Fibrosis inhibitor

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and medicine and concerns a fibrosis inhibitor containing 2-{4-N-(5,6-diphenyl-pyrazin-2-yl)-N-isopropylamino]butyloxy}acetic aci or a pharmaceutically acceptable salt thereof or 2-{4-[N-(5,6-diphenyl-pyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulphonyl)acetamide or a pharmaceutically acceptable salt thereof as an active ingredient for treating interstitial pneumonia, pulmonary fibrosis, scleroderma or hepatic cirrhosis.

EFFECT: inhibitor provides high efficacy.

6 cl, 0 dwg, 6 ex

 

The technical field to which the invention relates.

The present invention relates to an inhibitor of fibrosis containing heterocyclic derivative (in the present description hereinafter referred to as "the present heterocyclic derivative (1)")represented by the following General formula (1) or its pharmaceutically acceptable salt as an active ingredient;

In the formula (1) R1and R2are the same or different and each represents an optionally substituted aryl, the substituents are the same or different and from one to three substituents selected from the group consisting of halogen atom, alkyl, halogenoalkane, arylalkyl, alkoxy, alkylthio, alkoxyalkyl, alkylsulfonyl, hydroxy, amino, monoalkylamines, dialkylamino, carboxy, cyano and nitro;

R3and R4are the same or different and each represents a hydrogen atom or alkyl;

R5 represents a hydrogen atom, alkyl, or halogen atom;

Y is N or N→O;

And represents NR6, and R6represents a hydrogen atom, alkyl, alkenyl or cycloalkyl;

D represents alkylene or albaniles, which is optionally substituted by hydroxy, or a and D are joined together with formation of divalent group represented by the trail of the soup by the formula (2)

[in the formula (2) r is an integer from 0 to 2, q represents 2 or 3 and t is an integer from 0 to 4];

E is phenylene or a simple bond, or D and E combine with education divalent group represented by the following formula (3)

(--- represents a simple bond or double bond.)

[in the formula (3) u is an integer from 0 to 2, and v represents 0 or 1];

G represents O, S, SO or SO2; and

Q represents carboxy, alkoxycarbonyl, tetrazolyl, carbarnoyl, monoalkylammonium, dialkylammonium or a group represented by the following formula (4).

[In the formula (4), R7represents amino, monoalkylamines, dialkylamino, hydroxy, any of the following groups 1)to (4), which optionally is substituted by 1 to 3 substituents selected from the group consisting of halogen atom, alkyl, halogenoalkane, arylalkyl, alkoxy, alkylthio, alkoxyalkyl, alkylsulfonyl, hydroxy, amino, monoalkylamines, dialkylamino, carboxy, cyano and nitro;

1) alkyl,

2) aryl,

3) aryloxy and

4) heterocyclic group.]

The level of technology

Fibrosis of organs occurs so that the extracellular matrix is excessively accumulated in the organization who tries through invasion or damage to organs due to any reason. When the degree of damage to organs due to infestation or damage is light, the scar remains, and the bodies returned to normal. However, when the degree of damage to the organs because of infestation or damage is severe or persistent, fibrosis scar leads to deterioration of its inherent functions. In addition, he calls the new fibrosis and forms a vicious circle of fibrosis, and, in the end, there is dysfunction of organs.

As diseases caused by fibrosis of organs, was known interstitial pneumonia (pulmonary fibrosis), etc. Interstitial pneumonia is a disease in which inflammation occurs in the alveolar wall because of some reasons; fibroblasts proliferate in the intermediate fabric; easy compacted due to excessive deposition of collagen fibers; impaired gas exchange; and, in the end, there is respiratory distress. After the occurrence of the disease, the patient dies, on average, within three to five years. The detailed mechanism of the pathogenesis of interstitial pneumonia has not yet been explained and has not yet been installed and available treatment method.

It was recently reported that in a murine model of interstitial pneumonia caused by bleomycin, ONO-1301, which is an agonist of the receptor prostaglandin I2(hereafter referred to as PGI 2"), has an inhibitory effect on interstitial pneumonia (see, for example, Reputedly document 1).

That the present heterocyclic derivative (1) or its pharmaceutically acceptable salt suitable for treatment of pulmonary hypertension and obstructive atherosclerosis as an agonist of the PGI2 receptor, has been reported (see, for example, Patent document 1).

Patent document 1: international publication WO 02/088084

Reputedly document 1: American J. discrimination, 2006, vol. 290, pages 59 to 65

Description of the invention

The problem addressed by the invention

The main purpose of the present invention is the provision of new inhibitors of fibrosis.

Tools for problem solving

The authors of the present invention, it was found that the present heterocyclic derivative (1) has an inhibiting effect on the growth of fibroblasts, and achieved the present invention.

An example of the present invention is an inhibitor of fibrosis containing the present heterocyclic derivative (1) or its pharmaceutically acceptable salt as an active component.

The best option is the implementation of the present invention

In the present heterocyclic derivative (1), it is preferable that:

R1and R2are the same or different and each represents neobyazatel is substituted phenyl, and the substituents are the same or different and from one to three substituents selected from the group consisting of halogen atom, alkyl and alkoxy;

R3and R4are the same or different and each represents a hydrogen atom or alkyl;

R5represents a hydrogen atom;

Y represents N;

And represents NR6and R6represents alkyl;

D represents alkylene;

E represents a simple bond;

G represents O; and

Q represents carboxy or a group represented by the following formula (4), and R7represents amino, monoalkylamines, dialkylamino, hydroxy, or any of the following groups 1) to (4), which optionally is substituted by 1-3 substituents selected from the group consisting of halogen atom, alkyl, halogenoalkane, arylalkyl, alkoxy, alkylthio, alkoxyalkyl, alkylsulfonyl, hydroxy, amino, monoalkylamines, dialkylamino, carboxy, cyano and nitro;

1) alkyl,

2) aryl,

3) aryloxy, and

4) heterocyclic group.

More specifically, 2-{4-[N-(5,6-diphenyl-pyrazin-2-yl)-N-isopropylamino]butylochki}acetic acid (hereafter referred to as "compound a") and 2-{4-[N-(5,6-diphenylpyrazine-2-yl)-N-isopropylamino]butylochki}-N-(methylsulphonyl)ndimethylacetamide (hereafter referred to as "compound B") are the I, for example, preferred.

As for the "alkyl" in the present invention, which has an unbranched or branched chain, containing 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl or isohexyl can be given as an example. In particular, alkyl containing 1-4 carbon atoms, is preferred.

As for the alkyl group in the "halogenoalkane", "arylalkyl", "alkylthio", "alkoxyalkyl", "alkylsulfonyl", "monoalkylamines", "dialkylamino", "monoalkylbenzenes" and "diallylbarbituric" in the present invention, which is the same as the aforementioned alkyl, which may be described as an example.

As for the "alkoxy" in the present invention, which has an unbranched or branched chain, containing 1 to 6 carbon atoms, for example methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentylamine, n-hexyloxy or etexilate can be given as an example. In particular, alkoxy containing 1-4 carbon atoms, is preferred.

As for alkoxygroup "alkoxycarbonyl" and "alkoxyalkyl" which is the same as already mentioned alkoxy, which can be cast in quality is TBE example.

As for "alkenyl" in the present invention, which has an unbranched or branched chain containing 2 to 6 carbon atoms, e.g. vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl can be given as an example. In particular, alkenyl containing 3 or 4 carbon atoms, is preferred.

As for "cycloalkyl" in the present invention, which has 3-8 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl can be given as an example. In particular, cycloalkyl containing from 5 to 7 carbon atoms, is preferred.

As for the "halogen atom" in the present invention, a fluorine atom, chlorine atom, bromine atom and iodine atom can be given as an example.

As for the "aryl" in the present invention, which has 6 to 10 carbon atoms, for example, phenyl, 1-naphthyl or 2-naphthyl can be mentioned as an example. In particular, phenyl is preferred.

As for the aryl group in the "arylalkyl and aryloxy" in the present invention, which is the same as the above-mentioned aryl, which may be described as an example.

What concerning the Xia of alkylene" in the present invention, which is unbranched or branched chain, containing from 1 to 8 carbon atoms, for example, methylene, ethylene, 1-methylation, 2-mutilation, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethine or octamethylene can be given as an example. In particular, alkylene containing 3-6 carbon atoms is preferred, and alkylene containing 4 carbon atoms is more preferred.

As for "Alcanena" in the present invention, which has an unbranched or branched chain, containing 2-8 carbon atoms, for example, ethenylene, 1-propanole, 2-propanole, 1-butylen, 2-butylen, 3-butylen, 1-penttinen, 2-penttinen, 3-penttinen, 4-penttinen, 4-methyl-3-penttinen, 1-hexarelin, 2-hexarelin, 3-hexarelin, 4-hexarelin, 5-hexarelin, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-hoktanyan, 2-hoktanyan, 3-hoktanyan, 4-hoktanyan, 5-hoktanyan, 6-hoktanyan or 7-hoktanyan can be given as an example. In particular, albaniles containing 3-6 carbon atoms is preferred, and albaniles containing 4 carbon atoms is more preferred.

As for the "heterocyclic group" in the present invention, the following (1) or (2) can be given as an example.

(1) five or six-membered aromaticity the ring group, containing 1-4 heteroatoms selected from a nitrogen atom, oxygen atom and sulfur atom, or its condensed benzene ring and the nitrogen atom and the sulfur atom can form the oxide, when the atom contained in the ring is a nitrogen atom or a sulfur atom. Its examples include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-indolyl, 2-furanyl, 3-furanyl, 3-benzofuranyl, 2-thienyl, 3-thienyl, 3-benzothiazol, 1,3-oxazol-2-yl, 4-isooxazolyl, 2-thiazolyl, 5-thiazolyl, 2-benzothiazolyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 2-benzimidazolyl, 1H-1,2,4-triazole-1-yl, 1H-tetrazol-5-yl, 2H-tetrazol-5-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-pyrazolyl, 2-pyrimidinyl 4-pyrimidinyl, 2-pyrazinyl, and 1,3,5-triazine-2-yl.

(2) four to eight-membered saturated ring group which optionally has from one to four identical or different nitrogen atoms, oxygen atoms or sulfur atoms, or condensed benzene ring and the nitrogen atom and the sulfur atom can form the oxide, when the atom contained in the ring is a nitrogen atom or a sulfur atom. Its examples include piperidine, piperazinil, 3-methylpiperazin-1-yl, homopiperazin, morpholino, thiomorpholine, 1-pyrrolidinyl, 2-pyrrolidinyl and 2-tetrahydrofuranyl.

The present heterocyclic derivative (1) can be synthesized by the method mentioned in Patent document 1 (international public who acacia WO 02/088084).

Although the present heterocyclic derivative (1) can be used as a drug only in the form of free base or acid, it is also possible to use by converting them into the form of a pharmaceutically acceptable salt by known methods.

Examples of "salt", when the present heterocyclic derivative (1) shows basicity, include salts with inorganic acid, such as chloromethane acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid or Hydrobromic acid, and organic acid, such as acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonate, econsultation, benzosulfimide, p-toluensulfonate, naphthalenesulfonate or camphorsulfacid.

Examples of "salt", when the present heterocyclic derivative (1) shows the acidity include salts of alkaline metal such as sodium or potassium salts, and salts of alkaline earth metal such as calcium salts.

The present heterocyclic derivative (1) are geometrical isomers (Z and E substances), and each of the geometric isomers and their mixtures are also included in the present heterocyclic derivative(1). Some of the present heterocyclic derivative (1) have an asymmetric carbon(s), and each of the optical isomers and racemic substance is also included in the present heterocyclic derivative (1). The optical isomer can be obtained by exposure to racemic compounds, obtained as described above, the separation of optical isomers by using a known method using an optically active acid such as tartaric acid, bentivegna acid, mandelic acid or 10-camphorsulfacid), using the basicity or using the previously obtained optically active compound as a material.

Inhibitor of fibrosis according to the present invention can be used to treat the following diseases, for example, are involved in fibrosis of organs or tissues.

(1) Renal failure

Tubulointerstitial jade

(2) Respiratory diseases

Interstitial pneumonia (pulmonary fibrosis)

(3) gastrointestinal diseases

Cirrhosis of the liver, chronic pancreatitis and sirrodney stomach cancer

(4) cardiovascular diseases

Myocardial fibrosis

(5) Diseases of bones and joints

Fibrosis of the bone marrow and rheumatoid arthritis

(6) diseases of the skin

Postoperative scar, burn scar, keloid, hypertrophic scar and sclerodermia

(7) Obstetric diseases

Uterine fibroids

(8) Urologic diseases

Prostatic hypertrophy

(9) Other diseases

Alzheimer's disease, sclerosing peritonitis, diabetes type I and postoperative spike.

Inhibitor of fibrosis according to the present invention constitutes the present heterocyclic derivative (1) by itself or contains it in pharmaceutically acceptable, non-toxic and inert carrier in an amount in the range from 0.01 to 99.5% or, preferably, in the range from 0.5 to 90%.

Examples of the carrier include solid, semi-solid or liquid diluent, filler and other optional agents for pharmaceutical compositions. They can be used alone or as a mixture of two or more agents.

Inhibitor of fibrosis according to the present invention may be in any form of oral preparations such as powder, capsules, tablets, pills, coated with sugar, granules, diluted powder, suspension, liquid, syrup, elixir or the toffee and parenteral preparations such as injection solution or suppository in solid or liquid standard dose. It can also be in the form of a drug with a slow release. Among them, oral preparations such as tablets, are particularly preferred.

The powder can be made is here grinding the present heterocyclic derivative (1) into the corresponding small size.

The diluted powder it may be made so that the present heterocyclic derivative (1) make the corresponding small size and then mixed with a pharmaceutical carrier which likewise do a small size, such as food carbohydrate (such as starch and mannitol). Flavouring agent, preservative, dispersing agent, dye, perfume, etc. may be optionally added.

Capsules can be made so that the powder or diluted powder, which is made in the form of a powder, as mentioned above, or granules, which will be mentioned under the name of tablets, fill the shell of a capsule such as a gelatin capsule. They can also be manufactured in such a way that the powdered substance mixed with grease or thinning additive such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol, with the subsequent exposure operation of the filling. When the substance to improve raspadaemosti tablets or solubilizers component, such as carboxymethylcellulose, calcixerollic, hydroxypropylcellulose with a low degree of substitution, croscarmellose sodium, sodium carboximetilkrahmal, calcium carbonate or sodium carbonate, add, efficiency, l is drug money, if swallowed capsules, can be improved. It is also possible to fine powder of the present heterocyclic derivative (1) was suspended/dispersed in vegetable oil, polyethylene glycol, glycerin or a surface-active substance and Packed in sheet gelatin with reception of a preparation of soft capsules.

Tablets can be manufactured in such a way that a powder mixture obtained by adding a filler and turned into pellets or the workpiece, and then the substance to improve raspadaemosti tablets or grease, add to it and then convert into tablets.

Powdery mixture can be obtained by mixing appropriately converted into a powder substance with the above-described diluent or base. If necessary, you can add a binding component (such as carboxymethylcellulose sodium, methylcellulose, hypromellose, gelatin, polyvinylpyrrolidone or polyvinyl alcohol), an additive that slows the liquefaction process (such as paraffin), reabsorbable substance (such as a Quaternary salt), adsorbent (such as bentonite or kaolin), etc.

Powdery mixture can be converted into granules in such a way that it first makes crude, using, for example, syrup, starch paste, acacia, the solution is cellulose or polymer solution, mixed under stirring and then dried, followed by rubbing. Instead of turning the powder into granules as such, it is also possible to place the powder in a tablet machine, and the workpiece incomplete forms grind with obtaining granules. When a lubricant such as stearic acid, stearate, talc or mineral oil, is added to the granules obtained in this way, the adhesion of the granules to each other can be prevented.

Tablets can also be made so that the present heterocyclic derivative (1) is mixed with a liquid inert carrier and then directly produce tablets without conducting the above-described stages of obtaining granules or pieces.

The tablets thus obtained may be film-coated or sugar. It is also possible to apply a transparent or translucent protective coating comprising a tightly closed shellcove film, coating, including sugar or polymeric material or polished floor, including wax.

Other oral drugs, such as liquid, syrup, toffee or elixir, it is also possible to turn into a standard drug dose, in which a predetermined quantity contains a predetermined amount of the present heterocyclic derivative (1).

The syrup can be made by dissolved what I present heterocyclic derivative (1) in the corresponding aqueous solution of aromatic additives. The elixir can be made using non-toxic alcoholic media.

The suspension can be produced by dispersion of the present heterocyclic derivative (1) non-toxic carrier. If necessary, you can add solubilizers component or emulsifier (such as ethoxycarbonyl isostearoyl alcohol or polyoxyethylene ester of sorbitol), preservative or component that provides fragrance (such as peppermint oil or saccharin).

If necessary, a standard dosage form for oral administration can be turned into microcapsules. The above form can also be covered or filled polymer or wax with obtaining prolonged action or slow release of the active component.

The parenteral preparation can be in standard liquid dosage forms for subcutaneous, intramuscular or intravenous injection, such as in the form of a solution or suspension. The parenteral preparation can be made so that a predetermined number of the present heterocyclic derivative (1) is suspended or dissolved in a non-toxic liquid carrier, given the goal of introduction, such as aqueous or oily medium, and then the suspension or solution is sterilized. Non-toxic salt or Rast is the PR can add to to make solution for injection isotonic. It is also possible to add a stabilizer, a preservative, an emulsifier and the like.

The suppository can be produced by dissolution or suspension of the present heterocyclic derivative (1) in fusible and soluble or insoluble solid substance, such as polyethylene glycol, cocoa butter, semi-synthetic grease/oil (such as Witepsol (registered trademark)), higher esters (such as ministervalletta) or their mixture.

Despite the fact that the dose of the inhibitor of fibrosis according to the present invention may vary depending on the condition of the patient, such as body weight or age, route of administration or the degree of the symptom, the range from 0.01 mg to 1000 mg/day as the number of the present heterocyclic derivative (1) mainly suitable for adult, and range from 0.1 mg to 100 mg is preferable. In some cases, the dose is less than the above may be sufficient or, on the other hand, the dose more than the above may be necessary. It is also possible to introduce one or more times a day, or to enter with an interval of one or a few days.

Examples

The present invention will be further illustrated in more detail in the following test examples, although the present the invention is not limited by the scope of the claims, mentioned in the examples.

Test example 1

(1) Methods

The lung fibroblasts human (manufactured by Lonza Walkersville; next will be used the same product) was cultured in a nutrient medium, which consists of a basal medium for fibroblasts easy man (manufactured by Lonza Walkersville; next will be used the same product and is called "basal medium"), with a set of additional factors FGM-2 (manufactured by Lonza Walkersville; next will be used the same product) under conditions of 37°C and 5% CO2. Fibroblasts easy person were cultivated in 96-well plate at 1×103cells/well and incubated overnight in growth medium.

Fibroblasts were washed once with 100 μl of saline phosphate buffer (manufactured by Nissui Seiyaku; next will be used the same product), and it was added 100 μl of basal medium followed by incubation for 24 hours. They were additionally washed once with 100 µl of physiological solution with phosphate buffer and then added to the basal medium in the amount of 80 μl/well. Ten μl of a 100 μm solution of compound a or compound B was added to each well and incubated for 2 hours. Added them in the solution was obtained in such a way that the compound a or B was first dissolved in dimethyl sulfoxide (DMSO), and received a 10 mmol solution time is alali 100-fold the basal medium. For a group restimulating control and the control group used DMSO, which was diluted 100-fold the basal medium.

Then 100 ng/ml epidermal growth factor (EGF) (manufactured by Pepro Tech) was added in an amount of 10 μl to each well and incubated for 48 hours. For a group restimulating control used basal medium.

After incubation for 48 hours was measured by the absorbance at 490 nm using Cell Titer 96, AQueous Assay (manufactured by Promega) to analyze the activity of cell proliferation. For measuring the absorption of the used reader for microplates (Benchmark, manufactured by Bio-Rad; next will be used the same instrument).

(2) the Results

As shown in figure 1, the activity of cell proliferation of lung fibroblasts human has been greatly enhanced by stimulation with EGF. In contrast, in cells treated with compounds a or B, the activity of cell proliferation was significantly decreased compared to the control group.

Test example 2

(1) Methods

The lung fibroblasts human incubated in a nutrient medium under conditions of 37°C and 5% CO2. Fibroblasts easy person were cultivated in 96-well plate at 3×103cells/well and incubated overnight in growth medium.

Fibroblasts were washed once with 100 µl of the physical and the logical solution with phosphate buffer. Then it was added to the basal medium, and incubation was carried out for 24 hours. They additionally were washed in 100 μl of physiological solution with phosphate buffer once, and then was added to the basal medium in the amount of 80 μl/well. Ten μl of a 1 μm solution of the compound was added to each well and incubated for 2 hours. Used the solution was obtained in such a way that the connection is first dissolved in DMSO, and the resulting 1 mmol solution was diluted 100-fold the basal medium. For a group restimulating control and the control group used DMSO, which was diluted 100-fold the basal medium.

Then 100 ng/ml transforming factor-α(TGF-α) (manufactured by Chemicon) was added in an amount of 10 μl to each well and incubated for 48 hours. For a group restimulating control used basal medium.

After incubation for 48 hours was measured by the absorbance at 490 nm, in the same manner as in test example 1, using the reagent for analysis of cell proliferation. For measuring the absorption of the used reader for microplates.

(2) the Results

As shown in figure 2, the activity of cell proliferation of lung fibroblasts human has been greatly enhanced by stimulation with TGF-α. In contrast, in cells, processing the data connection And, the activity of cell proliferation was significantly decreased compared to that of the control group.

Test example 3

(1) Methods

Fibroblasts easy person were cultivated in 96-well plate at 5×103cells/well, and incubated in a nutrient medium in the same manner as in test example 1 all night. Fibroblasts were washed once with 100 μl of basal medium, and 100 μl of basal medium was added followed by incubation for 24 hours. They were additionally washed once with 100 μl of basal medium, and then 80 μl/well was added to the basal medium. of 0.1, 1, 10 or 100 μm solution of the compound was added in an amount of 10 μl to each well. Added them in the solution was obtained in such a way that the connection is first dissolved in DMSO and made up to a concentration of 10 µm, 100 µm, 1 µm or 10 µm, with a further 100-fold dilution of the basal medium. For a group restimulating control and the control group, DMSO, was diluted 100-fold the basal medium was added in an amount of 10 μl. After incubation for 2 hours, 10 μl of 100 ng/ml transforming factor β1 (TGFβ1) (manufactured by Pepro Tech; next will be used the same product) was added and, for a group restimulating control was added 10 μl of basal medium. After incubation for 48 hours, the medium was removed to change the value of concentration With terminal peptide of procollagen type I (PIP) in the environment, and after adding 100 μl of basal medium, we measured the activity of cell proliferation, using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT) (manufactured by Nacalai Tesque). Measurement of the activity of cell proliferation was performed so that 10 μl of 5 mg/ml MTT stock solution was added to each well, and after incubation for 4 hours, 100 μl of 0,04M isopropanolic solution chloroethanol acid was added to each well, followed by measurement of absorbance at 595 nm (reference wavelength: 655 nm), using the reader for microplates. The concentration of PIP after incubation for 48 hours was measured using EIA kit C-peptide of procollagen type I (PIP) (manufactured by Takara; next will be used the same set) in accordance with the attached instructions. Assessment this test was performed using the value (the index for the production of collagen), calculated as a relative value, where the average value of the group restimulating control was defined as 1 after correction of the measured value of the concentration of PIP values for the activity of cell proliferation (absorption).

(2) the Results

As shown in Figure 3, the production of collagen significantly increased in lung fibroblasts human due to stimulation with TGFβ1. In contrast, in cells treated joint is m a, the production of collagen was significantly decreased compared to that of the control group.

Test example 4

(1) Methods

Fibroblasts easy person were cultivated in 24-well plate at 1×105cells/well, and incubated in a nutrient medium in the same manner as in test example 1 all night. Fibroblasts were washed once with 500 μl of basal medium, and dobavlyali μl of basal medium followed by incubation for 24 hours. They were additionally washed once with 500 μl of basal medium and then 400 μl/well was added to the basal medium. 1, 10 or 100 μm solution of the compound obtained in the same manner as in test example 3, was added in the amount of 50 μl to each well. For a group restimulating control and the control group, DMSO, was diluted 100-fold the basal medium was added in a quantity of 50 μl. After incubation for 2 hours, 50 μl of 100 ng/ml TGFβ1 solution was added to each well, and for a group restimulating control was added 50 μl of basal medium. After incubation for 24 hours, RNA was extracted using the system for the allocation of the total RNA SV (manufactured by Invitrogen), and the first chain cDNA was synthesized from RNA using SuperScript III (manufactured by Invitrogen). Using cDNA obtained as described above as a template, the amount of mRNA expressed 1 chain of type I collagen (COL 1α1), 2 chain of type I collagen (COL 1α2), (smooth muscle actin (ACTA), TGF β1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured using the method of quantitative PCR performed in real time. Quantitative PCR performed in real time was performed using Platinum SYBR Green qPCR Super Mix-UDG with ROX (manufactured by Invitrogen) and primer specific to each gene in accordance with the instructions attached to the Platinum SYBR Green qPCR Super Mix-UDG with ROX, using ABI PRISM 7000 (Applied Biosystems). Assessment this test was performed using the value (the level of expression of mRNA), calculated as a relative value in which the average value of the group restimulating control was defined as 1 after correction values expressed mRNA for each gene downregulation of the number of GAPDH mRNA.

(2) the Results

As shown in Figures 4-7, in lung fibroblasts person, for each level of mRNA expression was increased by stimulation with TGF β1. In contrast, in cells treated with compound a, each mRNA level was decreased compared with that in the control group.

Test example 5

(1) Methods

Rat renal interstitiality (cells NRF 49F) were cultivated in 96-well plate at 1×104cells/well, and incubated in minimum essential medium (MEM medium; manufactured by Nippon Seiyaku; next, you will use the van the same product), containing 10% fetal bovine serum (manufactured by JRH Bioscience; next will be used the same product) under conditions of 37°C and 5% CO2the whole night. Cells were washed once with MEM medium containing no fetal bovine serum (hereafter referred to as "MEM medium without serum)and 100 μl of MEM medium without serum was added followed by incubation for 24 hours. After washing MEM medium without serum once with MEM medium without serum was added in the amount of 80 μl/well. a 100 μm solution of the compound obtained using the same method as in test example 1, was added in an amount of 10 μl to each well. For a group restimulating control and the control group, DMSO, was diluted 100 times with MEM medium without serum was added in an amount of 10 μl. After incubation for 2 hours, 10 μl of 100 ng/ml of platelet growth factor BB (PDGF BB; manufactured by Sigma) was added to each well, and, for a group restimulating control 10 μl of MEM medium without serum was added, followed by incubation. After incubation for 48 hours, the activity of cell proliferation (absorbance) was measured using MTT method in the same manner as in test example 3. Assessment this test was performed using the value (the level of cell proliferation), expressed as a relative value in which the average mn is an increase in the absorption estimulando control group was defined as 1.

(2) the Results

As shown in Fig, in the rat kidney interstitiality the level of cell proliferation was significantly increased by stimulation with PDGF BB. In contrast, in cells treated with compound a, the level of cell proliferation was significantly decreased compared with that in the control group.

Test example 6

(1) Methods

Fibroblasts of human skin (manufactured by Kurabo; next will be used the same product) were cultivated in 96-well plate at 5×103cells/well, and incubated in a modified way of Dulbecco environment Needle (DMEM medium; manufactured by Nippon Seiyaku; next will be used the same product)containing 10% fetal bovine serum, under conditions of 37°C and 5% CO2the whole night. Fibroblasts of human skin were washed once with DMEM medium containing no fetal bovine serum (hereafter referred to as "DMEM medium without serum")was added 100 μl of DMEM medium without serum, followed by incubation for 24 hours. After washing with DMEM medium without serum again, DMEM medium without serum was added in the amount of 80 μl/well. Ten μl of 0.1, 1, 10 or 100 μm solution of the compound obtained using the same method as in test example 3, was added to each well. For a group restimulating control and the control group, LCA is, which was diluted 100-fold with DMEM medium without serum was added in an amount of 10 μl into each well followed by incubation. After incubation for 2 hours was added 10 μl of 100 ng/ml TGF β1 solution to each well, and for a group restimulating control was added to each 10 μl of MEM medium without serum. After incubation for 48 hours, the activity of cell proliferation (absorbance) was measured using MTT method, and the concentration of PIP in the medium was measured in the same manner as in test example 3. Assessment this test was performed using the value (the index for the production of collagen), calculated as a relative value in which the average value in the group restimulating control was defined as 1 after correction of the measured value of the concentration of PIP, using the value of active cell proliferation (absorption).

(2) the Results

As shown in Fig.9, in fibroblasts of human skin producing collagen significantly increased by stimulation with TGF β1. In contrast, in cells treated with compound a, the production of collagen was significantly decreased compared to that of the control group.

Brief description of drawings

1

Figure 1 shows the inhibiting effects of compounds a and B on the proliferation of lung fibroblasts human-driven what's with EGF. The ordinate shows absorbance.

2

Figure 2 shows the inhibiting effects of compounds a and B on the proliferation of lung fibroblasts human stimulated with TGFα. The ordinate shows absorbance.

3

Figure 3 shows the inhibitory effect of the compounds And the production of collagen in lung fibroblasts human stimulated with TGF β1. The ordinate shows the value calculated as a relative value in which the average value in the group restimulating control was defined as 1 after correction of the measured value for the concentration of PIP using the activity values for cell proliferation (absorption).

4

Figure 4 shows the inhibitory effect of the compounds And on the expression of COL 1α1 mRNA in lung fibroblasts human stimulated with TGF β1. The ordinate shows the relative value in which the average value in the group restimulating control was defined as 1 after adjusting the downregulation of the number of mRNA using COL 1α1, using the amount of mRNA expressed using GAPDH.

5

Figure 5 shows the inhibitory effect of the compounds And on the expression of COL 1α2 mRNA in lung fibroblasts human stimulated with TGF β1. The ordinate shows the relative value in which the average value in the group nest is melirovanie control was defined as 1 after adjusting the downregulation of the number of mRNA using COL 1α2, using the amount of mRNA expressed using GAPDH.

6

Figure 6 shows the inhibitory effect of the compounds And on the expression of ACTA mRNA in lung fibroblasts human stimulated with TGF β1. The ordinate shows the relative value in which the average value in the group restimulating control was defined as 1 after adjusting the downregulation of the number of mRNA using ACTA, using the amount of mRNA expressed using GAPDH.

Fig.7.

7 shows the inhibitory effect of the compounds And on the expression of TGF β1 mRNA in lung fibroblasts human stimulated with TGF β1. The ordinate shows the relative value in which the average value in the group restimulating control was defined as 1 after adjusting the downregulation of the number of mRNA using TGF β1, using the amount of mRNA expressed using GAPDH.

Fig

On Fig shows the inhibiting effect of the compounds And on the proliferation of rat renal interstititial stimulated with PDGF BB. The ordinate shows the relative value at which the absorbance value of the group restimulating control was defined as 1.

Fig.9

Figure 9 shows the inhibitory effect of the compounds And the production of collagen in the fibroblasts of human skin, stimulated with POM is by TGF β1. The ordinate shows the value calculated as a relative value in which the average value in the group restimulating control was defined as 1 after correction of the measured value of the concentration of PIP using the activity values for cell proliferation (absorption).

1. Inhibitor of fibrosis containing 2-{4-[N-(5,6-diphenyl-pyrazin-2-yl)-N-isopropylamino]butylochki} acetic acid or its pharmaceutically acceptable salt as an active component.

2. Inhibitor of fibrosis according to claim 1 for the treatment of diseases selected from the group consisting of interstitial pneumonia, pulmonary fibrosis, scleroderma and cirrhosis of the liver.

3. Inhibitor of fibrosis according to claim 2 for the treatment of interstitial pneumonia.

4. Inhibitor of fibrosis containing 2-{4-[N-(5,6-diphenyl-pyrazin-2-yl)-N-isopropylamino]butylochki}-N-(methylsulphonyl)ndimethylacetamide or its pharmaceutically acceptable salt as an active component.

5. Inhibitor of fibrosis according to claim 4 for the treatment of diseases selected from the group consisting of interstitial pneumonia, pulmonary fibrosis, scleroderma and cirrhosis of the liver.

6. Inhibitor of fibrosis according to claim 5 for the treatment of interstitial pneumonia.



 

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SUBSTANCE: method refers to veterinary science and aims at maintaining the physiological status in newborn calves. The method involves using the biologically active substance coredon. Coredon is pre-mixed with ascorbic acid. This mixture is dissolved in water, fed to calves in the amount of 2 g, once a day, from the second after-birth day for 5 days.

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5 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: dermal flap is simulated in laboratory animals on the second day of the experiment. On the first and fourth day of the experiment, minoxidil is introduced intraperitoneally in a daily dose of 1.0 mg/kg in two stages.

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FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. The agent represents a mixture of peptides of molecular weight 500-900 Da prepared of the brain of cattle and/or pigs of one year old or younger, particularly the midbrain (mesencephalon), namely the central grey substance surrounding the Sylvian aqueduct (aquaeductus cerebri Sylvii) with the peptide mixture concentration in the agent of 5.0-10.0 mg/ml of the aqueous solution or the content of 5.0-10.0 mg/g in a witepsol suppository. The method involves grinding the cattle and/or porcine frozen midbrain, hydrolysis at +20°C in the presence of pepsin, extraction, separation of the extract from ballast substances by separation through materials with retention potential more than 2000 Da, separation of the purified extract by fluid extraction at critical temperature CO2 31.1°C and critical pressure 7.3±0.1 atm, separation of the fraction of 500-900 Da, dissolution in water and freeze drying. The agent is applied in the form of the solution in water in the active substance concentration of 5.0-10.0 mg/ml for single parenteral administration in a dose of 10-30 mg, or in the form of a witepsol suppository with the active substance concentration of 5.0-10.0 mg/ml for single rectal administration in a dose of 10-30 mg.

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3 cl, 5 ex, 10 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

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SUBSTANCE: invention relates to veterinary science and may be used to increase the radioactive irradiation sensitivity in animal tissues. That is ensured by the pre-radiation parenteral administration of gamma-aminobutyric acid lithium salt in a dose of 40 to 120 mg per 1 kg of body weight.

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FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely neurology, and concerns treating multiple sclerosis. That is ensured by the oral introduction of the S1P receptor modulator representing 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol or a pharmaceutically acceptable salt thereof in a daily dose of 0.5 mg.

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41 cl, 5 tbl, 9 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and aims at prevention of the purulent complications of pancreatonecrosis. Patient's intestinal mucosal permeability is evaluated by the mannitol/lactulose test. If the urine mannitol/lactulose relation falls within the range of 0.03 to 0.05, a nasojejunal catheter is used to introduce 0.25% Derinat 10ml dissolved in physiological saline 50 ml into the intestine. If the urine mannitol/lactulose relation exceeds 0.05, 0.25% Derinat 20ml dissolved in physiological saline 50 ml is introduced into the intestine using the nasojejunal catheter. The preparation is introduced once a day for 3-4 days.

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FIELD: medicine.

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FIELD: medicine.

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6 cl, 8 dwg, 16 tbl, 12 ex

FIELD: medicine.

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1 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

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14 cl, 2 ex

FIELD: medicine, pharmaceutics.

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Organic compounds // 2496479

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing an inhaled fine or crystalline glycopyrronium powder salt. The declared method involves suspending the crystalline glycopyrronium salt in acetone to prepare a suspension, homogenising the suspension at pressure 500-2000 bar to prepare glycopyrronium salt particles at average particle size less than 10 mcm, and drying the glycopyrronium salt particles to remove residual acetone if any.

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7 cl, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

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Synbiotic mixture // 2495927

FIELD: chemistry.

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6 cl, 1 tbl, 1 ex

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11 cl, 3 dwg, 6 ex

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8 cl, 5 dwg, 1 tbl

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16 cl, 2 tbl, 26 ex

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