavage fluid were measured. In wild-type mice and S1P ₂ -KO mice, the amount of soluble collagen in bronchoalveolar lavage fluid (Fig. 2A), total protein (Fig. 2B), and total number of cells (Fig. 2C) and the number of macrophages (FIG. 2D) were significantly increased or tended to increase. In S1P ₂ -KO mice, the amount of soluble collagen in bronchoalveolar lavage fluid (FIG. 2A), the total number of cells (FIG. 2C), and the number of macrophages (FIG. 2D) were significantly reduced compared to wild-type mice. Moreover, the total protein amount (FIG. 2B) showed a decreasing tendency.

From the above results, S1P ₂ was considered to be involved in the spread of inflammation to the alveolar space by intraperitoneal administration of bleomycin.

3. Results of expression analysis of S1P ₂ in lung tissue
S1P ₂ ^{LacZ / +} mice were used to confirm the cells expressing S1P ₂ in lung tissue.
X-gal staining was performed on lungs of S1P ₂ ^{LacZ / +} mice 33 days after intraperitoneal administration of bleomycin or physiological saline (FIGS. 3-1 and 3-2). In lung tissue, X-gal staining positive cells (blue) were detected in type II alveolar epithelial cells, macrophages, vascular endothelial cells, vascular smooth muscle cells and fibroblasts. In addition, accumulation of X-gal staining positive cells was observed in the fibrosis region (FIG. 3-1). In addition, when double staining was performed with X-gal staining and immunostaining of macrophage marker Mac3 (red), Mac3 positive macrophages were X-gal staining positive (FIG. 3-2).

These results, SlP ₂ in lung tissues to be expressed major constituent cells of the lung, including macrophages, and SlP ₂ expressing cells revealed that accumulate in fibrotic areas.

4). S1P ₂ expressed in bone marrow-derived cells is involved in pulmonary fibrosis.
In lung tissue, S1P ₂ is expressed in the major constituent cells of the lung, including macrophages, and therefore, S1P ₂ expressed in either bone marrow-derived cells or non-bone marrow-derived cells is involved in lung fibrosis induced by bleomycin administration I confirmed what to do.

Bone marrow chimera mice were prepared by transplanting wild-type bone marrow or S1P ₂ -KO bone marrow to a lethal dose of wild-type mice or S1P ₂ -KO mice, and a bleomycin solution was injected twice a week for 4 weeks for a total of 8 times peritoneal cavity. It was administered internally. The fibrosis region was quantified using the Sirius Red stained image of the lung of each bone marrow chimeric mouse on day 33 after administration (FIG. 4A). In the mice transplanted with S1P ₂ -KO bone marrow, a decrease in the fibrosis region was observed as compared with the mice transplanted with wild-type bone marrow (FIG. 4A). Further, bronchoalveolar lavage fluid of each bone marrow chimeric mouse on day 33 after bleomycin administration was collected, and the amount of soluble collagen, total protein, total number of cells and macrophage in bronchoalveolar lavage fluid were measured (FIGS. 4B and 4C). And 4D). In mice transplanted with S1P ₂ -KO bone marrow, the amount of soluble collagen in the bronchoalveolar lavage fluid (FIG. 4B), total protein (FIG. 4C), total number of cells and macrophages (in comparison with mice transplanted with wild type bone marrow) FIG. 4D) showed a significantly decreasing or decreasing tendency.

These results suggest that S1P ₂ expressed in bone marrow-derived cells is involved in lung fibrosis.

5. Macrophage growth / differentiation inhibitor (BLZ945) suppresses pulmonary fibrosis induced by bleomycin.

Since SlP ₂ expressed on bone marrow-derived cells was considered to be involved in pulmonary fibrosis, macrophages was confirmed whether involved in pulmonary fibrosis induced by bleomycin administration.

  To investigate whether macrophages are important for pulmonary fibrosis due to intraperitoneal administration of bleomycin, 20 mg / kg body weight of BLZ945 (CSF1 receptor kinase inhibitor) was orally administered once daily every day, and 0.035 mg / g The body weight bleomycin solution was administered intraperitoneally twice a week for a total of 7 times. Sirius Red staining of mouse lung tissue of BLZ945 administration group, solvent administration group, and non-administration group on the 25th day after administration was performed (FIG. 5-1). In the BLZ945 administration group, the Sirius Red positive area was reduced compared to the solvent administration group, and lung fibrosis was reduced (FIG. 5-1). When the fibrosis region was quantified, the BLZ945 administration group showed a decrease in the fibrosis region compared to the solvent administration group (FIG. 5-2A). In addition, bronchoalveolar lavage fluid from each group of mice 25 days after bleomycin administration was collected, and the amount of soluble collagen, total protein, total cells, and macrophage in bronchoalveolar lavage fluid were measured. BLZ945 administration group In FIG. 5-2B, 5-2C and 5-2D, there was a significant decrease compared to the solvent administration group.

  These results suggest that macrophages play an important role in pulmonary fibrosis induced by bleomycin administration.

6). Macrophage aging is advanced in S1P ₂ -KO mice.
Macrophages in the lung tissue of S1P ₂ -KO mice were confirmed to have been aging by bleomycin administration.

Since we observed that the size of macrophages in the lung tissue of S1P ₂ -KO mice on day 33 after bleomycin administration had increased, it was examined whether macrophages had undergone cellular senescence due to bleomycin administration. In order to examine cell aging, SA-β-gal staining was performed on lung tissues of wild-type mice and S1P ₂ -KO mice 33 days after bleomycin administration (FIGS. 6-1 and 6-2). By administration of bleomycin, SA-β-gal staining positive cells in lung tissue were observed (FIGS. 6-1 and 6-2). S1P ₂ -KO mice had more SA-β-gal staining positive cells in the lung tissue (blue: observed as a dark granular image in the monochrome diagram) compared to wild-type mice (FIG. 6-1). And 6-2). Many SA-β-gal staining positive cells were localized in the alveolar space (FIGS. 6-1 and 6-2). Next, cells were isolated from bronchoalveolar lavage fluid of wild-type mice and S1P ₂ -KO mice 33 days after bleomycin administration, and fluorescent immunostaining of macrophage marker F4 / 80 and cell aging marker p16 ^INK4a was performed. Performed (Figures 6-3 and 6-4) By administration of bleomycin, F4 / 80 positive (red); p16 ^INK4a positive (green) cells were observed, and large F4 / 80 positive macrophages were particularly p16 ^INK4a positive (FIGS. 6-3 and 6-4). ). In S1P ₂ -KO mice, more F4 / 80 positive; p16 ^INK4a positive cells were observed than in wild type mice (FIGS. 6-3 and 6-4). Expression of p16 ^INK4a in lung tissue of wild-type mice and S1P ₂ -KO mice 33 days after bleomycin administration was examined by immunostaining (FIGS. 6-5). By administration of bleomycin, p16 ^INK4a positive cells were observed in the alveolar space, and large cells were particularly positive for p16 ^INK4a (FIGS. 6-5). In S1P ₂ -KO mice, more p16 ^INK4a positive cells were observed than in wild-type mice (FIGS. 6-5).

These results suggest that S1P ₂ promotes fibrosis by inhibiting cell aging of macrophages, which is important for pulmonary fibrosis by bleomycin administration.

7). The phenotype of S1P ₂ -KO lung fibroblasts is different from wild type lung fibroblasts.
It was confirmed that pulmonary fibroblasts of S1P ₂ -KO mice were more aging by administration of bleomycin and increased mRNA expression of collagenase as compared with wild-type lung fibroblasts. In order to examine whether cells other than macrophages were also senescent after administration of bleomycin, fibroblasts were isolated from lungs of S1P ₂ -KO mice and wild-type mice 33 days after administration of bleomycin. In order to examine cell senescence, pulmonary fibroblasts isolated from each mouse were analyzed by expression of ^p16INK4a protein by Western blot and SA-β-gal staining (FIGS. 7-1 to 7-4). Expression of p16 ^INK4a protein was observed in lung fibroblasts isolated from bleomycin administration (FIGS. 7-1 and 7-2). In S1P ₂ -KO lung fibroblasts, the expression of p16 ^INK4a protein was increased as compared to wild-type lung fibroblasts (FIGS. 7-1 and 7-2). Many SA-β-gal staining positive cells were observed in lung fibroblasts isolated from mice administered with bleomycin (FIGS. 7-3 and 7-4). In S1P ₂ -KO lung fibroblasts, the number of SA-β-gal staining positive cells was increased compared to wild-type lung fibroblasts (FIGS. 7-3 and 7-4). Using quantitative PCR, mRNA expression of collagenolytic enzymes MMP1, MMP2 and MMP3 in lung fibroblasts isolated from S1P ₂ -KO mice and wild type mice 33 days after intraperitoneal administration of bleomycin was analyzed ( Fig. 7-5). In lung fibroblasts isolated from bleomycin administration, mRNA expression of MMP1, MMP2 and MMP3 was increased in S1P ₂ -KO lung fibroblasts compared to wild-type lung fibroblasts (FIG. 7-5). ).

From the above results, it was considered that S1P ₂ promotes fibrosis by suppressing cell senescence of lung fibroblasts by bleomycin administration.

Comparative Example Relationship between S1P ₁ and S1P ₃ and suppression of fibrosis
It was confirmed whether S1P ₁ and S1P _{3 which} are S1P ₂ related receptor molecules are involved in the suppression of fibrosis.

Specifically, using the pulmonary fibrosis model induced by bleomycin administration, SlP ₁ and S1P ₃ has confirmed what effect on pulmonary fibrosis.

0.05 mg / g body weight bleomycin solution was administered intraperitoneally twice a week for 4 weeks to wild type mice and S1P ₁ ^+/- mice (homo mice are heterozygous mice because of embryonic lethality) and S1P ₃ -KO mice did. On day 33 after administration, lungs were removed from each mouse and subjected to Sirius Red staining (collagen staining) (FIGS. 8A and B). The fibrosis area was quantified using the Sirius Red stained image of the lung on the 33rd day after administration, and it was found that it was between wild-type mice and S1P ₁ ^+/− mice (FIG. 8A) or S1P ₃ -KO (FIG. 8B). There was no significant difference.

The composition containing the sphingosine-1-phosphate type 2 receptor (S1P ₂ ) antagonist of the present invention as an active ingredient can be used as a pharmaceutical that promotes aging of macrophages in the fibrotic region and suppresses fibrosis.

SEQ ID NOS: 1-17 Primer

Claims (8)

A composition for promoting aging of macrophages in a fibrotic region, comprising a sphingosine-1-phosphate type 2 receptor (S1P ₂ ) antagonist as an active ingredient.   Furthermore, the composition of Claim 1 which promotes the expression of the collagenolytic enzyme in the fibroblast in a fibrosis area | region. The sphingosine-1-phosphate type 2 receptor (SIP ₂ ) antagonist is a pyrazolopyridine derivative represented by the following formula (1) or (2), or a pharmaceutically acceptable salt thereof: Or the composition according to 2:
(1) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(3,5- Dichloropyridin-4-yl) urea
Or (2) [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(2,6) represented by the following formula -Dichloropyridin-4-yl) urea
  The lung fibrosis inhibitor containing the composition of any one of Claims 1-3.   A pulmonary fibrosis inhibitor comprising, as an active ingredient, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that inhibits macrophage proliferation and differentiation. The lung fibrosis inhibitor according to claim 5, wherein the inhibitor of colony stimulating factor 1 receptor (CSF1R: Colony Stimulating Factor 1 Receptor) is any one of the following compounds (1) to (6):
(1)
BLZ945 (4- (2-((1R, 2R) -2-hydroxycyclohexylamino) benzo [d] thiazol-6-yloxy) -N-methylpicolinamide)
,
(2)
GW2580
(2,4-Pyrimidinediamine, 5-[[3-methoxy-4-[(4-methoxyphenyl) methoxy] phenyl] methyl])
,
(3)
Linigfanib (ABT-869)
(1- (4- (3-amino-1H-indazol-4-yl) phenyl) -3- (2-fluoro-5-methylphenyl) urea)
,
(4)
OSI-930
(3-[(4-quinolinylmethyl) amino] -N- [4- (trifluoromethoxy) phenyl] -2-thiophenecarboxamide)
,
(5)
CEP-32469
(Urea, N- [3-[(6,7-dimethoxy-4-quinazolinyl) oxy] phenyl] -N- [5- (2,2,2-trifluoro-1,1-dimethylethyl) -3-isoxazolyl] )
Or (6)
Pexidartinib (PLX3397)
(N- [5-[(5-chloro-1H-pyrrolo [2,3-b] pyridin-3-yl) methyl] -2-pyridinyl] -6- (trifluoromethyl) -3-pyridinemethanamine)
Contacting the cell with a candidate compound expressing SlP _2, screening methods fibrosis inhibitor, which comprises selecting the candidate compound that inhibits the activity or expression of SlP _2.   The screening method according to claim 7, wherein the candidate compound is selected using the inhibition activity of binding of radiolabeled S1P, protein kinase ERK activation, or intracellular signal transduction activation as an index.