Dermatologit or its salt, antithrombotic agent, the method of prevention or treatment of thrombosis, the method of prevention or treatment of syndrome of diffuse intravascular coagulation, the method of treatment of myocardial infarction

 

(57) Abstract:

Describes dermatologit or its salt, having a characteristic viscosity of 0.8 (100 ml/g or higher when determining the viscometer Ubbelohde using as solvent 0.2 M solution of sodium chloride at a temperature of 30 0,1oWith the specified dermatologit contains 0.15% or less of heparin or heparan sulfate when defining a method, comprising processing enzymes cleavage of heparin or heparan sulfate and liquid chromatography high resolution of 0.07% or less of heparin or heparan sulfate determined by the inhibitory effect of the active factor X in the presence of anti-thrombin III with heparin isolated from cow's intestines as a standard substance, or 0.05% or less of heparin or heparan sulfate determined by the inhibitory effect of the active factor II in the presence of anti-thrombin III when used as a standard substance heparin isolated from cow intestines. Dermatosurgery or their salts are to thrombotic means, to prevent the spread of blood clots and stimulate their lysis. Also describes antithrombotic agent, the method makes the 12 C.p. f-crystals, 8 tab., 6 Il.

The present invention relates to antithrombotic means, to prevent the spread of blood clots and stimulate their lysis.

Thrombosis is a disease caused by blood coagulation in living blood vessels due to imbalance between platelets, coagulation and fibrinolytic system, which basically should maintain the fluidity of blood in the blood vessels. Clots in the coronary arteries cause heart attacks, and blood clots in the veins in the lower limbs, and so on, cause deep phlebothrombosis. Abnormal activation of the coagulation system due to severe infections or tumors leads to blood clots in all capillaries and result in the syndrome of diffuse intravascular coagulation /hereinafter referred to as DIC/. DIC caused severe infectious disease is often complicated by disorders of many organs and a very poor prognosis. Developed various countermeasures for the treatment of thrombosis, which shows extremely varied symptoms depending on the location of its occurrence. For the treatment of myocardial infarction and deep vein thrombosis apply urokinase or tissue activator is dostatkom urokinase is a low affinity to fibrin, a t-PA is expensive and has a very short half-life. In addition, percutaneous transluminal coronary recanalization and reperfusion therapy by direct peripheral intravenous t-PA have a tendency to cause the recurrence of thrombosis. The problem of heparin is that to secure the necessary steps to enhance therapeutic effect requires a certain or higher concentration of anti-thrombin III. In addition, these drugs cause serious side effects - bleeding. Therefore, in order to cope with these difficulties, developing synthetic drugs. However, sufficiently address the issue with the side effect is not obtained, and remains unresolved is the problem of an extremely short half-life excretion.

It is reported that dermatosurgery increase anticoagulation activity through cofactor II heparin, but reported their fibrinolytic activity, accelerate the excretion of t-PA/Abbadini, M. et al., Blood 1987, 70, 1858-1860/, while it is reported that dermatosurgery not affect the number of t-PA or fibrinolytic activity /Tripodi, A. et al., Thromb. Res., 1991, 62, 663-672/, and the exact effect on the fibrinolytic system is not the medication, derived from pig or cow intestines or skin /Fareed, J. et al., Recent Advance in Blood Coagulation, 1993, 6, 169-187/. It is known that dermatosurgery from other sources do not have activity.

The authors of the present invention investigated the influence of dermatosurgical on the fibrinolytic system and found that thrombolytic activity increases in their presence when compared with the activity in their absence. In addition, the inventors have found that accumulated antithrombin activity dermatosurgical introduced into a living organism, including anticoagulation and fibrinolytic activity is particularly significant in the case of certain dermatosurgical having a characteristic viscosity of 0.8 (100 ml/g or higher, certain dermatosurgical derived from cocks ' combs, certain dermatosurgical, with its components of the disaccharides 2-7% Di-S, or in the case of certain dermatosurgical having an average molecular weight 25000-100000 daltons, determined by the method described hereinafter, and, in addition, especially in the case of dermatosurgical with a very low content of heparin or heparan sulfate among the above dermatosurgical. The present invention osushestvljaem, containing as an effective ingredient dermatosurgery, or to the combined use of dermatosurgical fabric plasminogen activator /t-PA/.

Dermatosurgery have the basic structure of the repeating disaccharide consisting of N-acetyl-D-galactosamine-4-sulfate and L-iduronovoy acid with a small amount of D-glucuronic acid, and have different content of sulfuric acid and D-glucuronic acid and various joining sulfuric acid and so on, depending on the source animal species and organs, etc.

Dermatosurgery can be obtained mainly from raw materials originating from mammals or other animals, such as bovine or porcine mucosa of the intestines or the skin or chicken combs,

The authors of the present invention have found that the activation of plasminogen one - or two-chain t-PA can be enhanced in the presence of dermatosurgical obtained from such raw materials or their pharmacologically acceptable salts, compared with a case of their absence. In addition, the authors present invention also found that specific dermatosurgery of the present invention, having a characteristic viscosity of 0.8 (100 ml/g) or more, prepact the Ried and with an average molecular weight 25000-100000, preferably 30000-60000, daltons, determined by means of gel filtration, as described in Biochem. Bicphys. Acta, 1117, 60-70 (1992), in particular those that have a low content of heparin or heparan sulfate, or their pharmacologically acceptable salts are introduced into a living organism may persist longer in the blood than other dermatosurgery, and discover pharmacological effect, resulting in a comprehensive antithrombotic activity not only with fibrinolytic action, but also c antikoaguliruyuschim action, and it is the basis of the present invention.

Pharmacologically acceptable salts dermatosurgical include salts, such as salts of sodium, potassium, lithium and calcium, but is usually injected sodium salt. Tissue plasminogen activators /t-PA/ used for the present invention include both endogenous and exogenous, single and double circuit, and natural and gene-recombinant products. In addition, t-PA analogs with partially incomplete amino acid composition or having excess amino acids can also be used as long as they discover the properties of the activation of plasminogen /see EP-A-112122].

Dermatosurgery and ogechukwu activity at single and the only or rare, or repeated introduction. Dermatosurgery can be administered by intravenous, intra-arterial or coronary-arterial injection and can be applied subcutaneously or intramuscularly. DC and t-PA can be administered simultaneously by the same ways, or may be introduced separately.

When it is expected that when the aforementioned coronary-arterial or intravenous concentration in the blood DC and t-PA exceeds the effective concentration, there may be adopted other than the above, methods of administration is intramuscular or subcutaneous administration.

Pharmaceutical drugs dermatosurgical can be appropriately prepared with conventional pharmaceutically acceptable adjuvants, such as stabilizers, emulsifiers, regulators, osmotic pressure, pH-correcting means, and so forth.

DC is entered once at doses of 50-3000 mg/day for adult patients and can be administered concomitantly with 10000-500000 M. E. /international unit/ t-PA. These effective ingredients can be used once or fractional - in the form of two or more parts per day with suitable intervals. In addition, the DC can be administered intravenously for the change to activate fibrinolytic activity as suitable thrombolytic accelerator. Particularly noticeable effect find specific DC of the present invention, having a characteristic viscosity of 0.8 to 2.0 (100 ml/g), produced from chicken combs with degree Di-OS 2-7% in its average of the disaccharide or having an average molecular weight 25000-100000 determined by the method described hereinafter, and particularly those who have low levels of heparin or heparan sulfate, or their pharmacologically acceptable salts. At the same time, DC, has a characteristic viscosity of more than 2,0 100 ml/g, or the average molecular weight of more than 100,000, in highly concentrated solutions show a high degree of viscosity and are not recommended due to violations of the speed of blood flow. In addition, the simultaneous introduction of the above-described DC or their pharmacologically acceptable salts and t-PA significantly increases thrombolytic activity and may decrease the dose of t-PA for the treatment or prevention of thrombosis as a suitable thrombolytic tools.

In particular, the above-mentioned specific DC or their pharmacologically acceptable salts of the present invention when introduced into a living organism maintain an effective concentration in the blood over a longer period than other DC or pharmacologists who bolis. Thus, they are perfectly against syndrome diffuse intravascular coagulation /DIC/, multiple lesions of organs associated with numerous blood clots and deep vein thrombosis, etc., Such effects can be observed not only in the case of the above-mentioned diseases, but also in all types of blood clots, which serves as a basis for use in the treatment and prevention of blood clots in the arteries, capillaries and veins. In addition, specific DC or their pharmacologically acceptable salts of the present invention can be applied to prevent blood clots in the artificial circulation, such as hemodialysis for patients suffering from renal diseases.

Heparin /hereinafter referred to HepB/ or heparan sulfate /hereinafter referred GS/ present as an impurity in DC, can be removed to obtain DC, free from Hep or HS /hereinafter referred DS-H(-)/. Intravascularly, subcutaneous or intramuscular injection of the resulting DS-H(-) reduces the possibility of side reactions such as bleeding. In particular, the introduction of DS-H(-) patients with a tendency to bleeding due to low platelet count and factors coagula

Brief description of drawings

In Fig. 1 shows the change in blood in rats concentration dermatosurgical sodium obtained from cocks ' combs /see reference example 1, RCDS/ and from the small intestines of the cow /see reference example 2, BIDS/ /see example 1/.

Designations in Fig. 1 correspond to: - the introduction of BIDS, o - the introduction of RCDS, the introduction of BIDS-H(-). - the introduction of RCDS-H(-), and x indicates the point of intersection a , o, u .

Fig. 2 shows degree of stimulation BIDS activation of plasminogen single tissue plasminogen activator /sc-tPA/.

Fig. 3 shows degree of stimulation BIDS activation of plasminogen two tissue plasminogen activator /tc-tPA/.

Fig. 4 shows degree of stimulation RCDS activation of plasminogen sc-tPA.

Fig. 5 shows degree of stimulation RCDS activation of plasminogen tc-tPA.

Fig. 6 shows a stimulating effect lysis BIDS on a bunch of human plasma.

The present invention will be explained, from a practical point of view, the next following reference examples and examples.

Reference example 1

1/ get the DC from chicken combs

One kilogram of chicken combs are crushed, boiled in hot water and fiverivers diatomaceous earth, bring the pH of the filtrate to 5.8-6.3 and boiled at 50oC for 3 hours with 1000 TRU hyaluronidase derived from Streptomyces/Seikagaku Corp. / Boiled in the mixture add NaCl to a concentration of 0.65 M, and pour it into the column with anion exchange resin /column 3 x 20 cm, DiaioRTMHPA-10, Mitsubishi Kasei Corp./, balanced 0.5 M NaCl. The column is sequentially washed with 0.5 l of 0.65 M NaCl and 0.3 l 1.1 M NaCl and then elute 1.8 M NaCl. Erwerbende fractions are collected and evaporated under reduced pressure. Consolidated condensed fraction is subjected to dialysis overnight against distilled water. Dialysate evaporated to 10 ml and mixed with 5 M NaOH to obtain a 0.5 M NaOH solution. The alkaline solution is maintained at 37oC for 90 minutes, cooled and neutralized glacial acetic acid. The neutralized solution is mixed with 2-fold volume of ethanol. The precipitate successively washed with 70% ethanol, pure ethanol and ether and dried over phosphorus pentoxide under reduced pressure.

Leave 5 g of dried sludge to stand in 125 ml of water during the night, and a small number of nerastvorimogo precipitate is removed by centrifugation at 10oC at 10,000 rpm for 15 minutes. Supernatant concentration of 45% and allowed to stand at 4oC for 20 hours. Precipitation from centrifugation sequentially washed with 90% ethanol, pure ethanol and ether and dried over phosphorus pentoxide under reduced pressure to obtain pure sodium salt of DC /hereinafter referred to as RCDS/.

Isolation and purification of dermatosurgical from chicken combs can be performed not only in the way described above, but also by the methods described in the above published patent applications in Japan NN 9042 (1985) and 21241 (1986).

2/ Remove from DC heparin /HepB/ and heparan sulfate /GS/

A small amount of HepB and GS contained in DC as impurities, it is possible to delete one of the following ways.

1/ a Method of ion-exchange chromatography

Fifty grams of RCDS, obtained by the preceding method /1/, is placed in a column 4.5 x 27 cm with anion-exchange resin Diaio RTMHPAII /Mitsubishi Kasei Corp./, pre-balanced 1.1 M NaCl, washed with 8 l of 1.1 M NaCl solution and elute 8 l of 1.5 M NaCl. United eluate condense on a rotary evaporator, dialist and precipitated by adding ethanol to 42%, or lyophilized, and get standard RCDS/RCDS-H ( -)/ free from Hep and HS.

2/ the Method of nitrous acid cleavage

The removal of the wasp is barium and 400 ml IN H2SO4prepared at -10oC, centrifuged at 3000 rpm for 2 minutes and receive 500 ml of supernatant. Mix 500 ml of 0.1 mg/ml RCDS and 500 ml of the supernatant and leave to stand for 10 minutes at room temperature. The mixture is neutralized with a solution of Na2CO3. After the condensation is carried out the same procedures as in example 1, and receive a standard RCDS /RCDS-H ( -)/ free from Hep and HS.

Reference example 2

Getting DC from the mucosa of the small intestines of a cow

Ten pounds of cow's small intestines are chopped into pieces with the subsequent removal of feces and fat and then separated mucosa. Mucosa mixed with NaOH, the volume was adjusted to 3 l of 2.5 N NaOH solution and extracted at 37oC for 2 hours. The extract is neutralized, filtered through diatomaceous earth, dialist and centrifuged, supernatant get. The supernatant mixed with an equal amount of ethanol and 5 g of sodium acetate, and get the precipitate. The collected precipitate is dissolved in 0.65 M NaCl, and then, just as do in reference example 1, was poured into a column with anion-exchange resin. Column consistently breaking 0.65 M and 1.1 M solutions of NaCl and elute 1.5 M NaCl. United faction eluates cialiswhat against disti the M, respectively. To the solution was added ethanol, and collect the fraction deposited in 15-30 vol.%. The collected precipitate is transferred in the form of sodium salt ion exchange resin, washed and dried in the same manner as in reference example 1, and obtain the sodium salt of DC /referred BIDS/.

Hep C and HS is removed from BIDS in the same manner as in reference example 1/2 /2/ and get BIDS-H(-).

Compare characteristics of dermatosurgical with characteristics of dermatosurgical obtained from mucosa gut pig /Celsus Lab. Inc., distributed Cosmobio Co., Ltd./ and from pig skin /Seikagaku Corp./.

Reference example 3

Comparison of physico-chemical properties of various dermatosurgical different origin

1/ determination of the average molecular weight

The average molecular weight of DC is determined in accordance with the method Arai et al. /Biochem. Biophys. Acta, 1117. 60-70, 1992/. I.e., apply the gel filtration liquid chromatography high resolution /IHVR/, using as a standard sample glycosaminoglycans with known molecular weight, and determine the molecular weight, on the basis of their respective buervenich fractions. Prepare a number of columns TSKgelG4000PWXL, G3000PWXLand G2500PWZL/TOSOH Corp./ with internal and carry out detection with a differential Refractometer.

2/ analysis of the disaccharides

Determine where particles of sulfuric acid in DS is carried out in accordance with New Biochemical Experiments 3 Saccaride II, pp 49-62 (1991) /publ. Tokyo Called Dozin Co., Ltd./. For this DS digested with chondroitinase ABC, and the resulting disaccharide having the unsaturated bond /unsaturated disaccharide/ analyze GHWR and compared with the analysis of the above-mentioned unsaturated disaccharide treated with chondro-6-sulfatase. Conditions of digestion with chondroitinase ABC, desulfuromonas with chondro-6-sulfatase and analysis GHUR below.

a/ a Digestion with chondroitinase ABC

In accordance with the method of Yoshida /Anal. Biochem., 77, 327-332 (1989)/, in 20 ál of 10 mg/ml aqueous solution of DC add 20 ál of 0.4 M Tris-HCl-buffer /pH 8.0/, 20 μl of 0.4 M sodium acetate solution, 20 μl of 0.1% bovine serum albumin and 120 μl of water, then add 20 ál of 5 u/ml of chondroitinase ABC and incubated at 37oC within 2 hours.

b/ Digestion with chondro-6-sulfatase

100 μl of the above mentioned product digestion with chondroitinase ABC add 20 ál of 5 u/ml of chondro-6-sulfatase dissolved in 20 mm Tris-explicitation buffer /pH 7.0 and incubated at 37oC within 2 hours.

with/ Analysis GHUR

Fifty microl the equipment for GHWR /Hitachi Ltd. /. Use of ion-exchange column /UMS-Pack PA-120S5, the inner diameter of 2.6 mm, length 250 mm, and determine the absorbance at 232 nm. Elution performed at a flow rate of 1.5 ml/min with a gradient concentration of 800 mm sodium hydrogen phosphate from 0% to 100% within 60 minutes. Identify peaks buervenich unsaturated disaccharides containing sulfuric acid remains in various places.

3/ Definition of the characteristic viscosity

Determination of characteristic viscosity is carried out in accordance with Japanese Pharmacopoeia XII automatic viscometer /Rigosha Co., Ltd. type VMC-052/. As the solvent used 0.2 M NaCl and used to determine the expiry time in a viscometer of Ubbelohde. Determination of the viscosity is carried out at 300.1%oC and to compute the characteristic strength using three consecutive result of the expiration time with a difference within 0.1 C. the expiry Time is rounded up to 0.01, and calculating characteristic values of the viscosity is carried out using the following equation:

red= (ts/to-1)C,

where ts= time to expiry of the solution, to= time to expiration of the solvent and C = concentration of sample (%, V/V). The values of reduced viscosity

4/ Results

The results of experiments (1)-(3) summarize in table. 1. The average molecular weight of Dc derived from chicken combs, is 38000 daltons at a characteristic viscosity of 1.21. In contrast, three other DC have completely different physical-chemical properties and show the average molecular weight of 14000-16000 and characteristic viscosity of 0.44 and 0.68, indicating a significant difference between DC derived from chicken combs, and DC obtained from other sources. The average molecular weight of DC derived from bovine and porcine small intestines, is 16000 18500 and respectively according to the method of the present invention, as seen from table. 1, although there are reports that she is 25000 /Thromb Res, 71, 417-422, 1993 and/ 35700 /Blood, 74, 1577-1582, 1989/, respectively.

Abbreviations for expressions of the structure of the disaccharide are given in table. 2.

The present invention will be explained more by the following reference example 4 and examples.

Reference example 4

The definition of Hep or HS in DC

1/ Method

The definition of Hep or HS in DC is carried out using the following three methods.

2(pH 7.0) and incubated at 37oC for 2 hours. The reaction mixture analyzed GHUR under the same conditions that were used to determine the average molecular weight, in order to detect and determine the resulting unsaturated disaccharides on the differential Refractometer.

b/ the Determination of the activity of inhibition of factor active type X Hep or HS due to the activation of the effectiveness of anti-thrombin III

Hep or HS possess inhibitory activity against factor active type X /Xa/ and is denoted by anti - Xa-activity/ - one of the factors of coagulation, due to the activation of anti-thrombin III, while DC does not have this activity /Tollfsen, "Heparin", pp. 257-273, Eds.: David A. Lane and Ulf Lindahl. Edward Arnold, London, 1989/. Using this property, determine the anti-Xa activity using different concentrations of HepB to detect the interval from dose dependence of the ratio between inhibiting inhibition believe 100%, and the degree of inhibition with only one antithrombin III in the absence of Hep accept for 0%. Build a graph of the ratio, to draw a calibration curve. Do similar definitions for DC samples of the present invention, and the number of Hep or HS is determined by comparing the obtained anti-Xa activity with a calibration curve.

Practically carry out the following procedure. Mixed at 4oC 0.3 ml of 50 mm Tris-HCl /pH 8.0/ 150 mm Na2Cl, 10 mm CaCl2(buffer), 0.1 ml 1 u/ml of anti-thrombin III /cow, Sigma/ and 0.1 ml Hep /0,03-2 µg/ml, cow intestine, Syntex/ or 0.1 ml samples DS /50-100 μg/ml obtained in the form of free of Hep or HS, as described above, and the mixture is then incubated at 37oC for 2 minutes. Then to the mixture is added 0.1 ml Xa /7,1 nkat /ml bovine, Chromogenic AB Co. Co., Ltd., distributor Seikagaku Corp./, and the mixture is incubated at 37oC. After 5 minutes, to this mixture, 0.1 ml of 100 μm of the synthetic substrate /Boc-Ile-Glu-Gly-Arg-MCA, code 3094V, Peptide Research Inst./, which is incubated at 37oC for 3 minutes, and the reaction being removed by adding 0.3 ml of 30% acetic acid. And, finally, to the mixture was added 1 ml of a solution containing 7 volumes of 50 mm above buffer and 3 volume of 30% acetic acid, to obtain the sample for determination. Oany fluorescence 444 nm.

In advance argues that anti-Xa activity of the anti-thrombin III is almost invisible in the absence of Hep or HS in terms of this definition. The degree of inhibition is calculated by the following equation:

The degree of inhibition

A value defined for the sample containing Hep or HS;

B value obtained in the presence of buffer instead of anti-thrombin III, HepB or DC and Xa in the above procedure;

C - value obtained in the presence of buffer instead of anti-thrombin III in the above procedure.

c/ determination of the activity of inhibition of factor active type II Hep or HS through activating effect on antithrombin III

Hep or HS has inhibitory activity against factor coagulation active type II/IIa/ - one of the coagulation factors is due to the activation of anti-thrombin III, i.e. the effect on antithrombin /hereinafter referred to as anti-IIa-action/, while DC does not possess such activity /Tollfsen, "Heparin", pp. 257-273, Eds.: David A. Lane and Ulf Lindahl, Edward Arnold, London, 1989/. Anti-IIa activity is determined at different concentrations and C, using the above-mentioned property to determine the interval, dependent on the dose ratio between inhibitory activity and doses of HepB. Find min is about the antithrombin III in the absence of HepB take 0%. Build a graph of the ratio in order to have a calibration curve. This determination is carried out for sample DC of the present invention, and the number of Hep or HS is determined by comparing the obtained anti-IIa activity and the calibration curve.

In practice, carry out the following procedure. Mixed at 4oC of 0.35 ml of 20 mm Tris-HCl /pH of 7.4/ 150 mm NaCl, 10 mm CaCl2buffer with 0.1% bovine serum albumin buffer and 0.1 ml of anti-thrombin III /1E/ ml, bovine, Sigma/ Hep /0,3-20 µg/ml, cow intestine, Syntex/ or DC /50-100 µg/ml/ received free of Hep or HS form, as described previously, and the mixture is then incubated at 37oC for 2 minutes. Then to the mixture is added 0.05 ml IIa /50 IU/ml, cow, Boehringer/, and the mixture is incubated at 37oC. After 5 minutes, to this mixture, 0.1 ml of 70 μm of the synthetic substrate of Boc-Val-Pro-Arg-MCA /code 3093V, Peptide Research Inst./ and incubated at 37oC for 3 minutes. The reaction being removed by adding to a mixture of 0.3 ml of 30% acetic acid. And, finally, to the mixture was added 1 ml of a solution containing 7 volumes of the above buffer and 3 volume 30% glacial acetic acid, and receive a sample to determine. Determination is performed with the help of fluorophotometry /Japan Spectroscopic Co., Ltd., FP-777/ wavelength excitation imaeda insignificant in the absence of Hep C and GS in terms of this definition. The degree of inhibition is calculated by the following equation:

The degree of inhibition

A value defined for the sample containing Hep or DS;

B value obtained in the presence of buffer instead of anti-thrombin III, HepB or DC, and IIa in the above procedure;

C - value obtained in the presence of buffer instead of anti-thrombin III in the above procedure.

2/ Results

The results obtained above three methods are given in table. 3,

Example 1

Differences in the duration of preservation of the concentration in the blood RCDS, BIDS, RCDS-H(-) and BIDS-H(-) after the introduction of the research on rats.

1/ Materials and the way

Use of male rats Sprague-Dawley /SD/ /Charles River Japan, Inc./ at the age of 6.5-7.5 weeks. As the test samples using RCDS, BIDS, RCDS-H(-) and BIDS-H(-). To give a rats anesthetized with ether and injected into the tail vein of 10 mg/kg of each test sample. Blood is withdrawn from the lower /inferior/ hollow veins through 5 and 120 minutes after administration under ether anesthesia in tubes containing 1 volume of 3.2% citric acid in 9 volumes of blood. Separate the plasma by centrifugation, and determine the number of DC in the plasma in accordance with the "New Biochemical Experiments", 3, Saccharides II, pp. 175-179. I.e. plasma absoluut through Kolya through the membrane, cut-off molecular weight of 5000, and the resulting filtrate analyze GHUR.

2/ Results

The results are expressed as the relative residual ratio (%) DC with respect to its quantity at time 0 /zero/, as shown in Fig. 1. Residual coefficients RCDS and RCDS-H(-) is approximately two times higher than the BIDS and BIDS-H(-), 5 minutes after injection. RCDS and RCDS-H(-) show the residual coefficients of the order of 10% after 120 minutes after administration, while BIDS barely detected. RCDS-H(-) indicates a slightly higher residual ratio than the RCDS through 5 and 120 minutes after injection.

Example 2

Explore the antithrombotic effect of DS on the model of thrombosis in the inferior Vena cava in rats.

1/ Materials

Use of male rats Sprague-Dawley /SD/ /Charles River Japan, Inc./ at the age of 6.5-7.5 weeks. As DC use RCDS and BIDS.

2/ the Method

Obtaining experimental models of thrombosis

The model of thrombosis in rats receive in accordance with the method Reyers et al. [Thromb. Res., 18, 669-674 (1980)]. For this purpose, rats produce laparotomy under General anesthesia of NembutalRTM/Dynabott-Dainippon Pharmaceutical Co., Ltd./, and are ligated just below the branches of the left renal vein inferior Vena cava surgical SAlg/kg for 1 minute before ligation. The control group given saline. Rats produce laparotomy in 3 hours after ligation, and excised vein, to completely remove the blood clot. Clots leave to stand over night at 37oC, and measure their dry weight. The obtained data is analyzed statistically by the method of Newman-Keuls.

3/ Results

The results are given in table. 3A. Relative dry weight of thrombi in both groups with DC, which entered 1 mg/kg to the weight of blood clots in the control group indicates the degree of inhibition of 60%. However, the weight of blood clots in the group with RCDS is reduced to 2.4% 0,0% at doses of 3 and 10 mg/kg or more, respectively, when compared with the weight of thrombi in the control group, while the weight of blood clots in the group with BIDS only be reduced to 34.1% and 2.4% respectively when compared with the control group, indicating the usefulness of RCDS.

The control group contains 14 rats, groups with DC contain 6 rats, respectively, and calculate averages.

Example 3

The effect of DC on the formation of blood clots and thrombosis research on models of thrombosis in the inferior Vena cava in rats.

1/ Materials

Use of male rats Sprague-Dawley /SD/ /Charles River Japan, Inc./ at the age of 6.5-7.5 weeks. As DC use RCDS and BIDS.

oC to determine their dry weight. The obtained data is analyzed statistically by the method of Newman-Keuls.

2/ Get euglobulin fractions

In a plastic test tube is placed 0.5 ml of plasma, and add to it a 9.5 ml of deeply chilled 0,01 N acetate buffer /pH 4,85/ all leave in the refrigerator for 1 hour, centrifuged at 3000 rpm for 5 min, and receive euglobulin faction. Precipitation mixed with 0.5 ml of Tris-HCl-buffer /pH 7,4/ and get a clear solution.

3/ Determination of fibrinolytic activity

In the pilot hole fibrin-tablet placed 10 ál euglobulin faction, leave in the incubator at 37oC for 18 hours, and measure fibrinolytic area /square diameter/. Fibrinolytics is the Newman-Keuls.

3/ Results

The results are given in table. 4.

In table. 4 "control, 6 hour" refers to the group with laparotomy under General anesthesia in 6 hours after ligation of the vein, followed by blood collection and complete extraction of the thrombus. "Control, 8 hour" refers to the group that was injected with saline solution after 6 hours after ligation of the vein with subsequent selection of blood and complete extraction of the thrombus after another 2 hours.

1/ impact on the formation of blood clots and thrombosis

Thrombi observed in all groups, but among groups find noticeable differences, as shown in the table. 4. The group, which is injected as RCDS, and BIDS show a dose-dependent weight loss blood clots and compared with a group of "control, 8 hour". In addition, the group, which is administered 30 mg/kg BIDS and 10 mg/kg or more RCDS, demonstrate less weight clots than in group Control, 6 hour", which indicates not only the inhibition of thrombus formation, but also on thrombolytic action. In other words, RCDS has a stronger inhibitory effect on thrombus formation and thrombolytic effect at lower doses than that observed in group C BIDS.

2/ effect on the fibrinolytic system

RCDS and enter BIDS at daswani/. The results are given in table. 4. The activity of plasmin in the group that received 10 mg/kg RCDS, and in the group receiving 30 mg/kg or more BIDS, is much higher than in the control group. These results indicate the excellent action RCDS.

The weight of blood clots expressed as the ratio, in %, to the "Control, 8 hour, the weight of which is taken for 100%. Under Control, 8 hours is 16 rats, and other groups contains 8-9 rats, respectively, and calculate averages.

Example 4

Comparative trials of antithrombotic actions on the model DIC induced by endotoxin rats

1/ Materials

Use rat Sprague-Dawley /SD/ /Charles River Japan, Inc./, males aged 5.5 to 6.5 weeks. Rats given anesthesia through intraperitoneally injection of NembutalRTMand make an incision in the left hip. Movement back insert polyethylene cannula /PE-10, Imamura Co., Ltd./ from the incision in the ventral vein at the distance of 4 cm, and fix it. After recovery from anesthesia continuously infuziruut endotoxin /Escherichia coli 055:B5; Difco Co. , Ltd/ infusion pump /model 22M, HARVARD/ via syringe at a speed of 3.75 mg/kg/h for 4 hours. The test sample - RCDS-H(-) or BIDS-H(-)- injected simultaneously with endotoxin, and Nesci solution in volume, equivalent to the volume of the sample and introduce it within 4 hours.

2/ Separate points test

After injection of endotoxin away the blood and remove kidney, and conduct definitions listed below.

1/ Number of platelets. Platelets consider using automatic hemocytometer /Cellac MEK-4500; Nippon Denko Co., Ltd /.

2/ Fibrinogen. Isolated plasma and carry out the determination with the kit for determination of fibrinogen /Sunassay FibrinogenRTMproduction NITTO BOSEKI Co., Ltd, the distributor of Sanko Junyaku Co., Ltd/.

3/ degradation Products of fibrinogen and fibrin /FDP/. Allocate serum, and determine the FDP with a latex reagent for determining FDP/FDPLRTMtest, Teikoku Hormone Mfg. Co. Ltd./.

4/ Degree sediments renal glomerular fibrin /% GFD/. Kidney fixed with 10% neutral buffer formalin solution, pour the wax, make the cut and stained with hematoxylin with phosphonoformate acid. The renal glomeruli observed under the microscope across the field observations of samples of the kidney, and then count the number of deposits of fibrin, and Express it in percentages. The obtained results are analyzed statistically by the method of Newman-Keuls.

3/ Results4μl, 35 mg/DL, to 34.3 µg/ml and 61%, respectively.

The group, which impose DS-(H) ( -) shows improvement in all these parameters, and the group with RCDS-H(-) indicates all the parameters of the results, better than in the case of BIDS-H(-). In particular, % GFD, which is due to renal glomeruli, remains at the level of 16.1 per cent, in case of BIDS-H(-), whereas in the group with RCDS-H(-) there is a noticeable improvement - drop to 1%, which indicates the suitability of RCDS-H(-).

The experiment carried out on 19 rats in the normal group, 20 rats in the control group, and 7 rats in each group, which impose DS, and calculate the average values.

Example 5

Stimulating effect of DS on the activation of Glu-plasminogen and normal plasminogen, hereinafter referred to as the plasminogen/ t-PA

1/ Method

Mix 50 microliter sample of each concentration DS /BIDS, RCDS/ or chondroitin sulphate /control panel/, 25 μl of 1.6 μm of plasminogen and 25 μl of 20 nm one - or two-chain t-PA, and 100 μl of 0.6 mm synthetic substrate S-2251 /H-D-Val-L-Leu-L-Lys-p-2HCl/, and determine the absorbance at 405 nm CAoC in a buffer consisting of 50 mm Tris-HCl and twinRTM-80.

The initial degree of plasminogen activation is calculated as follows. The ratio of the slope of the graphs according to A405the absorption at 405 nm, from time to time square (A405/t2) divide 40,000 /Chibber, B. A. K., et al., Biochemistry, 1985, pp. 3429-3434/. The degree of stimulation is calculated from the correlation with the results obtained in the absence of DS or chondroitin sulphate, when the initial degree is 1 /unit/.

2/ Results

Results for BIDS is given in Fig. 2 and 3 DC, depending on the doses, stimulates the activation of plasminogen 1.5-4.5 times in the case of single t-PA /Sc-PA/, and 1.5-3.5 times in the case of two-chain t-PA /tc-tPA/, as shown in these figures. At the same time, a control group with chondroitin sulfate shows a weaker, compared with the group with DC, the degree of stimulation - a maximum of 2.5 times in the presence of one-chain t-PA and a maximum of 1.5 times in the presence of two-chain t-PA. In Fig. 4 and 5 the results for RCDS, RCDS also stimulates the activation of plasminogen in the 2.0-4.5 times in the presence of one-chain t-PA (sc-tPA) and 2.0-4.0 times in the presence of two-chain t-PA (tc-tPA), respectively.

Example 6

1/ Materials and method

Examine the impact of aminopentylRTM/Kyoritsu Shoji Co. Co., Ltd./, and injected them subcutaneously in the back 40 mg/kg DS/BIDS/. After 1, 6, 12 and 24 hours take away blood with a syringe containing citric acid. Prepared from plasma euglobulin fraction in the same manner as described in example 3, and fibrinolytic activity assessed by plating test definition lysis of fibrin (method with fibrin tablet). The obtained data is analyzed statistically by the method of Newman-Keuls.

Determine the number of DS in the plasma and euglobulin faction /New Biochemical Experiments", 3, Saccharide II pp. 175-179/. In the control group using saline solution. These experiments are performed at a predetermined time, taking into account the circadian rhythm of activity of coagulation and fibrinolysis in rats.

2/ Results

The results obtained are given in table. 6 and 7.

DS shows fibrinolytic activity up to 12 hours after injection, as shown in the table. 6. About 70-80% DS in the plasma are euglobulin fractions, and show fibrinolytic activity, as shown in the table. 7. RCDS also gives similar results.

As in the control group and in groups with DS present 4-5 rats, and calculate the average values.

As in the control group Explore the stimulating effect of DS on the lysis of the clot human plasma.

1/ Method

In 96-cell tablet for micrometrology put in 40 ál of DS of each of the different concentrations /BIDS/ /boundary concentration of 50.0-200 μg/ml, 20 ál-chain t-PA /the final concentration of 166.7 E/, 40 μl of the thrombin /final concentration of 20 u/ml/ 100 ál human plasma and mix. Immediately after cooking to determine the absorption at 340 nm, and then determine the absorption every 10 minutes, to obtain the minimum absorption /min/. Fibrinolytic activity is expressed as the ratio of DS-PLTI/2//minutes/ obtained by dividing by 2 the sum of the absorption maximum /max/ min and in the presence of DC to Cont-PLTI/2 in the control group.

2/ Results

The results are given in table. 6. DC, depending on the dose, stimulates fibrinolytic activity of blood plasma, as shown in Fig. 6, RCDS gives similar results.

Example 8

The toxicity testing of dermatosurgical after a single intravenous injection to mice

1/ Materials and methods

Use the mouse Crj: CD-1 both sexes /Charles River Japan, Inc./ at the age of five weeks. Prepare isotonic solutions RCDS-H(-) and BiDS-H(-) with sterile distilled water and sterile saline, and mice in the tail vein, respectively, adnocrine 14 days.

2/ Results

In none of the groups that received DC not see death, even at such a high dose as 2000 mg/kg, as shown in the table. 8. It has been proved that the lethal dose in the conditions of these tests exceeds 2000 mg/kg

Example 9

Pharmaceutical

1/ In sterile saline or sterile distilled water dissolve 500-5000 mg of sterilized sodium salt DS[RCDS or RCDS-H(-)] from cocks ' combs, received in accordance with reference example 1, and receive isotonic solutions, which volume is brought to 100 ml. of the Solution is poured into 10-ml ampoules and receive drugs for intravascular, subcutaneous or intramuscular injection.

2/ Packed separately 250 mg of sodium salt of DC [RCDS or RCDS-H(-)], obtained from chicken combs in accordance with the reference example 1, and 1.000.000 ME t-PA, and get the drugs for injection, which prepare the solution immediately before use. Such injections are used for intra-coronaryartery or intravenous injection.

Industrial application

Dermatosurgery or their pharmaceutically acceptable salts stimulate fibrinolytics the imposition of dermatosurgical, including their sodium salts, fabric plasminogen activators /t-PA/ considerably increases the thrombolytic effect and may reduce the dose of t-PA.

In addition, specific dermatosurgery of the present invention, i.e., dermatosurgery having a characteristic viscosity of 0.8 (100 ml/g or higher, obtained from cocks ' combs containing 2-7% Di-OS in its forming the disaccharide having an average molecular weight 25000-100000 daltons, as determined by gel-filtration in accordance with the method in Biochem. Biophys. Acta, 1117, 60-70 (1992), or the above-mentioned specific dermatosurgery containing a very small amount of heparin or heparan sulfate, or their pharmaceutically acceptable salts, retain their effective concentration in the blood for a long time and prevent the development of thrombosis, inhibit the development of blood clots, etc.

Therefore, antithrombotic means of the present invention is also suitable for the treatment and prevention of myocardial infarction, syndrome diffuse intravascular coagulation /DIC/, disorders of many organs that accompany multiple thrombosis, deep vein thrombosis, etc. in Addition, these tools can be shown for preduprezhdeny the renal failure, and so on, in Addition, DC have fewer side effects, such as bleeding, etc. in comparison with the side effect of heparin. DS-H(-), replacement removing HepB and GS from DC, has increased security at the same time with much less adverse reactions such as bleeding, etc., Thus, the resulting product can be preferably indicated in patients with a tendency to bleeding or risk of platelets and coagulation factors.

1. Dermatologit or its salt, having a characteristic viscosity of 0.8(100 ml/g or higher when determining the viscometer of Ubbelohde from use as a solvent of 0.2 M solution of sodium chloride at a temperature of 30 0,1oWith this specified dermatologit contains 0.15% or less of heparin or heparan sulfate when defining a method, comprising processing enzymes cleavage of heparin or heparan sulfate and liquid chromatography high resolution of 0.07% or less of heparin or heparan sulfate determined by the inhibitory effect of the active factor X in the presence of anti-thrombin III with heparin isolated from cow's intestines as a standard substance, or 0.05% or less of heparin or heparan is the whether as a standard substance heparin, isolated from cow's guts.

2. Dermatologit or its salt under item 1, characterized in that the characteristic viscosity is in the range of 0.9 to 2.0 (100 ml/g).

3. Dermatologit or its salt under item 1 or 2, characterized in that dermatologit isolated from chicken combs.

4. Dermatologit or its salt under item 1, characterized in that dermatologit or its salt contains 2-7% (2-acetamido-2-deoxy-3-0-4-deoxy-threo-Gex - L-4-andoperational acid)-D-galactose (Di-OS) components of the disaccharides when defining a method, comprising enzymatic cleavage and liquid chromatography high resolution.

5. Dermatologit or its salt under item 1, characterized in that dermatologit has an average molecular weight of 25000 to 100000 daltons when determining the method of gel filtration.

6. Antithrombotic agent, containing as an active ingredient dermatologit or its pharmaceutically acceptable salt in combination with one or more pharmaceutically acceptable adjuvant, wherein the active ingredient contains dermatologit or its salt according to any one of the preceding paragraphs in an effective amount.

7. When such compositions, containing dermatologit or its pharmaceutically acceptable salt together with a pharmaceutically acceptable adjuvant, patients with probable or developing thrombosis, according to any one of paragraphs.1 - 5.

8. The method according to p. 7, characterized in that the objects of the introduction are patients with possible or developing thrombosis or patients with thrombosis due to bleeding caused by low platelet counts or decreased coagulation factors.

9. The method of prevention or treatment of syndrome of diffuse intravascular coagulation, characterized in that it includes an introduction to pharmaceutical dosage forms, containing dermatologit or its pharmacologically acceptable salt, together with a pharmaceutically acceptable adjuvant, patients with probable or developing syndrome diffuse intravascular coagulation or patients with a syndrome of diffuse intravascular coagulation, where the use of dermatooncology or its pharmaceutically acceptable salt according to any one of paragraphs.1 - 5.

10. Antithrombotic agent containing an effective amount of tissue plasminogen activator (t-PA) and dermatooncology or its pharmacologically als characterized in that that dermatologit or its pharmacologically acceptable salt is recovered from chicken combs.

12. Antithrombotic funds under item 10, wherein dermatologit or its pharmacologically acceptable salt contains 2 to 7% Di-OS in the components of the disaccharides when defining a method, comprising enzymatic cleavage and liquid chromatography high resolution.

13. Antithrombotic funds under item 10, wherein dermatologit or its pharmacologically acceptable salt has an average molecular weight of 25000 to 100000 daltons when determining the method of gel filtration.

14. Antithrombotic funds under item 10, wherein dermatologit or its pharmacologically acceptable salt have a characteristic viscosity of 0.8 (100 ml/g or higher when determining the viscometer of Ubbelohde using as a solvent of 0.2 M solution of sodium chloride and at a temperature of 30 0,1oWith .

15. Antithrombotic funds under item 10, wherein dermatologit or its pharmacologically acceptable salt have a characteristic viscosity of 0.9 to 2.0 (100 mg/g).

16. Antithrombotic means on one of the PP.10 to 15, characterized in that dermatosurgery method, includes treatment with enzymes cleavage of heparin or heparan sulfate and liquid chromatography high resolution of 0.07% or less of heparin or heparan sulfate - when determining on the inhibitory action of the active factor X in the presence of anti-thrombin III used as a standard substance heparin from bovine intestines, or 0.05% or less of heparin or heparan sulfate - when determining on the inhibitory activity of factor II in the presence of anti-thrombin III used as a standard substance heparin from bovine intestines.

17. A method of treatment of myocardial infarction, characterized in that the patients with myocardial infarction administered pharmaceutical dosage form, comprising a fabric plasminogen activator (t-PA) and dermatooncology or its pharmacologically acceptable salt together with a pharmaceutically acceptable adjuvant.

18. Method of treatment for p. 17. wherein dermatologit or its pharmacologically acceptable salt is dermatologit according to any one of paragraphs.1 - 5.

 

Same patents:

The invention relates to a method for production of modified bitumen and can be used in road construction, as well as in the construction of industrial and civil structures, namely when creating roofs, waterproofing, sealants

The invention relates to polymer-mineral compositions, mainly for construction purposes, used, for example, during installation and repair of building structures and components on the basis of cement, concrete and other silicate materials, such as putties, for warmth and waterproofing of buildings, tanks and their individual parts, pipelines, etc

The invention relates to stapling powder mixture for connecting the means for textile materials, and the method of production associated with the polymer, textile molded or paintings using the mixture for connecting the means for textile materials

The invention relates to a block copolymer composition, curable by ultraviolet radiation (hereinafter UV block copolymer composition)
The invention relates to elastogram, to a method for producing such Anastasya, to the way these Anastasya and to the products derived from them

The invention relates to the processing of rubber for construction materials

The invention relates to agriculture and food industry

The invention relates to a medical, cosmetic and food industry and can be used for the production of medicines, cosmetics and dietary supplements
The invention relates to methods of producing inulin from inulinsoderzhathego raw materials
The invention relates to methods of producing inulin from inulinsoderzhathego raw materials

The invention relates to a method for anticoagulant compositions, and compositions obtained thereby

The invention relates to the field of chemical technology, in particular to methods for chitosan food

The invention relates to the field of production of water-soluble chitosan and relates to the synthesis of succinate sodium salt of chitosan, which can be used in veterinary medicine, medicine and cosmetics

The invention relates to medicine and related methods of use antifibrin antibodies for inhibition of in vivo blood clots, as well as pharmaceutical compositions and a kit containing a pharmaceutical composition for use in such methods
Up!