The means strengthening anti-inflammatory effect of the immunosuppressant (options), the method of strengthening the anti-inflammatory action of immunosuppressant, a method of treating inflammation

 

(57) Abstract:

The invention can be used in medicine for the treatment of such inflammatory processes, as prolonged inflammation (chronic progressive inflammation or inflammation, which is involved in the immunological mechanism. The means strengthening anti-inflammatory effect of immunosuppressant represents at least one of glycosaminoglycan from a group that includes dermatooncology, heparin, heparansulfate, chondroitin sulfate, keratinolytic and their pharmaceutically acceptable salts. Method of strengthening anti-inflammatory action of the immunosuppressant is to use a combination of it with glycosaminoglycans selected from the above group. A method of treating inflammation includes an introduction to the mammal an effective amount of an anti-inflammatory agent containing as an effective ingredient dermatologit or its pharmaceutically acceptable salt, and immunosuppressant. The stated use of dermatosurgery or its pharmaceutically acceptable salt as a means of enhancing the anti-inflammatory effect of immunosuppressant when receiving anti-inflammatory drugs is vospalitelnoe action of the immunosuppressant, to reduce its quantity. The result can be weakened by the severe adverse effects of immunosuppressant. 5 S. and 11 C.p. f-crystals, 3 tab., 10 Il.

This invention relates to an anti-inflammatory agent, particularly an anti-inflammatory agent against inflammatory processes, as prolonged inflammation (chronic progressive inflammation or inflammation, which involved immunological mechanism.

In particular, this invention relates to a potentiating substance immunosuppressant, reinforcing its anti-inflammatory effect, or it means strengthening anti-inflammatory effect.

According to this invention can be prevented or cured the beginning and/or development of diseases such as glomerulonephritis, collagen disorders, or related diseases, intractable anghit, immunological abnormalities, accompanied by inflammation, or autoimmune diseases, etc.

Background of the invention

The inflammatory process by involving the immune mechanism, for example, glomerulonephritis, collagen disorders, or related diseases, intractable Eski progress, giving repeated sharp aggravation, and without treatment, causes organic disturbances, leading to kidney failure or other organic disorders and leads, ultimately, to death.

It is assumed that most of these chronic progressive inflammation intractable, for the reason that, as is, they are associated with immunological abnormalities, and so on. are accompanied by continuous activation of the mediator of the inflammatory process. Researched therapies focused on these factors. For example, in relation to immunological abnormalities, applied in the usual way or by way of saving intravenous infusion therapy (pulse therapy) such an immunosuppressant, as adrenocorticosteroid, cyclophosphamide, mizoribine, methotrexate or azathioprine. However, adrenocorticosteroid has dangerous adverse effects, such as the overwhelming effects on adrenal function, ease of communicable infection, peptic ulcer, hypertension, osteoporosis, etc. in Addition, other immunosuppressive drugs also are extremely dangerous adverse effects, such as bone marrow depression, the occurrence of gastrointestinal resstr is so In addition, due to the fact that this type of pharmacotherapy is no causal therapy is the administration of a medicinal product for a long period of time, which leads to dangerous adverse effects, there are particularly serious problems. In addition, some of these diseases are resistant to the use of medicines. In diseases in which the main pathological changes are jade or anguita, can be used protivotromboznoe funds or anticoagulants. However, even the use of anticoagulant such as heparin or warfarin (brand name; common name, kalyanpuri) may be accompanied as adverse effects of bleeding. As for protivotromboznoe funds, it is considered that there is also a risk of bleeding. Because among patients with collagen disorders many patients with low platelets, as in the case of systemic lupus erythematosus (SLE), or many patients with pulmonary hemorrhage, as in the case of the syndrome of Good pasture, followed by glomerulonephritis, in the above cases apply protivotromboznoe funds. Further, although recently it was reported that the gap is Osadcha examination of the patent application of Japan 40327/1990, Arthritis Rheum. 33, 1554, 1990), experimental results obtained by the applicants of this application, the effect is not reliable enough, as described below.

Description of the invention

The inflammatory process by involving the immune mechanism, such as glomerulonephritis, collagen disorders, or related diseases, intractable anghit, immunological abnormalities, followed by inflammation or autoimmune disease, and so on, includes the above-described SLE. Considering the above situation, and investigating the effect of dermatosurgery or its pharmaceutically acceptable salt in these inflammatory diseases using MRL (lpr/lpr) mouse (Izui, S., et al. Mechanism of genetic control of murine systemic lupus lupus., In systemic Lupus Lupus, pp. 3-12, Ed. Peter A. Miescher, Springer-Verlag, Berlin, 1995) as a model of SLE on animals, the applicants have found that the connection not only reduces inflammation and slows the progression of the disease, but also very safe and can be used for long-term use. In addition, the effect of dermatosurgery investigated using an animal model of human multiple sclerosis and rats with experimental allergic encephalomyelitis in another example of the inflammatory process, when the Redi various kinds of dermatosurgery specific type of dermatosurgery with specific physical properties or a specific origin or its pharmacologically acceptable salt and maintain concentration in the serum for a long time and are preferred. It is found that the practical application of this invention suitable dermatologit or its pharmaceutically acceptable salt, which include 2-9%, preferably 3-8%, Di-OS (HexAl ---> 3GalNAc); the characteristic viscosity of 0.8 to 2.0 (100 ml/g) and molecular weight 25.000-100.000, preferably 30.000-60.000, which was determined by gel permeation GHUR, and fractions of known molecular weight as standard (see reference example 1 (1) Determination of molecular weight, as described in Biochim. Biophys. Acta, 60-70, 1992); or dermatologit obtained from crest. This invention is made on the basis of these observations.

The purpose of this invention is to obtain anti-inflammatory drugs with significant anti-inflammatory effect on the inflammatory process and use dermatologit or its salts are especially effective for prevention or treatment of refractory inflammatory disease, such as glomerulonephritis, which is an inflammatory process associated with an immunological mechanism, or prolonged inflammation, collagen disorders, or related diseases, intractable anguita, immunological abnormalities, accompanied by inflammation or autonoleggiochiesavalmalenco action of immunosuppressant in combination with glycosaminoglycans, having a sulfate group.

The present invention relates to an anti-inflammatory agent, comprising as an active ingredient dermatologit or its pharmaceutically acceptable salt, as described above. In particular, this invention relates to an anti-inflammatory agent, comprising as an active ingredient dermatologit that have anti-inflammatory effects on prolonged inflammation or an inflammatory process with an immunological mechanism, such as inflammation associated with autoimmune disease. More specifically, the present invention relates to an anti-inflammatory agent, useful in inflammation involving multiple sclerosis, collagen disorders or related disease, glomerulonephritis, intractable anguita and the like. In addition, the present invention relates to an anti-inflammatory agent, including dermatologit with specific physical properties or a specific origin or its pharmaceutically acceptable salt. Further, the present invention relates to a synergistic effect of the agent that increases the anti-inflammatory effect immunodepressants as an effective ingredient. In addition, this invention relates to the agent, reinforcing anti-inflammatory effect of the immunosuppressant, which includes glycosaminoglycan having a sulfate group, or its pharmaceutically acceptable salt and immunosuppressive compound (immunosuppressant).

As a specific example of glycosaminoglycans and collagen, with a sulphate group to be used in combination with immunosuppressant may be dermatooncology, heparin, heparansulfate, chondroitin sulfate and keratinolytic. Among them, preferred dermatologit and more desirable, as indicated above, dermatologit with specific physical properties or a specific origin or its pharmaceutically acceptable salt.

Brief description of drawings

The figure 1 presents the results of the analysis of concentration of dermatosurgery in the blood (residual level) over time after administration to rats of dermatosurgery of different origin. The figure indicates dermatologit obtained from the comb, and means dermatologit derived from bovine intestine, x denotes overlapping and .

The figure 2 shows the effect of dermatosurgery on blood clotting mouse is.

The figure 3 shows the effect of dermatosurgery level in the serum component of serum, reflecting renal function mouse in example 2.

"a" denotes the results of the analysis BUN (Atomoxetine in the blood), and "b" denotes the results of the analysis of creatinine. * "b" indicates significant deviation from p<0.05 using research by Bonferroni.

The figure 4 shows a micrograph showing the results of the autopsy kidneys from mice treated with dermatosurgical (magnification: x500, - (bar is 10 μm).

"a" denotes the normal tissue of the kidney, "b" refers to the tissue of the kidney with MS (minimal change), and "c" refers to the tissue of the kidney with DP corresponding to the proliferative glomerulonephritis among diffuse glomerulonephritis defined by WHO (who) and subject to dysfunction).

The figure 5 shows the level of abnormal glomeruli with the introduction of mouse dermatosurgery in example 2.

"a" denotes the level number MS (minimally modified) glomeruli of the total number of glomeruli, and "b" denotes the level number DP glomeruli of the total number of glomeruli.

The figure 6 shows histological micrograph showing the renal artery when the mice entered dermatologit is, and "b", "c" and "d" represent the histology weak, medium and heavy anguita, respectively.

The figure 7 shows the results of the analysis of fibrinogen in the blood when administered to mice of dermatosurgery in example 2.

The figure 8 shows the impact of dermatosurgery (25 mg/kg) and each dose of endoksana (brand cyclophosphamide), with separate introduction or joint introduction, the level of creatinine in the serum of mice in example 3.

DS denotes dermatooncology, and End means Endoxan. * indicates a significant deviation from p<0.05 using research by Bonferroni.

The figure 9 shows the impact of dermatosurgery (25 mg/kg) and each dose of endoksana, the separate introduction or joint application of the weight of the spleen in mice in example 3.

DS denotes dermatooncology, and End means Endoxan.

The figure 10 shows the impact of dermatosurgery (25 mg/kg) and each dose of endoksana, the separate introduction or joint application of the weight of the mesenteric lymph node in mice in example 3.

** indicates a significant deviation from p<0,01 in the study by Bonferroni.

The best option practical embodiment of the invention

Chosenin-4-sulfate and L-iduronovoy acid and small amounts of D-glucuronic acid. The content of sulfate, the provisions of the accession of sulfate groups and the content of D-glucuronic acid dermatosurgery vary depending on the species, tissue or organ.

Usually dermatologit produced from such raw materials as the mucous membrane of the intestine, skin, lung, kidney, liver, pancreas, artery, spleen, brain, thymus gland, cartilage or the crest of animals such as cow, pig, horse, sheep, goat, rabbit, etc. or such birds, like chicken, duck, goose, etc.

Although the present invention is used dermatologit or its pharmaceutically acceptable salt (hereinafter, unless otherwise stated, it is implied that "dermatooncology" includes pharmaceutically acceptable salt) can be used any already known in this field connection and its physical properties and nature is not limited.

Typical examples include compounds obtained, for example, from organs (intestine, skin, kidneys, blood vessel) animal (mammal and birds and so on), more specifically, scallop, bovine intestine, pig intestine, pig skin, bovine kidney, pork kidney, bovine aorta, and physical properties, see, for example, in tables a relatively low molecular weight 1.600-10.000 known (Dol, F. et al. , J. Lab. Clin. Med., 1990, vol. 115, 43-51 or Bianchini, P. et al. Thrombosis and Haemostasis, 1991, vol. 65, 1315, or Barbanti, M. et al. Thrombosis and Haemostasis, 1993, vol. 69, 147-151) and can be used in this invention. The disaccharide composition of this invention is determined by the composition (%) of unsaturated disaccharide obtained known in this area by way of dermatooncology split with chondroitinase ABC to unsaturated disaccharide and fractionary cleavage products by GHWR using ion-exchange media (see, "2.8 Structure analysis by glycosaminoglycan degrading enzyme in association with HPLC" Shin-seikagaku-jikken-koza 3, Tou-situ II, p. 49-62 (1991) Tokyo-Called-doujin, especially, see reference example 1). Unsaturated disaccharide is usually represented by the signals shown in table 1, and the structure of the isomer represented by each signal, is illustrated by the following formula [1] and table 2.

The structure designated by abbreviation code in tables 1 and 2, sometimes referred to as follows: Di-OS: HexAl ---> 3GalNAc, Di-6S: HexAl ---> 3GalNAc (6S), Di-4S: HexAl ---> 3GalNAc (4S), Di-diSD: HexA (2S)1 ---> 3GalNAc (6S), Di-diSE: HexAl ---> 3GalNAc (4,6-diS), Di-diSH: HexA (2S)1 ---> 3GalNAc (4S), Di-triS: HexA (2S)1 ---> 3GalNAc (4,6-diS).

In the above formula HexA denotes unsaturated hexuronic acid and GalNAc represents N-acyl-D-galaxylife can be used according to this invention, but preferably use dermatosurgery with the following physical properties and origin, characterized by a large life time in the blood and minor adverse effects, such as bleeding and so on:

(1) the Content of Di-OS (HexAl ---> 3GalNAc) is 2-9%, preferably 3-8%; (2) 0,8-2,0 characteristic viscosity (100 ml/g); (3) 25.000-100.000, preferably 30.000-60.000, average molecular weight, determined by gel GHWR and fractions of known molecular weight as standard (method described in Biochim. Biophys. Acta, 1117, 60-70, 1992); (4) or dermatologit obtained from crest. More desirable dermatologit with the above four properties, or equivalent. A typical example of such dermatosurgery is the sodium salt of dermatosurgery (see table 1) obtained from the comb according to the method described by the present applicants in the description of the patent application (see PCT international publication WO 95/09188) and confirmed by the examples.

Dermatologit obtained from the crest, and dermatooncology derived from bovine intestine (see reference example 2 in WO 95/09188), injected into the tail vein of male SD rats, measured changes of the residual level of sulfate at the time of introduction (see example 1 in the above international publication). The figure 1 shows the changes of the residual level over time after administration of the two compounds in the blood. The figure indicates dermatologit obtained from the comb, and means dermatologit derived from bovine intestine. As shown in figure 1 results in 5 minutes after the introduction of the residual level of dermatosurgery obtained from the crest, more than double the amount for dermatosurgery derived from bovine intestine, and even after 120 minutes after administration of dermatosurgery obtained from crest remains about 10%, whereas dermatologit derived from bovine intestine, is determined with difficulty. So, as dermatologit obtained from crest, or dermatologit with equivalent physical properties has a long life period in blood and for a long time retains an anti-inflammatory effect, the treatment does not require a large dose, and dermatooncology obtained from crest or equivalent preferred. As anti-inflammatory agents according to this invention can be used pharmaceutically acceptable salt dermatosurgery.

In cachia salt, and usually enter the sodium salt.

Dermatologit usually injected, but if necessary, it can be injected intraarterially, intramuscularly or transdermally. Containing eye drops can be obtained in combination with the corresponding acceptable auxiliary ingredient and used for backfilling. His ointments or creams can also be obtained in combination with a reasonable basis and applied on the skin or mucous membrane. In addition, oral medication can also be obtained by the development of dosage forms.

With the aim of obtaining anti-inflammatory drugs or tools that enhance the anti-inflammatory effect, or a pharmaceutical preparation according to this invention can accordingly be used dermatologit or containing a sulfate group glycosaminoglycan and/or immunosuppressive compound (immunosuppressant) with such auxiliary ingredients as conventional stabilizer, emulsifier, an isotonic agent and/or agent that creates a certain pH acceptable to the medicine.

Dose dermatosurgery or its pharmaceutically acceptable salt for an adult per day is 50-3.000 mg. Rst is a few days.

In addition, containing a sulfate group glycosaminoglycan in combination with immunosuppressant can significantly improve the anti-inflammatory effect of immunosuppressant and reduce the number of immunosuppressant. As a result of severe adverse effects of immunosuppressant, caused by its introduction, can be weakened. Thus, the pharmaceutical agents comprising as an effective ingredient containing sulfate group of glycosaminoglycan, may enhance the anti-inflammatory effect of immunosuppressant and used as a means of improving the anti-inflammatory effect of immunosuppressant and reducing its dose, as a means of reducing adverse effects of immunosuppressant or in combination with immunosuppressant as a means of improving the anti-inflammatory effect and reduce the undesirable effects of the specified immunosuppressant. As containing the sulfate group of glycosaminoglycans and collagen, used in combination with immunosuppressant, preferably using dermatosurgery, especially dermatosurgery with the above physical properties and specified origin or its pharmaceutically acceptable salt.

The dose of immunosuppressant in combination with containing a sulfate group by glycosaminoglycans depends on the type of immunosuppressant, the condition of the patient (age, gender, type of illness, severity and so on), the route of administration (route of administration, dosage forms and so on) and generally cannot be precisely specified, but usually it is desirable to enter for a reception 30-70% of the dose of the immunosuppressant.

As immunosuppressive remedies or immunosuppressant used in sachetana can be used any immunosuppressant, it has anti-inflammatory effect produced by any industry or known immunosuppressant (see "Today's remedy (1994)", 1994, Nankodo publ; p 157-161. "Immunosuppressants" and "Adrenocortical steroid"). Typical examples of immunosuppressant or immunosuppressive compounds are adrenocorticosteroid, cyclophosphamide, azathioprine, mizoribine, cyclosporine, methotrexate, takrai-asherat etc. Typical example of adrenocorticosteroid are prednisolone, methylprednisolone, betamethasone, dexamethasone, paramethasone, triamcinolone, hydrocortisone and corticonuclear.

Anti-inflammatory agent according to this invention has an anti-inflammatory effect against inflammatory processes accompanying the following diseases, and can prevent, treat and alleviate these diseases in mammals, including humans (primates, including humans; domestic animals such as dogs, cats, etc.; livestock, such as cows, pigs, horses, etc) and birds (chickens, and so on).

(1) Glomerulonephritis

According to the WHO classification (who) (Churg, J. & Sokin, LH: Renal disease classification and atlas of glomerular disease. Tokyo and New York, Ikagu-shoin, 1982), glomerulonephritis is divided into

1) primary glomerular disease, 2) as glama the facilities in diseases of metabolism, 5) hereditary nephropathy, 6) versatile glomerular disease, 7) end-stage kidney, and 8) glomerular damage, accompanying (as a consequence) transplantation. Anti-inflammatory agents according to this invention is effective in primary glomerular disease at the above paragraph 1). If (A. small glomerular abnormalities, B. focal/segmental lesions, C. Diffuse glomerulonephritis and D. Unclassified glomerulonephritis) and specific diseases (lupus-nephritis, allergic purpura, lgA nephropathy (disease Berger), syndrome Goodpasture'a) related to glomerulonephritis, systemic disease, paragraph 2). In addition, they are effective against glomerular damage due to vascular disease in paragraph (3) (periarteritis nodosa, granulomatous Wegener'a, thrombotic microangiopathy, glomerular thrombosis, arteriosclerosis, arteriolosclerosis nephrosclerosis, scleroderma) and glomerular damage accompanying the transplantation under item 8).

(2) collagen disorders and related diseases

The collagen disorders and related diseases belong to the diffuse connective tissue diseases classified American Roar of the authorized categories, include rheumatoid arthritis, juvenile arthritis, lupus-erythematous, scleroderma, diffuse fasciitis, polymyositis, necrotizing vasculitis, and other forms of vasculopathy (polyarteritis polyarteritis, allergic Wegener, allergic anghit, granulomatous Takayasu, Kawasaki disease, Behcet's disease, and so on), Sjogren syndrome, overlap syndromes and other (polymyalgia of rheumatica, relapsing panniculitis, receivername polyhedric, lymphomatosis Wegener, nodular erythema and so on). In addition, there are also glomerulonephritis and vasculitis caused by these diseases. Anti-inflammatory agents according to this invention is effective in these diseases. In addition, they are also effective in human autoimmune lymphoproliferative syndrome (Cell 81, 935-946 (1995), Science 268, 1347-1349 (1995)).

(3) Intractable vasculitis

According to Fauci, AS The vasculitis syndrome. Harrison''s Principles of Internal Medicine, 11th ed., McGraw-Hill) intractable vasculitis is classified into systemic necrotizing vasculitis (classical multiple Takayasu, allergic granulomatous of angia (syndrome Churg-Strauss'a), polyarthritis nodular complicated syndrome), allergic vasculitis (exogenous stimulation proved or preliminaries drug vasculitis, associated with infectious diseases and endogenous antigens involved in the pathological process in this way, vasculitis, vasculitis associated with tumors, vasculitis, associated with disease of connective tissue, vasculitis associated with other diseases, vasculitis associated with congenital deficiencies of the complement system), Wegener's granulomatosis, giant cell arteritis diagnostics (temporal arteritis diagnostics, Takayasu's arteritis), other vasculitis syndromes (illness Kawasaki'a, isolated vasculitis of the Central nervous system, Buerger's disease (thromboangiitis obliterans) and anti-inflammatory agents according to this invention is effective in these diseases.

(4) Immunological abnormalities or autoimmune (autoimmune) disease

To immunological abnormalities or autoimmune diseases include inflammatory processes, followed by rejection after transplantation of various organs; nervous diseases, including multiple sclerosis, Guillain-Barre syndrome, HTLV-1 induced myelopathy and the like; gastrointestinal diseases, including inflammatory diseases of the gastrointestinal tract (clonal disease, nespetsificheskuyu autoimmune thyroid disease, such as goiter Hashimoto's, graves ' disease, insulin-dependent diabetes mellitus (Insula) and the like; diseases of the cardiovascular system, including idiopathic myocarditis, Chagas disease, dilated cardiomyopathy, endocardial myofibrosis folds, idiopathic benign pericarditis, eosinophilic endocarditis, postcardiotomy syndrome, postmyocardial infarction syndrome, antiphospholipid antibodies, and the like; respiratory diseases, including idiopathic interstitial pneumonia, pulmonary fibrosis and the like; renal diseases including interstitial nephritis, and the like; cutaneous diseases, including hand, foot, pemphigoid, immunogenic wet acquired bullous bullosa and the like; ophthalmic diseases, including caused by lenses uveitis syndrome Vogt-Koyanagi-Harada'a and the like (Autoimmune diseases and immunodeficiency, Recent internal medicine 22. Hiroo Imura, ed. Nakayama-shoten, 1993, pp. 91-206). Anti-inflammatory agents according to this invention is effective for these diseases.

The present invention disclosed in the following characteristic reference examples, examples and comparative examples:

Reference example 1

Getting dermatan the brand), and incubated for carrying out the process (digestive) cleavage at the 50oC for 12 hours. Digested solution is filtered through diatomaceous earth and bring the pH of the filtrate to 5.8 and 6.3. Add hyaluronidase (1.000 TRU), obtained from streptomyces griceus (Seikagaku-Corporation), and incubated for the flow of the digestive process of splitting at the 50oC for 3 hours. To the solution add sodium chloride to obtain a 0.65 M solution, which is applied on a column (3 x 20 cm) with anion-exchange resin Diaion HPA-10 (Mitsubishi chemical, trade name), equilibrated with 0.5 M salt solution. Then the column is washed sequentially with 0.5 l of a 0.65 M solution of salt and 0.3 l 1.1 M solution of salt, fractions, washed 1.8 M solution of salt, is collected and concentrated under reduced pressure. The concentrate is subjected to dialysis against distilled water overnight and concentrated to 10 ml, to which add 5 M solution of sodium hydroxide so that the final concentration was 0.5 M. After keeping alkaline solution at 37oC for 90 minutes, it is cooled and neutralized with acetic acid. Then to the neutralized solution add double the amount of ethanol, the precipitate is washed successively with 70% ethanol, cheseny product (5 g) leave overnight in 125 ml of water, a small amount of insoluble matter is separated by centrifugation (10oC, 10.000 rpm for 15 minutes), add 125 ml of an aqueous solution of sodium acetate and ethanol, so that its final concentration was 45%, and cooled in an ice bath. The solution was kept at 4oC for 20 hours and centrifuged, receiving the precipitate, which is washed successively with 90% ethanol, pure ethanol and diethyl ether and dried under reduced pressure in the presence of phosphorus oxide (V), obtaining the purified sodium salt of dermatosurgery (denoted by DS).

Average molecular weight, characteristic viscosity and composition of the disaccharide are shown in table 1. Of course, the isolation and purification of DS is not limited to the above, and can be used the method described in published the examined patent application Japan 9042/1985 or published in the examined patent application Japan 21241/1986.

(1) Determination of average molecular weight

The average molecular weight of DS is determined by the way Arai'and et al. (Biochim. Biophys. Acta, 1117, 60-70, 1992). It is defined (using as a standard of glycosaminoglycan with known molecular weight determined on permissions (IHVR), using connected in series (one after another) three columns: TSK gel G4000PWXL, TSK gel G3000PWXLand TSK gel G2500PWXL(anion h,8 mm in inner diameter, Toso).

As an eluting solution using 0.2 M salt solution at a flow rate of 0.6 ml/min, the determination is performed by differential refractometry.

(2) Analysis of disaccharide

According to known methods (see, "2-8 Structural analysis by HPLC using glycosaminoglycan degrading enzyme", described in Shin-seikagaku-jikken-kouza 3, Tousitu, II, p. 49-62, (1991, Tokyo-Kazaki-dojin)), the analysis of the location of sulfate groups in DS performed, as described below. DS split chondroitinase ABC, and the resulting disaccharide fraction (unsaturated disaccharide), including unsaturated disaccharide, analyze liquid chromatography high resolution (IHVR), and the result is compared with GHUR analytical result of the product obtained by processing said fraction of unsaturated disaccharide chondro-6-sulfatase. Experimental conditions food cleavage using chondroitinase ABC and desulphurization with chondro-6-sulfatase and GHUR analysis of the following:

a) Food cleavage using chondroitinase ABC

By way Yoshida'a et al. (Anal. Biochem., 77, 372-333 (1989)), 20 ál of 0.4 M tanago solution DS (10 mg/ml) and 20 μl chondroitinase ABC (5 u/ml) and incubated at 37oC within 2 hours.

b) Food split under the action of chondro-6-sulfatase

To 100 ml of the above product splitting chondroitinase ABC add 20 ál of chondro-6-sulfatase (5 u/ml), dissolved in 20 mm solution of Tris-acetate buffer (pH 7.0), and incubated at 37oC within 2 hours.

c) Analysis method GHUR

50 μl of the above solution of the product of cleavage by chondroitinase ABC or solution of the product of cleavage of the chondro-6-sulfatase analyze GHUR (Hitachi Seisakusho) using ion-exchange column (YMC-Pack PA-120-S5 column, 250 x 2.6 mm inner diameter) and determine the absorbance at 232 nm. Elution carry 800 mm acidic phosphate with a gradient of 0-100% for 60 min at a flow rate of 1.5 ml/min. Determine peaks corresponding unsaturated disaccharides with different position of sulfate groups.

(3) Measurement of the characteristic viscosity

Characteristic viscosity DS measured according 12th ed. Japanese Pharmacopeia.

Analytical instruments: instrument for automatic measurement of viscosity, VMC-052 (Rigo) when using the viscometer Ubbelohde.

Solvent: 0.2 M sodium chloride solution (the same solution that was used on the manual measurements: characteristic viscosity is determined by segment, clip on vertical axes, where the decreasing viscosity ( red) lay on the vertical axis and the concentration of the sample along the horizontal axis.

red = (ts/t0-1)/C

in the above equation, ts: flow rate 0.2 M sodium chloride solution containing DS, to: the flow rate of solvent (0.2 M sodium chloride solution containing no DS), C: concentration of the sample (wt.%).

Table 1 summarizes the results of measurements of the physical properties of the above DS obtained from the comb, and other well-known DS (bovine intestine, pork intestine, pork skin). DS derived from bovine intestine, porcine intestine and pig skin obtained by the method described in patent application WO 95/09188.

Example 1

We studied the effects of DS and low molecular weight heparin (LMWHep; trade name, Fragmin, Pharmacia, Kissei-yakuhin-kogyo) on the coagulation of blood.

(1) Experimental materials

Using male C57BL/6 mice at the age of ten weeks (each group consists of 3 mice). DS and LMWHep dissolved in sterile physiological solution.

(2) Experimental method

After the introduction of the DS (25 mg/kg) and LMWHep (1 mg/kg) in the tail vein of mice selected blood samples with defined p the>The figure 2 shows changes in APTT after administration of each compound over time, where denotes the case of the introduction of LMWHep and refers to the case DS (mean standard error (sec)). As follows from figure 2, APTT in LMWHep () is still above 300 seconds, even after 30 minutes after administration, this means that LMWHep inhibits the activity of coagulation factor and the risk of bleeding is very high. In contrast, after 60 minutes after introduction of the APTT-prolonging activity is not observed, which means that LMWHep is removed from the blood for a short time and you cannot expect that it will last for a long time. On the other hand, after 5 minutes after the introduction of the DS is observed only weak APTT, prolonging the activity of DS (), which means no risk of bleeding. In addition, this activity can be registered even after 240 minutes after administration, this means that DS has a safe and more sustainable impact than LMWHep. As described above, since more than 1 mg/kg LMWHep may cause unwanted effects bleeding maximum dose LMWHep in subsequent experiments was 1 mg/kg Because even LMWHep, improved in relation to antikoaguliruyuschey activity, can cause cu is necessary.

Example 2

Research using the SLE model-1 in mice

Confirmation impacts separately applied dermatosurgery

(1) Experimental materials

As SLE models use male MRL (lpr/lpr) mice at the age of 17 weeks, but as a normal control using C57BL/6 mice of the same age and sex. As medicines, DS or heparin or low molecular weight (LMWHep, Fragmin), obtained by the method of the standard example 1, is dissolved before use in sterile physiological solution.

(2) Experimental method

Take blood samples from mice at the age of 17 weeks, selected only the MRL mice with nephritis, which have a high content of urea nitrogen, blood (BUN) was at approximately the same level, and divided into 4 groups, each group consisting of 5 mice. One group (untreated control group) was injected with a sterile saline solution, 3 other groups in the tail vein injected once a day and five times per week 5 mg/kg DS 25 mg/kg DS and 1 mg/kg LMWHep. After 4 weeks draw samples of blood during anesthesia with Nembutal and after the killing of animals by blood-letting excised kidneys. Taken whey and presledovaniya.

(3) the Results (see figure 3-6)

1) the Effect on renal function

The results of the analysis of BUN and creatinine is shown in figure 3a and figure 3b, respectively (figures shows the level in serum (mg/DL) of each substance). In figure 3a, PRE (dotted line) shows the average value of the BUN to the beginning of the introduction of agents (the age of 17 weeks). As shown in the figure, this value is considerably higher than the corresponding value for normal control group (C57BL/6), this means that MRL mice developed nephritis. In figure 3b, * indicates that there is a significant difference with p<0,05 from the untreated control group when analysed by Bonferroni. As the level of BUN and creatinine levels untreated control group is higher than the corresponding values for the normal control group (C57BL/6), from which it is clear that the functioning of kidney deterioration. Among the treated groups, BUN level slightly improved in the 5 mg/kg DS group, 25 mg/kg DS group and 1 mg/kg LMWHep group. Among the treated groups, the creatinine level is also slightly improved in the 5 mg/kg DS group and 1 mg/kg LMWHep group and significantly improved in the 25 mg/kg DS group.

2) Impact on the results of histopathological examination of kidneys

Figures 4a-c represent histologic kidney and dannyh mice. Figure 4a shows the histological picture of the normal control, and the rest of the relevant data for MRL mice (magnification x 500). The size of the straight horizontal line the base of each photo 10 microns.

Figure 5a represents the number of MILLISECONDS of glomeruli/total number of glomeruli (%), and figure 5b gives the number of DP glomeruli/total number of glomeruli (%).

According to the results of microscopic examination obtained by autopsy kidney of the mouse, the picture is clear glomerulonephritis was observed in the untreated control group, where no normal glomeruli (figure 4a), (1) based on the who classification refers to minimally modified (MS) type with slightly exaggerated loop and cell proliferation (figure 4b), (2) the glomeruli, which have almost completely lost the cavity of the capsules Bowman'and vascular cavity and filled with cellular components and sediment whose functions are impaired (according proliferative glomerulonephritis (hereinafter denoted DP) diffuse glomerulonephritis according to the who classification, figure 4c) and (3) illustrates the situation between (1) and (2). These results are gradual pathological changes in renal glomeruli MRL (lpr/lpr) mice from MS to DP, in which the cavity of the capsules Bowman'and vascular lesions is and immune complex.

The measurement of the number MS of glomeruli/total number of glomeruli (%) and number of DP glomeruli/total number of glomeruli (%), shows that the level of MS glomeruli increases significantly in the 25 mg/kg DS group and slightly increased in 5 mg/kg DS group and 1 mg/kg LMWHep group (figure 5a). On the other hand, the level of DP

glomeruli decreases depending on the dose of 5 mg/kg DS group and 25 mg/kg DS group, whereas in LMWHep group such effect is not observed (figure 5b).

As described above, histopathological study found that DS has an effect, prevent the development of nephritis. LMWHep has little effect, while maintaining a minimally modified (MS) group, but no effect on the already progressive nephritis.

3) Impact on histopathological changes in renal artery

Figure 6a represents the histological picture of the renal artery of normal control mouse, and figures 6b-d represent the histological picture of the renal artery MRL mouse, showing histopathological changes in renal artery at each stage (magnification: x 204). The size of the straight horizontal line the base of each pictures 50 μm. In the microscopic examination of the following histopathological Okrug blood vessel (figure 6b), medium: inflammatory hepatocellular infiltration around the blood vessel and the collapse of the outer shell and the middle part of the shell (degeneration vacuoles, deposition of fibrin) (figure 6c), severe inflammatory hepatocellular infiltration around the blood vessel and the collapse of the outer shell and the middle part of the shell (degeneration vacuoles, deposition of fibrin, proliferation of smooth muscle cells) and inflammatory infiltration of the inner and middle part of the membrane (figure 6d).

Anghit in each group evaluate histopathological using as parameters these histological picture. As a result, 25 mg/kg DS group reduced the number of States, characterized by severe degree, and the number of States increases medium and low severity (table 3). There is a big difference between 1 mg/kg LMWHep group, 5 mg/kg DS group and the untreated group (table 3). Of these histopathological observations, it follows that DS has a vast impact on anyit.

4) impact on the level of fibrinogen in the blood

Fibrinogen is one of the highly reactive compounds and its level increases not only with glomerulonephritis and anguita, but also when different, .A. and Cole, E. H. CRC Press, Boca Raton 1994). As shown in figure 7, this variable parameter for the untreated group MRL (lpr/lpr) mice are significantly higher than normal control group, indicating a severe inflammatory process in the untreated group. 5 mg/kg and 25 mg/kg DS group this increase was suppressed depending on the dose, but such action is barely expressed in LMWHep.

Example 3

Research using the SLE model-2

Confirmation of joint action dermatosurgery with cyclophosphamide

(1) Experimental materials

As the use of experimental animals male MRL (lpr/lpr) mice at the age of ten weeks, and as agent - DS obtained in reference example 1 by dissolving it before use in sterile physiological solution.

In each vial are dissolved for injection Endoxan (Cyclophosphamide, Shionogi) (100 mg/vial) by adding 5 ml of distilled water for injection, and then add sterile saline so that the concentration of endoksana was 4, 6 and 8 mg/ml In the negative control group (untreated group) was injected with a sterile saline solution.

(2) Experimental method

Determine the weight of the body is eatenin serum. Animals are divided into 5 groups of 6 mice each, separated by a continuous randomization, so that the average value and standard deviation in each group was approximately the same as in the other groups. DS impose daily 5 times a week (except Saturday and Sunday) in the tail vein of mice using a 1 ml disposable syringes at a dose of 25 mg/kg/day. Cyclophosphamide is administered intraperitoneally once a week at any of the doses: 20 mg/kg, 15 mg/kg or 10 mg/kg, as described in the literature, where the same agent was studied in mice (Shiraki, M. et al., Clin. Exp. Immunol., 55, 333-339 (1984)). During the introduction of animals every day thoroughly inspected. After 8 weeks of taking the blood samples under anesthesia with Nembutal and kill animals by blood-letting. Then measure the level of serum creatinine, weight, spleen weight and mesenteric lymph node.

(3) the Results (see figures 8-10)

1) the Effect on renal function

The figure 8 shows the results of the analysis of the level of serum creatinine (mg/DL). Compared with negative control group one-time introduction of 20 mg/kg of endoksana not has no effect on creatinine levels. However, the joint introduction of endoksana (10 mg/kg and 20 endoxana with DS significantly improves the level (p<0,05). Especially should pay attention to the fact that both joint injection, 20 mg/kg of endoksana with 25 mg/kg DS and 15 mg/kg endoksana with 25 mg/kg DS, give better results than the separate introduction of 20 mg/kg of endoksana. Next, group the joint introduction of 15 mg/kg endoksana with 25 mg/kg DS gives much better results than a group of separate injection of 20 mg/kg of endoksana, and also found that DS reduces the toxicity of endoksana and is effective.

2) Impact on the weight of the spleen

The weight of the spleen in MRL mice abnormally heavier than normal mice. Been tested for the treatment of enlarged spleen in MRL mice. The figure 9 shows the results of measuring the weight of the spleen. Compared with negative control group, a group of separate injection of 10 mg/kg of endoksana no effect, but all other groups give effective results in splenomegaly. Dose DS in all cases is 25 mg/kg noteworthy that the result is that if you use the same number of endoksana, the group of the joint influence of endoksana with DS gives much better results than a group of separate introduction of endoksana.

3) Effect on the weight of mesenteric lymph node

The weight m on the treatment of this anomaly. The figure 10 shows the results of the weighing of the mesenteric lymph node. Compared with negative control group, there was a decrease of weight of the lymph node in all other groups. It is important that the group of combined effects of 10 mg/kg of endoksana with DS gives better results than a group of separate injection of 10 mg/kg of endoksana. Dose DS in all cases is 25 mg/kg

Based on the results of example 3, it is established that the combination of DS with immunosuppressant increases anti-inflammatory effect of immunosuppressant and gives the same or better effect than the individual use of immunosuppressant at a certain dose level (hereinafter referred to as A dose). That is, it is found that combining a smaller number of immunosuppressant than the dose A, s DS (DS dose not limited) gives a better effect than a single dose of A immunosuppressant. Since DS is one of glycosaminoglycans containing a sulfate group, a combination of other, containing sulfate group fractions with immunosuppressant, will give the same effect.

Example 4

A study using a model of multiple sclerosis (MS) in rats

Study the impact of DS on experimetrix, which is autoimmune and represents a characteristic disease with immunologic abnormalities of the Central nervous system (Clinical immunology illustrated, R. 112-117, Brostoff, Scadding, Male, Roitted., Shun-ichi Hirose, Shogo Kano, Tornio Tada, transl., Nanko-do, 1994).

(1) Experimental materials

As experimental animals using 4-week-old female Lewis rats and divided into two groups, each of 10-12 rats. As an immunizing agent used myelin-basic protein of Guinea pigs (hereinafter referred to GPMBP) with a full additive Freund'a, including Mycobacterium tuberculosis (MTB). As agent for use DS obtained by the standard method of example 1 by dissolving it before use in sterile saline. Next, the group entered DS is referred to as the DS group. The group of negative control was injected with saline.

(2) Experimental methods

Each rat immunized injection up to 12.5 µg GPMBP and 200 µg MTB. Starting from the third day after immunization administered intraperitoneally injected once daily with a sterile saline solution in the DS group and negative control group, respectively. Clinical symptoms that characterize EAE, expressed in digits, as follows: (hereinafter called the clinically whom she 3; paralysis of the upper extremities - 4; death - 5.

The symptoms of each rat daily numerically evaluate, and in each group a set day of onset and accumulated clinical evaluation. Significant deviation determined by Student test.

(3) Results

Called clinical assessments statistically significant deviation (p<0,05) between the DS group (12,41,7) and control group (16,35,9), confirming that DS suppress symptoms. In addition, the average day of onset in the DS group is 9,50,5, and in the control group 9,10,3, this suggests that the expression of symptoms tend to slow down in the DS group, and that the DS suppresses the symptoms. From these results it follows that DS is an effective agent for the treatment of MS, because EAE is a model of MS on animals.

Example 5

Test toxicity by single intravenous dermatosurgery

(1) Experimental method

As the use of experimental animals five-week females and males Crj; CD-I mice (Japanese Charles-river (n = 5 each). The DS solution obtained by the method of reference example 1, make isotonic, using sterile distilled water and sterile fiziologicheskii within 14 days.

(2) Results

After 14 days of observation is not observed deaths among 5 female and 5 male mice. Accordingly, the lethal dose DS in these conditions, estimate above 2000 mg/kg

Example 6

Pharmaceutical drug

(1) Injection, including dermatologit as an effective ingredient

DS (5000 mg) is dissolved in 100 ml of physiological saline, sterilized and injected into vials (5 ml each), which are used for intravascular, subcutaneous, or intramuscular injections.

(2) Joint use of immunosuppressant

Different types of immunosuppressants at the following dose dissolved in 100 ml of physiological saline, sterilized and injected into the ampoules. Depending on the type of immunosuppressant dose is defined as follows: cyclophosphamide, 30 mg; azathioprine, 30 mg; mizoribine, 150 mg, methotrexate, 3 mg; tacrolimus.html, 3 mg; cyclosporine, 15 mg.

These immunosuppressants is mixed with DS-injection, obtained above under item 1.

Drugs DS can be entered before or after the introduction of the above immunosuppressants. For example, you can use 25 mg/day of prednisolone, 30 mg/day of cyclophosphamide or AZ="ptx2">

The impact of these immunosuppressive drugs for various diseases can be enhanced by the introduction of drugs DS before or after their application. In addition, this dose may vary depending on the severity of the patient's condition. Of these agents, prednisolone, cyclophosphamide, methotrexate and cyclosporine can be entered intravascular or oral. The best way to introduce other agents per os (by mouth).

Industrial applicability

Dermatologit or its pharmaceutical salt can be used as a useful anti-inflammatory agents that do not have adverse effects for the suppression of refractory or persistent inflammation or inflammation that involve immunological mechanism.

In addition, when you use containing a sulfate group of glycosaminoglycans and collagen or its pharmaceutically acceptable salt in combination with an immunosuppressant, such as adrenal cortical steroid, cyclophosphamide, azathioprine, mizoribine, cyclosporine, methotrexate, tacrolimus.html and so on, fewer immunosuppressant than in the case of separate administration of immunosuppressant, can lead to the same effect, reducing not the positive effect of the immunosuppressant, representing at least one of glycosaminoglycan selected from the group including dermatooncology, heparin, heparansulfate, chondroitin sulfate and keratinolytic and their pharmaceutically acceptable salts.

2. Means under item 1, representing dermatologit.

3. Vehicle, p. 1, wherein said immunosuppressant includes at least one immunosuppressant selected from the group comprising adrenocorticosteroid, cyclophosphamide, azathioprine, mizoribine, cyclosporine, methotrexate and tacrolimus.html.

4. Means, including immunosuppressant and glycosaminoglycan selected from the group including dermatooncology, heparin, heparansulfate, chondroitin sulfate and keratinolytic or their pharmaceutically acceptable salts.

5. Means, under item 4, in which glycosaminoglycan represents dermatologit.

6. Means under item 4 or 5, wherein said immunosuppressant includes at least one immunosuppressant selected from the group consisting of adrenocorticosteroids, cyclophosphamide, azathioprine, mizoribine, cyclosporine, methotrexate and tacrolimus.html.

7. Method of strengthening anti-inflammatory action immunod the Ohm, selected from the group including dermatooncology, heparin, heparansulfate, chondroitin sulfate and keratinolytic and their pharmaceutically acceptable salts, with the time injection mode or route of administration may vary.

8. The method according to p. 7, where the specified glycosaminoglycan or its pharmaceutically acceptable salt is administered in the form of injections, and the specified immunosuppressant in the form of oral medications or injections.

9. The method according to PP.7 and 8, where the specified glycosaminoglycan represents dermatologit.

10. The method according to PP.7 - 9, where the specified immunosuppressant includes at least one immunosuppressant selected from the group consisting of adrenocorticosteroids, cyclophosphamide, azathioprine, mizoribine, cyclosporine, methotrexate and tacrolimus.html.

11. The method of treatment of inflammation, including the introduction of the immunosuppressant and anti-inflammatory effective amount means, including dermatologit or its pharmaceutically acceptable salt, a mammal suffering from an inflammatory disease.

12. The method according to p. 11, where the specified inflammatory disease is prolonged inflammatory process.

13. FPIC is an immunological mechanism.

14. The use of dermatosurgery or its pharmaceutically acceptable salt as a means of enhancing the anti-inflammatory effect of immunosuppressant.

15. Application under item 14, in which immunodepressant is intended for the treatment of prolonged inflammation.

16. Application under item 14, in which immunodepressant is intended for the treatment of an inflammatory process involving the immune mechanism.

 

Same patents:

The invention relates to the field of pharmacology, and relates to a wound healing drug for the treatment of purulent wounds, burns and skin diseases

The invention relates to medicine, in particular to solid dosage forms quick release containing therapeutic active substances or their concentrates, in particular lipid conjugates of nucleosides, which are characterized by gelling properties in aqueous media

The invention relates to pharmaceutical compositions containing as active ingredient at least one substance inhibiting NO-synthetase, and at least one substance that traps the reactive forms of oxygen, and, optionally, a pharmaceutically acceptable carrier
The invention relates to medicine, specifically to medicines, analgesic action

The invention relates to new, containing diazepambuy group of compounds with biological activity, and more particularly to a derivative of diazepinone, their racemic forms, their isomeric configurations defined by the carbon in position 3 diazepinone-4-about the kernel and their pharmacologically acceptable salts, intermediate compounds and pharmaceutical compositions inhibiting phosphodiesterase IV activity

The invention relates to chemical-pharmaceutical industry, namely the non-narcotic analgesic and antipyretic drug and method thereof

The invention relates to new ascomycin General formula I, where Y represents a phenylene; Z is selected from carboxyl and physiologically hydrolyzable of oxycarbonyl or alkyl, CNS, alkylamino or dialkylamino bearing from 1 to 4 carboxyl or physiologically hydrolyzable oxycarbonyl group; Q is O or S; R1Is H, alkyl or aryl; R2is hydrogen or hydroxyl; R3is methyl, ethyl, propyl or allyl; R4is hydroxyl or alkoxyl; R5-oxoprop or (H, OH), R6- oxoprop, H, HE H, alkoxyl); n is an integer 1 or 2, in free form or in the form of a pharmaceutically acceptable salt

The invention relates to the medical industry, namely the production of medicines, containing four, pathcomponent mixture of acetylsalicylic acid, paracetamol, ascorbic acid, organic salts of calcium, Dimedrol, and(or) routine used for relief of acute manifestations of influenza
The invention relates to the use of chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, as agent, capable of providing stimulated the regeneration of hard tissues
The invention relates to medicine, namely to ophthalmology, and relates to a composition for pain relief and reduce inflammation

The invention relates to medicine, in particular for Allergology and dermatology, and for the treatment of atopic dermatitis

The invention relates to medicine, namely to surgery
The invention relates to medicine, more specifically to trauma, and can be used to accelerate the consolidation of diaphyseal fractures of the tibia after a stable immobilization of the fragments

The invention relates to the field of biology, in particular biochemistry

The invention relates to a new anti-adhesion agent
The invention relates to the use of chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, as agent, capable of providing stimulated the regeneration of hard tissues

The invention relates to biology, in particular biochemistry
Up!