Fibrosis inhibitor

Abstract

[Object] The main object of the present invention is to provide a fibrosis inhibitor. [Solving Means] The present invention relates to a fibrosis inhibitor containing the heterocyclic derivative represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient; ##STR00001##
In the formula (1), R.sup.1 and R.sup.2 are the same or different and each represents an optionally substituted aryl; R.sup.3 and R.sup.4 are the same or different and each represents hydrogen atom or alkyl; R.sup.5 represents hydrogen atom, alkyl or halogen atom; Y represents N or N.fwdarw.O; A represents NR.sup.6, and R.sup.6 represents hydrogen atom, alkyl, etc.; D represents alkylene or alkenylene which is optionally substituted with hydroxy; E represents phenylene or a single bond; G represents O, S, etc.; and Q represents carboxy, alkoxycarbonyl, etc.

Claims

1. A method for treating interstitial pneumonia comprising administering a fibrosis inhibitor containing 2-{4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}acetic acid, 2-{4[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulfonyl)acetamide or a pharmaceutically acceptable salt thereof as an active ingredient to a patient in need of such treatment.

.Iadd.2. The method according to claim 1, wherein the active ingredient is 2-{4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}acetic acid or a pharmaceutically acceptable salt thereof..Iaddend.

.Iadd.3. The method according to claim 2, wherein what is treated is pulmonary fibrosis..Iaddend.

.Iadd.4. The method according to claim 1, wherein the active ingredient is 2-{4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulfonyl)acetamide or a pharmaceutically acceptable salt thereof..Iaddend.

.Iadd.5. The method according to claim 4, wherein what is treated is pulmonary fibrosis..Iaddend.

Description

EXAMPLES

(1) The present invention will now be illustrated in more detail by way of the following test examples although the present invention is not limited to the scope mentioned in the examples.

Test Example 1

(2) (1) Methods

(3) Human Lung fibroblasts (manufactured by Lonza Walkersville; hereinafter, the same product will be used) were cultured in growth medium, which consisted of basal medium for human lung fibroblasts (manufactured by Lonza Walkersville; hereinafter, the same product will be used and referred to as basal medium) with FGM-2 additional factor set (manufactured by Lonza Walkersville; hereinafter, the same product will be used) under the condition of 37 C. and 5% CO.sub.2. The human lung fibroblasts were seeded in 96-well plate at 110.sup.3 cell/well and incubated overnight in growth medium.

(4) The fibroblasts were washed once with 100 L of phosphate-buffered saline (manufactured by Nissui Seiyaku; hereinafter, the same product will be used) and 100 L of basal medium was added thereto followed by incubation for 24 hours. They were further washed with 100 L of phosphate-buffered saline once and then basal medium was added in an amount of 80 L/well. Ten L of a 100 M solution of the compound A or the compound B was added to each well and incubated for 2 hours. The solution added thereto was prepared in such a manner that the compound A or B was previously dissolved in dimethyl sulfoxide (DMSO) and the resulting 10 mM solution was diluted 100-fold with basal medium. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with basal medium was used.

(5) Then a 100 ng/mL solution of epidermal growth factor (EGF) (manufactured by Pepro Tech) was added in an amount of 10 L to each well and incubated for 48 hours. For a non-stimulated control group, the basal medium was used.

(6) After incubating for 48 hours, absorbance at 490 nm was measured using Cell Titer 96, AQueus Assay (manufactured by Promega) for the analysis of cell proliferation activity. For the measurement of the absorbance, a microplate reader (Benchmark, manufactured by Bio-Rad; hereinafter, the same one will be used) was used.

(7) (2) Results

(8) As shown in FIG. 1, cell proliferation activity of the human lung fibroblasts significantly increased by stimulation with EGF. On the contrary, in the cells treated with the compounds A or B, the cell proliferation activity significantly decreased as compared with the control group.

Test Example 2

(9) (1) Methods

(10) Human lung fibroblasts were incubated in growth medium under the condition of 37 C. and 5% CO.sub.2. The human lung fibroblasts were seeded in 96-well plate at 310.sup.3 cell/well and incubated overnight in growth medium.

(11) The fibroblasts were washed once with 100 L of phosphate-buffered saline. Then basal medium was added thereto and incubation was conducted for 24 hours. They were further washed with 100 L of phosphate-buffered saline once and then basal medium was added in an amount of 80 L/well. Ten L of a 1 M solution of the compound A was added to each well and incubated for 2 hours. The solution used therefor was prepared in such a manner that the compound A was previously dissolved in DMSO and the resulting 1 mM solution was diluted 100-fold with basal medium. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with the basal medium was used.

(12) Then a 100 ng/mL solution of transforming growth factor-(TGF-) (manufactured by Chemicon) was added in an amount of 10 L to each well and incubated for 48 hours. For a non-stimulated control group, the basal medium was used.

(13) After incubating for 48 hours, absorbance at 490 nm was measured, in the same way as in Test Example 1, using a reagent for the analysis of cell proliferation activity. For the measurement of the absorbance, a microplate reader was used.

(14) (2) Results

(15) As shown in FIG. 2, cell proliferation activity of the human lung fibroblasts significantly increased by stimulation with TOF-. On the contrary, in the cells treated with the compound A, the cell proliferation activity significantly decreased as compared with that of the control group.

Test Example 3

(16) (1) Methods

(17) Human lung fibroblasts were seeded in 96-well plate at 510.sup.3 cells/well and incubated in the growth medium in the same way as in Test Example 1 overnight. The fibroblasts were washed once with 100 L of basal medium and 100 L of basal medium was added thereto followed by incubating for 24 hours. They were further washed once with 100 L of basal medium and then 80 L/well of basal medium was added. A 0.1, 1, 10 or 100 l solution of the compound A was added in an amount of 10 L to each well. The solution used therefor was prepared in such a manner that the compound A was previously dissolved in DMSO and adjusted to a concentration of 10 M, 100 M, 1 mM or 10 mM, followed by a 100-fold dilution with the basal medium. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with basal medium was added in an amount of 10 L. After incubating for 2 hours, 10 L of 100 ng/mL transforming growth factor 1 (TGF1) (manufactured by Pepro Tech; hereinafter, the same product will be used) was added and, for the non-stimulated control group, 10 L of basal medium was added. After incubating for 48 hours, the medium was recovered for measuring the concentration of the type I pro-collagen C terminal peptide (PIP) in the medium, and after an addition of 100 L of the basal medium, the cell proliferation activity was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (manufactured by Nacalai Tesque). Measurement of the cell proliferation activity was conducted in such a manner that 10 L of a 5 mg/mL MTT stock solution was added to each well, and after incubation fur 4 hours, 100 L of a 0.04M isopropanol solution of hydrochloric acid was added to each well, followed by the absorbance measurement at 595 nm (reference wavelength: 655 nm) using a microplate reader. The PIP concentration after incubation for 48 hours was measured using a type I pro-collagen C-peptide (PIP) EIA kit (manufactured by Takara; hereinafter, the same one will be used) in accordance with the manual attached thereto. The evaluation of this test was carried out using the value (index for collagen production) calculated as a relative value where a mean value of the non-stimulated control group was defined as 1 after correcting the measured value of PIP concentration for the cell proliferation activity value (absorbance).

(18) (2) Results

(19) As shown in FIG. 3, production of collagen significantly increased in the human lung fibroblasts by stimulation with TGF 1. On the contrary, in the cells treated with the compound A, the collagen production significantly decreased as compared with that of the control group.

Test Example 4

(20) (1) Methods

(21) Human lung fibroblasts were seeded in 24-well plate at 110.sup.5 cells/well and incubated in growth medium in the same way as in Test Example 1 overnight. The fibroblasts were washed once with 500 L of basal medium, and added with 500 L of basal medium followed by incubation for 24 hours. They were further washed once with 500 L of basal medium and then 400 L/well of basal medium was added. A 1, 10 or 100 M solution of the compound A prepared by the same manner as in Test Example 3 was added in an amount of 50 L to each well. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with basal medium was added in an amount of 50 L. After incubating for 2 hours, 50 L of a 100 ng/mL TGF 1 solution was added to each well and, for the non-stimulated control group, 50 L of basal medium was added. After incubating for 24 hours, RNA was extracted using an SV Total RNA Isolation System (manufactured by Invitrogen) and the 1st strand cDNA was synthesized from the RNA using a Superscript III (manufactured by Invitrogen). Using the cDNA prepared as above as a template, expressed amount of mRNA for type I collagen 1 chain (COL 11), type I collagen 2 chain (COL 12), smooth muscle actin (ACTA), TGF 1 and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were measured by means of a real-time quantitative PCR method. The real-time quantitative PCR was conducted using Platinum SYBR Green qPCR Super-Mix-UDG with ROX (manufactured by Invitrogen) and a primer being specific to each gene in accordance with the manual attached to the Platinum SYBR Green qPCR Super-Mix-UDG with ROX using ABI PRISM 7000 (Applied Biosystems). The evaluation of this test was carried out using the value (mRNA expression level) calculated as a relative value where a mean value of the non-stimulated control group was defined as 1 after correcting the expressed value of mRNA of each gene for the expressed amount of mRMA of GAPDH.

(22) (2) Results

(23) As shown in FIGS. 4 to 7, in the human lung fibroblasts, each mRNA expression level increased by stimulation with TGF l. On the contrary, in the cells treated with the compound A, each mRNA level decreased as compared with that of the control group.

Test Example 5

(24) (1) Methods

(25) Rat renal interstitial cells (NRF 49F cells) were seeded in 96-well plate at 110.sup.4 cells/well and incubated in an Minimum Essential Medium (MEM medium; manufactured by Nippon Seiyaku; hereinafter, the same product will be used) containing 10% of bovine fetal serum (manufactured by JRH Bioscience; hereinafter, the same product will be used) under the condition of 37 C. and 5% CO.sub.2 overnight. The cells were washed once with an MEM medium containing no bovine fetal serum (hereinafter, referred to as serum-free MEM medium), and 100 L of the serum-free MEM medium was added thereto followed by incubation for 24 hours. After washing with serum-free MEM medium once, serum-free MEM medium was added in an amount of 80 L/well. A 100 M solution of the compound A prepared by the same method as in Test Example 1 was added in an amount of 10 L to each well. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with serum-free MEM medium was added in an amount of 10 L. After incubating for 2 hours, 10 L of a 100 ng/mL platelet-derived growth factor BB (PDGF BB; manufactured by Sigma) solution was added to each well and, for the non-stimulated control group, 10 L of serum-free MEM medium was added followed by incubating. After incubating for 48 hours, cell proliferation activity (absorbance) was measured by an MTT method in the same way as in Test Example 3. The evaluation of this test was carried out using the value (cell proliferation level) expressed as a relative value where a mean value of absorption of the non-stimulating control group was defined as 1.

(26) (2) Results

(27) As shown in FIG. 8, in the rat renal interstitial cells, cell proliferation level significantly increased by stimulation with PDGF BB. On the contrary, in the cells treated with the compound A, the cell proliferation level significantly decreased as compared with that of the control group.

Test Example 6

(28) (1) Methods

(29) Human skin fibroblasts (manufactured by Kurabo; hereinafter, the same product will be used) were seeded in 96-well plate at 510.sup.3 cells/well and incubated in a Dulbecco's modified Eagle's medium (DMEM medium; manufactured by Nippon Seiyaku; hereinafter, the same product will be used) containing 10% of bovine fetal serum under the condition of 37 C. and 5% CO.sub.2 overnight. The human skin fibroblasts were washed once with an DMEM medium containing no bovine fetal serum (hereinafter, referred to as serum-free DMEM medium), and 100 L of serum-free DMEM medium was added thereto followed by incubation for 24 hours. After washing with serum-free DMEM medium once more, serum-free DMEM medium was added in an amount of 80 L/well. Ten L of the 0.1, 1, 10 or 100 M solution of the compound A prepared by the same method as in Test Example 3 was added to each well. For the non-stimulated control group and the control group, DMSO which was diluted 100-fold with serum-free DMEM medium was added in an amount of 10 L each and followed by incubating. After incubating for 2 hours, 10 L of a 100 ng/mL TGF 1 solution was added to each well and, for the non-stimulated control group, each 10 L of serum-free MEM medium was added. After incubating for 48 hours, cell proliferation activity (absorbance) was measured by the MTT method, and PIP concentration in the medium was measured in the same way as in Test Example 3. The evaluation of this test was carried out using the value (index for production of collagen) calculated as a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the measured value of PIP concentration using the cell proliferation activity value (absorbance).

(30) (2) Results

(31) As shown in FIG. 9, in the human skin fibroblasts, production of collagen significantly increased by stimulation with TGF 1. On the contrary, in the cells treated with the compound A, production of collagen significantly decreased as compared with that of the control group.

BRIEF DESCRIPTION OF DRAWINGS

(32) FIG. 1 shows the inhibitive effects of the compound A and the compound B on the proliferation of human lung fibroblasts stimulated by EGF. An ordinate shows absorbance.

(33) FIG. 2 shows the inhibitive effects of the compound A and the compound B on the proliferation of human lung fibroblasts stimulated by TGF. An ordinate shows absorbance.

(34) FIG. 3 shows the inhibitive effect of the compound A on the production of collagen in human lung fibroblasts stimulated by TGF 1. An ordinate shows the value calculated as a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the measured value for PIP concentration by the cell proliferation activity value (absorbance).

(35) FIG. 4 shows the inhibitive effect of the compound A on COL 11 mRNA expression in human lung fibroblasts stimulated by TGF 1. An ordinate shows a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the expressed amount of mRNA by COL 11 using the expressed amount of mRNA by GAPDH.

(36) FIG. 5 shows the inhibitive effect of the compound A on COL 12 mRNA expression in human lung fibroblasts stimulated by TGF 1. An ordinate shows a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the expressed amount of mRNA by COL 12 using the expressed amount of mRNA by GAPDH.

(37) FIG. 6 shows the inhibitive effect of the compound A on ACTA mRNA expression in human lung fibroblasts stimulated by TGF 1. An ordinate shows a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the expressed amount of mRNA by ACTA using the expressed amount of mRNA by GAPDH.

(38) FIG. 7 shows the inhibitive effect of the compound A on TGF 1 mRNA expression in human lung fibroblasts stimulated by TGF 1. An ordinate shows a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the expressed amount of mRNA by TGF 1 using the expressed amount of mRNA by GAPDH.

(39) FIG. 8 shows the inhibitive effect of the compound A on the proliferation of rat renal interstitial cells stimulated by PDGF BB. An ordinate shows a relative value where a mean value of absorbance in the non-stimulated control group was defined as 1.

(40) FIG. 9 shows the inhibitive effect of the compound A on the production of collagen in human skin fibroblasts stimulated by TGF 1. An ordinate shows a value calculated as a relative value where a mean value in the non-stimulated control group was defined as 1 after correcting the measured value of PIP concentration by the cell proliferation activity value (absorbance).