CA1186646A - Anti-thromboticum based on polysacharides, method for its preparation and pharmaceutical compositions - Google Patents
Anti-thromboticum based on polysacharides, method for its preparation and pharmaceutical compositionsInfo
- Publication number
- CA1186646A CA1186646A CA000403376A CA403376A CA1186646A CA 1186646 A CA1186646 A CA 1186646A CA 000403376 A CA000403376 A CA 000403376A CA 403376 A CA403376 A CA 403376A CA 1186646 A CA1186646 A CA 1186646A
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- CA
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- Prior art keywords
- content
- thromboticum
- process according
- meq
- daltons
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
Abstract
ABSTRACT
A new anti-thromboticum based on a mixture of oligo- and polysaccharides is provided. The invention also relates to methods for preparation thereof and to pharmaceutical compositions including the said anti-thromboticum.
The new product is of natural origin and can be obtained from mammal tissue, particularly intestinal mucous. The product is useful owing to its high "benefit/risk" ratio (i.e. the ratio between the anti-thrombotic activity and the haemorrhagic activity).
A new anti-thromboticum based on a mixture of oligo- and polysaccharides is provided. The invention also relates to methods for preparation thereof and to pharmaceutical compositions including the said anti-thromboticum.
The new product is of natural origin and can be obtained from mammal tissue, particularly intestinal mucous. The product is useful owing to its high "benefit/risk" ratio (i.e. the ratio between the anti-thrombotic activity and the haemorrhagic activity).
Description
6~6 New anti-thromboticum based on polysaccharides, method for its preparation and pharmaceutical compositions.
The invention relates to a new anti-thromboticum based on a mixture of oligo- and polysaccharides and to methods for preparation thereof. The invention also relates to pharmaceutical compositions which include the said mixture.
It is known that certain mucopolysaccharides influence the coagulation properties of blood. The best known mucopolysaccharide is heparin, a sulfated mucopolysaccharide1 which is employed in prophylaxis and the treatment of venous thrombosis and thrombo-embolism. The anti-thrombotlc effect of heparin is based on acceleration of the inhibition of blood-coagulating factors by anti-thrombin III~ One major difficulty in using heparin for the prevention o-f and the therapy for thrombosis and thrombo-embolism is the bleeding inducing capacity of heparin. By optimizing the dose, manner and frequency of application the risk of bleeding can to some extent be reduced, but the intrinsic ratio between -the anti-thrombotic properties and the bleeding-inducing properties of heparin cannot be influenced.
In consequence thereof the prophylactic application of heparin has been restricted to those indications in which the coagulation system is activated only moderately such as in the case of mild forms of deep S vein thrombosis. A further short-coming of heparin is its relatively short period of action, so that for efficient prophylaxis, normally a dosage has to be administered at least twice per day.
Various attempts have already been made to produce "improved heparins", or to produce heparinoids with improved properties. For example British patent publication 2 002 406 relates to an oligo-heteropoly-saccharine mixture with a mow weight distribution between 2000 and 5000 Dalton, which is obtained by lo depolymerisa-tion of heparin and/or is isolated from the mother liquor of the normal heparin preparation, after which the material obtained is further sulphatised. It is stated that the ratio between the anti-thrombotic effect (in viva activity) and anti-coagulation effect (in vitro activity) of the material thus obtained is more favorable than for heparin.
A new anti-thrombotica71y~effective heparinoid has now been discovered which is a mixture of oligo- -and heteropolysaccharides with a striking dissociation between the anti~thrombotic activity and thehae~orrhagic activity (bleeding inducing capacity).
During various bleeding tests on rats (muscle bleeding test, capillary bleeding test) the new heparinoid appears to cause no bleeding or hardly any more bleeding than a placebo, whilst the hemorrhagic action over a wide dosage range (10-250 mg/kg) increased only to a slight extent.
Admittedly the anti-thrombotic action per unit of weight is less than that of heparin, but the benefit risk ratio (i.e. the ratio between the anti-thrombotic activity and the hemorrhagic activity) is 10-40 times more favorable than that of heparin~
This will be illustrated below in further detail by means of comparative examples.
The new anti-thromboticum is of natural origin and consists of a mixture of oligo~ and polysaccharide~
which are based on hoaxes derivatives such as glucuronic acid, iduronic acid, glucosamine, g~lactosamine and sulfated and acetylated derivatives thereof The new product is a white, amorphous, slightly hydroscopic powder having the following characteristics:
a) a mow weight distribution (determined by comparison with dextran by means of gel permeation chromatography on a macro porous silica matrix (Nucleosil*50-S) with 0.5 molar ammonium acetate buffer (pi = 5.0) as eluant) between 2000 and 40,000 Dalton with a main peak between 2500 and 15~000 Dalton, more specifically between 4,000 and 10,000 Dalton, and averaged between 5000 and 8000 Dalton, and usually an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 Dalton, more I;: specifically between Tao and 50,000 daltonst with an average molecular weight of roughly 40,000 Dalton;
b) a specific rotation ([aye) between +25 and +809 more specifically between ~30 and ~70 ;
c) a nitrogen content between 1.5 and 4% by weight, preferably between 2.5 and 3.5% by weight;
do a Selfware content between 5 and 7.5% by weight, preferably between 5.5 and 6.5% by weight;
e) a content of ionic groups in Meg between 3 and 5, preferably between 3.5 and 4.5;
, ', * Trade Mar Jo ., f) a content of sulphamido groups in Meg between 0.5 and 1.5, preferably between 0.5 and 1.0; g) a glucose amine content in Meg of 0.5 to 1.5; h) a galactosamine content in Meg of 0.0 to 0.6;
and i) an dose (iduronic acid glucose (.glucuronic acid) ratio of 0.5 to 3, move especially from 1 to 3.
According to the present invention, a process for preparing an antithrombotically effective heparinoid which is a mixture of oligo- and pol~saccharides based on hoaxes derivatives, said heparinoid having parameters as defined above characterized in that (a) a molecular weight between 2000 and 40,000 Dalton with main peak between 2500 and 15,000 Dalton and optionally an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 Dalton; (b) a specific rotation t.[~]20) between ~25 and ~80; (c) a nitrogen content between 1.5 and 4% by weight; (d) a Selfware content between 5 and 7.5% by weight; (e) a content of ionic grounds in Meg between 3 and 5; (f) a content of sulphamido groups in Meg between 0.5 and 1.5; (g) a glucose amine content in Meg of 0.5 to 1.5; (ho a galactosamine content in ego of 0.0 to 0.6; and (i) an dose ~:induronic acid)/ylucose (glucuronic acid)-ratio of 0.5 to 3, characterized in that mammal tissue is subjected to atlases or to the action of proteolytic enzymes, the resultant mixture is filtered, said heparinoid is isolated from the filtrate by fractional precipitation by adding methanol up to 50% v/v, removing the precipitate, adding more methanol up to 75% v/v and isolating the precipitate thus obtained, or by treating the filtrate with a qua ternary aliphatic ammonium base or with a basic ion exchanger, equating the separated base or ion exchanger with Jo .; - 4 -' aqueous salt solutions and subjecting the equate to said fractional precipitation .
Even though it is not possible Jo exactly specify the oligo- and polysaeeharide composition of the new product, the above-mentioned parameters do eharaeterise the product to a satisfactory extent, particularly in conjunction with its forum-ecological profile.
The pharmacological profile of the new heparinoid is characterized by: I an anti-coagulation activity (CUSP) of less than 10 international units per my (IU/mg), thus only a fraction (usually less thaw 5%) of that of heparin US; 2) a negligible anti-thrombin activity (less than 1% of that of heparin US);
3) an antics activity of less than 20~ of that of heparin, usually between 2.5 and 15%, 4) an anti-thrombotic activity (Umetsu model) with a IDEA of roughly 2 to 8 mg/kg ivy. (IDEA of heparin US is roughly 0.4 to I mg/kg ivy.); 5) a bleeding activity which scarcely increases over a wide dosage range (up to 300 mg/kg ivy whilst that of heparin US at a dosage of 1 mg/kg ivy. is clearly : observable and increases rapidly with higher dosages; 6) a benefit/risk ratio which is 10-40 times more favorable than that for heparin US having regard to the anti-thrombotie activity compared with the hemorrhage activity, and - pa -.
"'I
I
7) a half-value time which is certainly twice as long as that of heparin US.
This interesting profile renders the new product extremely suitable for the prophylaxis and treatment of venous thrombosis and thrombo-embolism. Thus product appears particularly suitable above all for the prevention of DOT (deep vein thrombosis) in patients who are being subjected to or have undergone hip surgery. Hitherto no adequate prophylactic measures have been feasible against the frequent occurrence of DOT and lung embolisms in the case of hip surgery Low doses (sac.) of heparin are not effective and higher ivy. doses are contra-indicated because of the high risk of bleeding.
The new product appears to possess no mutagenic and no toxic properties, even at high doses (up to 400 mg/kg/day).
The new product can be obtained in different ways. Mammal tissue such as lungs, pancreas, liver, intestines and particularly intestinal mucous can serve as the basic material. From the mammal tissue the product is initially liberated by atlases or with the aid of proteolytic enzymes (e.g. enzymes from pig pancreas or bacterial enzymes, such as ; 25 pro teases from Bacillus subtile The ~rodtuct it subsequently isolated by precipitation with orgy it solvents, usually by means of solvents miscible with water such as alcohols, e.g. methanol. Other methods of isolation are the binding of the product to a qua ternary aliphatic ammonium base or a basic ion exchanger and subsequent equation of the product with aqueolls salt solutions n Further purification of the product can be undertaken by fractional precipitation.
, ;
More detailed investigations have shown that by treating the product obtained in this manner with chondroitinase ARC, the greatest proportion of the high-molecular galactosamine-containing polyp saccharides present is removed The remaining product (about 70 -to 75% of the initial product) however appears to have almost the same surprising pharmacological profile as the untreated product, in other words as a result of the said enzymatic lo treatment a preponderantly high-molecular inactive fraction (25-30~ by weight) is removed.
The physical properties of this product obtained after treatment with chondroitinase ABC remain within the characteristics specified above for the new heparinoid, except that in the mow weight distribution the peak and/or shoulder of the high-molecular fraction has almost disappeared, the specific rotation appears to be increased to a value between ~45 and ~75 j and the galactosamine content is reduced practically to zero. It is clear that the invention also comprises this product.
The new product 5 ) can be processed in the manner conventionally employed for heparin into a pharmaceutical dosage form, e.g. by dissolution in water suitable for injection purposes, to which if required further pharmaceutically acceptable auxiliary substances are added (preservation agent, certain salts). Clinical application is by means of subcutaneous or intravenous (possibly intermittent) injection or by infusion. Other methods of dosing are also possible, such as intra-pulmonary application via spray inhalation or administration by means of a suppository.
The invention will be described in greater detail by means of the following examples without these restricting its scope.
~8~6~
Example I
Bovine lung ~100 kg) was treated with proteolytic enzymes from pig pancreas. Aster 15 hours incubation at pi 8.5, 40 C,the mixture was filtered off The clear filtrate was brought into contact with a strongly basic ion exchanger (RAE Sephadex*A50) and stirred for 15 hours. Then the ion exchanger was filtered off and equated with an aqueous solution of Nail (200 g/l).
Methanol was added -to the equate up to 50% v/v.
The resultant precipitate was removed, after which methanol was added to the mother liquor up to 75% v/v. The precipitate was recovered, washed with 100% methanol and dried. The white amorphous powder (22.7 g) obtained had a galactosamine content of 0.~5 mmol/g, a glucosamine content of 0.54 mmol/g, an average molecular weight of 6600 Dalton with an auxiliary peak at an average of 38,000 Dalton (determined with respect to dextrane) 9 a Do of +34.2, a Selfware content of 5.7%, a nitrogen content of 2.6%~ a content of ionic groups of 3.90 Meg a content of sulphamido groups of 0.69 mmol/g and an idose/glucose ratio of 2.1.
The product exhibited an electrophoresis pattern as shown in fig. 1 (agrees gel, equilibrated in 0.2 molar calcium acetate, pi = 7.2, buffer 0.2 molar calcium acetate; 300 V max., 7 ma 30 minutes).
Example II
Pig intestinal mucous (100 kg) was treated with pro teases of bacillus subtilis at 35 C and pi 8.2 for 24 hours. The mixture was filtered and the clear filtrate was processed in a manner similar to -that described in Example I. The yield was 3.2 g of white amorphous powder with a galactosamine content of * Trade Mark 6~6 0.38 mmol/g, a glucosamine con-tent of 0.82 mmol/g, an average molecular weight of 6100 Dalton with an auxiliary peak at an average of 42,000 Dalton (determined with respect to dextrane),an Audi of +35~1, a Selfware content of 5 9%, a nitrogen content of 2.7%, a content of ionic groups of 3.70 Meg a content of sulphamido groups of 0~73 mmol/g and an idose/glucose ratio of 1.9.
The product exhibited a NOR spectrum (4% in D20 at 50 C, 270 MHz) as shown in fig. 2.
Example III 3 Bovine intestinal mucous (1.0 m ) was processed with pancreatine (40 C, pi 8.5, 20 Horace The mixture was filtered and the filtrate processed in the manner as described in Example I. Yield 335 g of white amorphous powder with a galactosami.ne : content of 0~28 mmol/g, a glucosamine content of 0.90 mmol/g, an average molecular weight of 5600 Dalton with an auxiliary peak at on average 41,000 Dalton (determined with respect to dextrane), a Do of ~3609 , a Selfware content of 6.1%, a Jo nitrogen content of 2.8%, a content of ionic groups of 4.0 Meg a content of sulphamide groups of 0.75 mmol/g and an idose/glucose ratio of 1.8.
Example IV
The product from Example III (].00 g) was treated with chondroitinase ABC. Via methanol.
precipitation (50-75% v/v) -the remaining product was isolated. 72 g of product was obtained (white ; amorphous powder) with a galactosamine content of ; 0.05 mmol/g, a glucosamine content of 1.20 mmol/g, an average molecular weight of 5400 Dalton with an extremely small auxiliary peak at on average 40,000 .
~36~
Dalton (determined with respect to dextrane), a []20 of +61~2, a Selfware content of 6.2%, a nitrogen content of 2.75%, a content of ionic groups of 4.1 Meg 9 a content of sulphamido groups of 0.91 mmol/g, and an idose/glucose ratio of 1.9.
Pharmacological tests The products obtained in Examples I to IV
were subjected to a number of pharmacological tests in comparison with heparin US.
Influence on blood coagulation Product Anti- Anti- Anti-X
coagulation thrombin- a activity activity activity (Iamb) (Iamb) (Iamb) .
heparin 179 164 167 Example I 1 0.5 4.5 " II 1 0.1 5 " III 3 1.0 6 " IV 4 1.3 8 The anti coagulation activity was determined in accordance with the US method The anti-thrombin activity and the antics activity were ascertained ; using a chromogenic substrate method, whereby purified cow anti-thrombin III and the substrates (Cobb ABE Sweden) S-2238 and S-2222 respectively were employed.
Anti-thrombotic activity Anti-thrombotic activity was determined in the Umetsu model (Thrombos. Homesites. 39, 74-83, 1978) in rats In this model thrombi are made to occur in arterio-veinous shunts by allowing blood to flow along a silken thread for 15 minutes. The placebo effect and the influence of the substances to be tested in differing doses on thrombus formation is measured From the relationship between dose and the inhibition of thrombus formation with respect to the placebo effect it is possible to derive a IDEA dose required for 50% inhibition of the thrombus formation).
Results _ Product ID in McKee - I --heparin US 0,5 example I 6.0 " II 5.0 " III 4.0 " IV 5.3 Hemorrhagic activity a) Capillary bleeding test in rats Placebo or the products to be tested were dosed via the dorsal vein of the penis of anesthetized rats After one minute a strip of skin was pulled away manually from the shaven abdomen along two previously applied incisions. The wound was covered with a gauze bandage and the strip of skin was laid back over the gauze. After lo minutes the gauze was removed and the blood present therein was extracted with 20 ml water.
The hemoglobin concentration in the water was measured spectrophotometrically and employed as parameter for 25 the loss of blood (see Thrombos. Homesites. 42, 466, 1979).
During this test the product from Example II and heparin were compared at different dosages, consideration being given to the difference in anti-thrombotic activity as between the new product and heparin, The result is shown in fig. I Corresponding ; results were obtained with the products from Examples I, III and IV.
;
;
' I
b) Muscle bleeding tests in rats q The biceps fPmoris in each paw of an anesthetized rat was out longitudinally with a scalpel, one minute after the placebo or product to be tested had been administered intravenously via the dorsal vein of the penis. Each wound was covered with a gauze bandage. After 30 minutes -the bandages were removed. The loss of blood was determined in the same way as during the capillary bleeding test. The product from Example II and heparin were compared in this test The result is shown in fig. I Corresponding results were obtained with the products from Examples I, III and IV.
Figures 3 and 4 show clearly the much greater tendency towards bleeding of heparin, whilst that of the new product does not differ or hardly differs ; significantly at all from that of the placebo over a wide dosage range. The benefit/risk ratio is quite obviously in favor of the new product 20 Half-value time anti-thrombotic effect The duration of the anti-thrombotic effect was determined in the Umetsu model for heparin and for the product from Example II, whereby heparin and the new product were tested in the ratio of their IDEA
25 (heparin 2 mg/kg ivy., product II, 20 mg/kg ivy.).
The result is shown in fig. 5.
< The new product is obviously effective for a longer period than heparin. Corresponding results were obtained with the products from Examples I, III
30 and IV.
The invention relates to a new anti-thromboticum based on a mixture of oligo- and polysaccharides and to methods for preparation thereof. The invention also relates to pharmaceutical compositions which include the said mixture.
It is known that certain mucopolysaccharides influence the coagulation properties of blood. The best known mucopolysaccharide is heparin, a sulfated mucopolysaccharide1 which is employed in prophylaxis and the treatment of venous thrombosis and thrombo-embolism. The anti-thrombotlc effect of heparin is based on acceleration of the inhibition of blood-coagulating factors by anti-thrombin III~ One major difficulty in using heparin for the prevention o-f and the therapy for thrombosis and thrombo-embolism is the bleeding inducing capacity of heparin. By optimizing the dose, manner and frequency of application the risk of bleeding can to some extent be reduced, but the intrinsic ratio between -the anti-thrombotic properties and the bleeding-inducing properties of heparin cannot be influenced.
In consequence thereof the prophylactic application of heparin has been restricted to those indications in which the coagulation system is activated only moderately such as in the case of mild forms of deep S vein thrombosis. A further short-coming of heparin is its relatively short period of action, so that for efficient prophylaxis, normally a dosage has to be administered at least twice per day.
Various attempts have already been made to produce "improved heparins", or to produce heparinoids with improved properties. For example British patent publication 2 002 406 relates to an oligo-heteropoly-saccharine mixture with a mow weight distribution between 2000 and 5000 Dalton, which is obtained by lo depolymerisa-tion of heparin and/or is isolated from the mother liquor of the normal heparin preparation, after which the material obtained is further sulphatised. It is stated that the ratio between the anti-thrombotic effect (in viva activity) and anti-coagulation effect (in vitro activity) of the material thus obtained is more favorable than for heparin.
A new anti-thrombotica71y~effective heparinoid has now been discovered which is a mixture of oligo- -and heteropolysaccharides with a striking dissociation between the anti~thrombotic activity and thehae~orrhagic activity (bleeding inducing capacity).
During various bleeding tests on rats (muscle bleeding test, capillary bleeding test) the new heparinoid appears to cause no bleeding or hardly any more bleeding than a placebo, whilst the hemorrhagic action over a wide dosage range (10-250 mg/kg) increased only to a slight extent.
Admittedly the anti-thrombotic action per unit of weight is less than that of heparin, but the benefit risk ratio (i.e. the ratio between the anti-thrombotic activity and the hemorrhagic activity) is 10-40 times more favorable than that of heparin~
This will be illustrated below in further detail by means of comparative examples.
The new anti-thromboticum is of natural origin and consists of a mixture of oligo~ and polysaccharide~
which are based on hoaxes derivatives such as glucuronic acid, iduronic acid, glucosamine, g~lactosamine and sulfated and acetylated derivatives thereof The new product is a white, amorphous, slightly hydroscopic powder having the following characteristics:
a) a mow weight distribution (determined by comparison with dextran by means of gel permeation chromatography on a macro porous silica matrix (Nucleosil*50-S) with 0.5 molar ammonium acetate buffer (pi = 5.0) as eluant) between 2000 and 40,000 Dalton with a main peak between 2500 and 15~000 Dalton, more specifically between 4,000 and 10,000 Dalton, and averaged between 5000 and 8000 Dalton, and usually an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 Dalton, more I;: specifically between Tao and 50,000 daltonst with an average molecular weight of roughly 40,000 Dalton;
b) a specific rotation ([aye) between +25 and +809 more specifically between ~30 and ~70 ;
c) a nitrogen content between 1.5 and 4% by weight, preferably between 2.5 and 3.5% by weight;
do a Selfware content between 5 and 7.5% by weight, preferably between 5.5 and 6.5% by weight;
e) a content of ionic groups in Meg between 3 and 5, preferably between 3.5 and 4.5;
, ', * Trade Mar Jo ., f) a content of sulphamido groups in Meg between 0.5 and 1.5, preferably between 0.5 and 1.0; g) a glucose amine content in Meg of 0.5 to 1.5; h) a galactosamine content in Meg of 0.0 to 0.6;
and i) an dose (iduronic acid glucose (.glucuronic acid) ratio of 0.5 to 3, move especially from 1 to 3.
According to the present invention, a process for preparing an antithrombotically effective heparinoid which is a mixture of oligo- and pol~saccharides based on hoaxes derivatives, said heparinoid having parameters as defined above characterized in that (a) a molecular weight between 2000 and 40,000 Dalton with main peak between 2500 and 15,000 Dalton and optionally an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 Dalton; (b) a specific rotation t.[~]20) between ~25 and ~80; (c) a nitrogen content between 1.5 and 4% by weight; (d) a Selfware content between 5 and 7.5% by weight; (e) a content of ionic grounds in Meg between 3 and 5; (f) a content of sulphamido groups in Meg between 0.5 and 1.5; (g) a glucose amine content in Meg of 0.5 to 1.5; (ho a galactosamine content in ego of 0.0 to 0.6; and (i) an dose ~:induronic acid)/ylucose (glucuronic acid)-ratio of 0.5 to 3, characterized in that mammal tissue is subjected to atlases or to the action of proteolytic enzymes, the resultant mixture is filtered, said heparinoid is isolated from the filtrate by fractional precipitation by adding methanol up to 50% v/v, removing the precipitate, adding more methanol up to 75% v/v and isolating the precipitate thus obtained, or by treating the filtrate with a qua ternary aliphatic ammonium base or with a basic ion exchanger, equating the separated base or ion exchanger with Jo .; - 4 -' aqueous salt solutions and subjecting the equate to said fractional precipitation .
Even though it is not possible Jo exactly specify the oligo- and polysaeeharide composition of the new product, the above-mentioned parameters do eharaeterise the product to a satisfactory extent, particularly in conjunction with its forum-ecological profile.
The pharmacological profile of the new heparinoid is characterized by: I an anti-coagulation activity (CUSP) of less than 10 international units per my (IU/mg), thus only a fraction (usually less thaw 5%) of that of heparin US; 2) a negligible anti-thrombin activity (less than 1% of that of heparin US);
3) an antics activity of less than 20~ of that of heparin, usually between 2.5 and 15%, 4) an anti-thrombotic activity (Umetsu model) with a IDEA of roughly 2 to 8 mg/kg ivy. (IDEA of heparin US is roughly 0.4 to I mg/kg ivy.); 5) a bleeding activity which scarcely increases over a wide dosage range (up to 300 mg/kg ivy whilst that of heparin US at a dosage of 1 mg/kg ivy. is clearly : observable and increases rapidly with higher dosages; 6) a benefit/risk ratio which is 10-40 times more favorable than that for heparin US having regard to the anti-thrombotie activity compared with the hemorrhage activity, and - pa -.
"'I
I
7) a half-value time which is certainly twice as long as that of heparin US.
This interesting profile renders the new product extremely suitable for the prophylaxis and treatment of venous thrombosis and thrombo-embolism. Thus product appears particularly suitable above all for the prevention of DOT (deep vein thrombosis) in patients who are being subjected to or have undergone hip surgery. Hitherto no adequate prophylactic measures have been feasible against the frequent occurrence of DOT and lung embolisms in the case of hip surgery Low doses (sac.) of heparin are not effective and higher ivy. doses are contra-indicated because of the high risk of bleeding.
The new product appears to possess no mutagenic and no toxic properties, even at high doses (up to 400 mg/kg/day).
The new product can be obtained in different ways. Mammal tissue such as lungs, pancreas, liver, intestines and particularly intestinal mucous can serve as the basic material. From the mammal tissue the product is initially liberated by atlases or with the aid of proteolytic enzymes (e.g. enzymes from pig pancreas or bacterial enzymes, such as ; 25 pro teases from Bacillus subtile The ~rodtuct it subsequently isolated by precipitation with orgy it solvents, usually by means of solvents miscible with water such as alcohols, e.g. methanol. Other methods of isolation are the binding of the product to a qua ternary aliphatic ammonium base or a basic ion exchanger and subsequent equation of the product with aqueolls salt solutions n Further purification of the product can be undertaken by fractional precipitation.
, ;
More detailed investigations have shown that by treating the product obtained in this manner with chondroitinase ARC, the greatest proportion of the high-molecular galactosamine-containing polyp saccharides present is removed The remaining product (about 70 -to 75% of the initial product) however appears to have almost the same surprising pharmacological profile as the untreated product, in other words as a result of the said enzymatic lo treatment a preponderantly high-molecular inactive fraction (25-30~ by weight) is removed.
The physical properties of this product obtained after treatment with chondroitinase ABC remain within the characteristics specified above for the new heparinoid, except that in the mow weight distribution the peak and/or shoulder of the high-molecular fraction has almost disappeared, the specific rotation appears to be increased to a value between ~45 and ~75 j and the galactosamine content is reduced practically to zero. It is clear that the invention also comprises this product.
The new product 5 ) can be processed in the manner conventionally employed for heparin into a pharmaceutical dosage form, e.g. by dissolution in water suitable for injection purposes, to which if required further pharmaceutically acceptable auxiliary substances are added (preservation agent, certain salts). Clinical application is by means of subcutaneous or intravenous (possibly intermittent) injection or by infusion. Other methods of dosing are also possible, such as intra-pulmonary application via spray inhalation or administration by means of a suppository.
The invention will be described in greater detail by means of the following examples without these restricting its scope.
~8~6~
Example I
Bovine lung ~100 kg) was treated with proteolytic enzymes from pig pancreas. Aster 15 hours incubation at pi 8.5, 40 C,the mixture was filtered off The clear filtrate was brought into contact with a strongly basic ion exchanger (RAE Sephadex*A50) and stirred for 15 hours. Then the ion exchanger was filtered off and equated with an aqueous solution of Nail (200 g/l).
Methanol was added -to the equate up to 50% v/v.
The resultant precipitate was removed, after which methanol was added to the mother liquor up to 75% v/v. The precipitate was recovered, washed with 100% methanol and dried. The white amorphous powder (22.7 g) obtained had a galactosamine content of 0.~5 mmol/g, a glucosamine content of 0.54 mmol/g, an average molecular weight of 6600 Dalton with an auxiliary peak at an average of 38,000 Dalton (determined with respect to dextrane) 9 a Do of +34.2, a Selfware content of 5.7%, a nitrogen content of 2.6%~ a content of ionic groups of 3.90 Meg a content of sulphamido groups of 0.69 mmol/g and an idose/glucose ratio of 2.1.
The product exhibited an electrophoresis pattern as shown in fig. 1 (agrees gel, equilibrated in 0.2 molar calcium acetate, pi = 7.2, buffer 0.2 molar calcium acetate; 300 V max., 7 ma 30 minutes).
Example II
Pig intestinal mucous (100 kg) was treated with pro teases of bacillus subtilis at 35 C and pi 8.2 for 24 hours. The mixture was filtered and the clear filtrate was processed in a manner similar to -that described in Example I. The yield was 3.2 g of white amorphous powder with a galactosamine content of * Trade Mark 6~6 0.38 mmol/g, a glucosamine con-tent of 0.82 mmol/g, an average molecular weight of 6100 Dalton with an auxiliary peak at an average of 42,000 Dalton (determined with respect to dextrane),an Audi of +35~1, a Selfware content of 5 9%, a nitrogen content of 2.7%, a content of ionic groups of 3.70 Meg a content of sulphamido groups of 0~73 mmol/g and an idose/glucose ratio of 1.9.
The product exhibited a NOR spectrum (4% in D20 at 50 C, 270 MHz) as shown in fig. 2.
Example III 3 Bovine intestinal mucous (1.0 m ) was processed with pancreatine (40 C, pi 8.5, 20 Horace The mixture was filtered and the filtrate processed in the manner as described in Example I. Yield 335 g of white amorphous powder with a galactosami.ne : content of 0~28 mmol/g, a glucosamine content of 0.90 mmol/g, an average molecular weight of 5600 Dalton with an auxiliary peak at on average 41,000 Dalton (determined with respect to dextrane), a Do of ~3609 , a Selfware content of 6.1%, a Jo nitrogen content of 2.8%, a content of ionic groups of 4.0 Meg a content of sulphamide groups of 0.75 mmol/g and an idose/glucose ratio of 1.8.
Example IV
The product from Example III (].00 g) was treated with chondroitinase ABC. Via methanol.
precipitation (50-75% v/v) -the remaining product was isolated. 72 g of product was obtained (white ; amorphous powder) with a galactosamine content of ; 0.05 mmol/g, a glucosamine content of 1.20 mmol/g, an average molecular weight of 5400 Dalton with an extremely small auxiliary peak at on average 40,000 .
~36~
Dalton (determined with respect to dextrane), a []20 of +61~2, a Selfware content of 6.2%, a nitrogen content of 2.75%, a content of ionic groups of 4.1 Meg 9 a content of sulphamido groups of 0.91 mmol/g, and an idose/glucose ratio of 1.9.
Pharmacological tests The products obtained in Examples I to IV
were subjected to a number of pharmacological tests in comparison with heparin US.
Influence on blood coagulation Product Anti- Anti- Anti-X
coagulation thrombin- a activity activity activity (Iamb) (Iamb) (Iamb) .
heparin 179 164 167 Example I 1 0.5 4.5 " II 1 0.1 5 " III 3 1.0 6 " IV 4 1.3 8 The anti coagulation activity was determined in accordance with the US method The anti-thrombin activity and the antics activity were ascertained ; using a chromogenic substrate method, whereby purified cow anti-thrombin III and the substrates (Cobb ABE Sweden) S-2238 and S-2222 respectively were employed.
Anti-thrombotic activity Anti-thrombotic activity was determined in the Umetsu model (Thrombos. Homesites. 39, 74-83, 1978) in rats In this model thrombi are made to occur in arterio-veinous shunts by allowing blood to flow along a silken thread for 15 minutes. The placebo effect and the influence of the substances to be tested in differing doses on thrombus formation is measured From the relationship between dose and the inhibition of thrombus formation with respect to the placebo effect it is possible to derive a IDEA dose required for 50% inhibition of the thrombus formation).
Results _ Product ID in McKee - I --heparin US 0,5 example I 6.0 " II 5.0 " III 4.0 " IV 5.3 Hemorrhagic activity a) Capillary bleeding test in rats Placebo or the products to be tested were dosed via the dorsal vein of the penis of anesthetized rats After one minute a strip of skin was pulled away manually from the shaven abdomen along two previously applied incisions. The wound was covered with a gauze bandage and the strip of skin was laid back over the gauze. After lo minutes the gauze was removed and the blood present therein was extracted with 20 ml water.
The hemoglobin concentration in the water was measured spectrophotometrically and employed as parameter for 25 the loss of blood (see Thrombos. Homesites. 42, 466, 1979).
During this test the product from Example II and heparin were compared at different dosages, consideration being given to the difference in anti-thrombotic activity as between the new product and heparin, The result is shown in fig. I Corresponding ; results were obtained with the products from Examples I, III and IV.
;
;
' I
b) Muscle bleeding tests in rats q The biceps fPmoris in each paw of an anesthetized rat was out longitudinally with a scalpel, one minute after the placebo or product to be tested had been administered intravenously via the dorsal vein of the penis. Each wound was covered with a gauze bandage. After 30 minutes -the bandages were removed. The loss of blood was determined in the same way as during the capillary bleeding test. The product from Example II and heparin were compared in this test The result is shown in fig. I Corresponding results were obtained with the products from Examples I, III and IV.
Figures 3 and 4 show clearly the much greater tendency towards bleeding of heparin, whilst that of the new product does not differ or hardly differs ; significantly at all from that of the placebo over a wide dosage range. The benefit/risk ratio is quite obviously in favor of the new product 20 Half-value time anti-thrombotic effect The duration of the anti-thrombotic effect was determined in the Umetsu model for heparin and for the product from Example II, whereby heparin and the new product were tested in the ratio of their IDEA
25 (heparin 2 mg/kg ivy., product II, 20 mg/kg ivy.).
The result is shown in fig. 5.
< The new product is obviously effective for a longer period than heparin. Corresponding results were obtained with the products from Examples I, III
30 and IV.
Claims (14)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing an antithrombotically effective heparinoid which is a mixture of oligo- and polysaccharides based on hexose derivatives, said heparinoid having (a) a molecular weight between 2000 and 40,000 daltons with main peak between 2500 and 15,000 daltons and optionally an auxiliary peak and/or shoulder in the range between 15,000 and 60,000. daltons;
(b) a specific rotation ([.alpha.]?0 ) between +25° and +80°;
(c) a nitrogen content between 1.5 and 4% by weight;
(d) a sulphur content between 5 and 7.5% by weight;
(e) a content of ionic grounds in meq/g between 3 and 5;
(f) a content of sulphamido groups in meq/g between 0.5 and 1.5;
(g) a glucoseamine content in meq/g of 0.5 to 1.5;
(h), a galactosamine content in meg/g of 0.0 to 0.6; and (i) an idose (induronic acid)/glucose (glucuronic acid)-ratio of 0.5 to 3, characterised in that mammal tissue is subjected to autolysis or to the action of proteolytic enzymes, the result-ant mixture is filtered, said heparinoid is isolated from the filtrate by fractional precipitation by adding methanol up to 50% v/v, removing the precipitate, adding more methanol up to 75% v/v and isolating the precipitate thus obtained, or by treating the filtrate with a quaternary aliphatic ammonium base or with a basic ion exchanger, eluating the separated base or ion exchanger with aqueous salt solutions and subjecting the eluate to said fractional precipitation.
(b) a specific rotation ([.alpha.]?0 ) between +25° and +80°;
(c) a nitrogen content between 1.5 and 4% by weight;
(d) a sulphur content between 5 and 7.5% by weight;
(e) a content of ionic grounds in meq/g between 3 and 5;
(f) a content of sulphamido groups in meq/g between 0.5 and 1.5;
(g) a glucoseamine content in meq/g of 0.5 to 1.5;
(h), a galactosamine content in meg/g of 0.0 to 0.6; and (i) an idose (induronic acid)/glucose (glucuronic acid)-ratio of 0.5 to 3, characterised in that mammal tissue is subjected to autolysis or to the action of proteolytic enzymes, the result-ant mixture is filtered, said heparinoid is isolated from the filtrate by fractional precipitation by adding methanol up to 50% v/v, removing the precipitate, adding more methanol up to 75% v/v and isolating the precipitate thus obtained, or by treating the filtrate with a quaternary aliphatic ammonium base or with a basic ion exchanger, eluating the separated base or ion exchanger with aqueous salt solutions and subjecting the eluate to said fractional precipitation.
2. A process according to claim 1 wherein said hexose derivatives are selected from the group consisting of glucoronic acid, iduronic acid, glucosamine, galactosamine, and sulfated and acetylated derivaties thereof.
3. A process according to claim 1 wherein said mammal tissue is selected from the tissue group consisting of lungs, pancreas, liver, intestines and intestinal mucous.
4. A process according to claim 1, 2 or 3 characterised in that the isolated product is subjected to the action of chondroitinase ABC in order to remove the greatest part of the high-molecular polysaccharides, and the remaining product is isolated.
5. An anti-thromboticum based on a mixture of oligo- and polysaccharides with:
(a) a molecular weight between 2000 and 40,000 daltons with main peak between 2500 and 15,000 daltons and optionally an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 daltons;
(b), a specific rotation ([.alpha.]?0) between +25°and +80°;
(c), a nitrogen content between 1.5 and 4% by weight;
(d) a sulphur content between 5 and 7.5% by weight;
(e) a content of ionic grounds: in meq/g between 3 and 5;
(f) a content of sulphamido groups in meq/g between 0.5 and 1.5;
(g) a glucoseamine content in meq/g of 0.5 to 1.5;
(h) a galactosamine content in meq/g of 0.0 to 0.6; and (i) an idose (induronic acid)/glucose (glucuronic acid)-ratio of 0.5 to 3, whenever prepared by the process of claim 1, or by an obvious chemical equivalent thereof.
(a) a molecular weight between 2000 and 40,000 daltons with main peak between 2500 and 15,000 daltons and optionally an auxiliary peak and/or shoulder in the range between 15,000 and 60,000 daltons;
(b), a specific rotation ([.alpha.]?0) between +25°and +80°;
(c), a nitrogen content between 1.5 and 4% by weight;
(d) a sulphur content between 5 and 7.5% by weight;
(e) a content of ionic grounds: in meq/g between 3 and 5;
(f) a content of sulphamido groups in meq/g between 0.5 and 1.5;
(g) a glucoseamine content in meq/g of 0.5 to 1.5;
(h) a galactosamine content in meq/g of 0.0 to 0.6; and (i) an idose (induronic acid)/glucose (glucuronic acid)-ratio of 0.5 to 3, whenever prepared by the process of claim 1, or by an obvious chemical equivalent thereof.
6. A process according to claim 1 wherein the anti-thromoboticum has a bimodal molecular weight distribution with a main peak between 4000 and 10,000 daltons and an average molecular weight between 5000 and 8000 daltons, and an optional peak between 30,000 and 50,000 daltons, with an average molecular weight of substantially about 40,000 daltons.
7. A process according to claim 1 wherein the anti-thromboticum has a [.alpha.]?0 between +40° amd +70°.
8. A process according to claim 1 wherein the anti-thromboticum has a nitrogen content between 2.5 and 3.5% by weight.
9. A process according to claim 1 wherein the anti-thromboticum has a sulphur content between 5.5 and 6.5% by weight.
10. A process according to claim 1 wherein the anti-thromboticum has a content of ionic groups between 3.5 and 4.5 meq/g.
11. A process according to claim 1 wherein the anti-thromboticum has a content of sulphamido groups between 0.5 and 1.0 meq/g.
12. A process according to claim 1 wherein the anti-thromboticum has a galactosamine content of 0.0 to 0.1 meq/g.
13. A process according to claim 1 wherein the anti-thromboticum has an idose (iduronic acid)/glucose (glucuronic acid) ratio of 1 to 3.
14. A process according to claim 1 wherein the anti-thromboticum has 1) an anti-coagulation activity (USP) of less than 10 international units per mg;
2) substantially no thrombin activity (less than 1% of that of heparin USP);
3) an anti-Xa activity of less than 20% of that of heparin;
4) an anti-thrombotic activity (Umetsu model) with an ID50 of substantially 2 to 8 mg/kg i.v.;
5) a bleeding activity which is substantially constant over a wide dosage range up to 300 mg/kg i.v.;
6) a benefit/risk ratio which is 10-40. times more favourable than that for heparin USP having regard to the anti-thrombotic activity as compared with haemorrhagic activity; and 7) half value time which is certainly twice as long as that of heparin USP.
2) substantially no thrombin activity (less than 1% of that of heparin USP);
3) an anti-Xa activity of less than 20% of that of heparin;
4) an anti-thrombotic activity (Umetsu model) with an ID50 of substantially 2 to 8 mg/kg i.v.;
5) a bleeding activity which is substantially constant over a wide dosage range up to 300 mg/kg i.v.;
6) a benefit/risk ratio which is 10-40. times more favourable than that for heparin USP having regard to the anti-thrombotic activity as compared with haemorrhagic activity; and 7) half value time which is certainly twice as long as that of heparin USP.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL8102514 | 1981-05-21 | ||
| NL81.02514 | 1981-05-21 |
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| Publication Number | Publication Date |
|---|---|
| CA1186646A true CA1186646A (en) | 1985-05-07 |
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ID=19837549
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000403376A Expired CA1186646A (en) | 1981-05-21 | 1982-05-20 | Anti-thromboticum based on polysacharides, method for its preparation and pharmaceutical compositions |
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| Country | Link |
|---|---|
| US (1) | US4438108A (en) |
| EP (1) | EP0066908B1 (en) |
| JP (1) | JPS57197221A (en) |
| AT (1) | ATE15142T1 (en) |
| AU (1) | AU550317B2 (en) |
| CA (1) | CA1186646A (en) |
| DE (1) | DE3265781D1 (en) |
| DK (1) | DK157061C (en) |
| ES (1) | ES512464A0 (en) |
| FI (1) | FI75491C (en) |
| GR (1) | GR76794B (en) |
| HU (1) | HU190687B (en) |
| IE (1) | IE53255B1 (en) |
| NZ (1) | NZ200688A (en) |
| PT (1) | PT74928B (en) |
| ZA (1) | ZA823248B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4696816A (en) * | 1985-11-07 | 1987-09-29 | Brown Mark D | Method for treating intervertebral disc displacement with enzymes |
| US4745106A (en) * | 1986-08-20 | 1988-05-17 | Griffin Charles C | Heparin derivatives having improved anti-Xa specificity |
| US4942156A (en) * | 1986-08-20 | 1990-07-17 | Hepar Industries, Inc. | Low molecular weight heparin derivatives having improved anti-Xa specificity |
| DE3639561A1 (en) * | 1986-11-20 | 1988-06-01 | Baumann Hanno | METHOD FOR PRODUCING NON-THROMBOGEN SUBSTRATES |
| IT1213384B (en) * | 1986-11-24 | 1989-12-20 | Lab Derivati Organici Mediolan | PROCESS FOR THE CONTROLLED PREPARATION OF LOW MOLECULAR WEIGHT GILCOSAMINOGLICANS. |
| EP0333243A3 (en) * | 1988-03-10 | 1989-09-27 | Akzo N.V. | Sulphated k5 antigen and sulphated k5 antigen fragments |
| EP0337327A1 (en) * | 1988-04-09 | 1989-10-18 | Bioiberica, S.A. | Process for the preparation of new oligosaccharide fractions by controlled chemical depolimerization of heparin |
| IT1234826B (en) * | 1989-01-30 | 1992-05-29 | Alfa Wassermann Spa | HEPARIN DERIVATIVES AND PROCEDURE FOR THEIR PREPARATION |
| ATE98656T1 (en) * | 1989-10-04 | 1994-01-15 | Akzo Nv | SULPHATED GLYCOSAMINOGLYCURONANS WITH ANTITHROMBOTIC ACTIVITY. |
| USRE38743E1 (en) | 1990-06-26 | 2005-06-14 | Aventis Pharma S.A. | Mixtures of particular LMW heparinic polysaccharides for the prophylaxis/treatment of acute thrombotic events |
| FR2663639B1 (en) * | 1990-06-26 | 1994-03-18 | Rhone Poulenc Sante | LOW MOLECULAR WEIGHT POLYSACCHARIDE BLENDS PROCESS FOR PREPARATION AND USE. |
| US5306711A (en) * | 1992-06-24 | 1994-04-26 | Georgetown University | Organ preservative solution |
| FR2723847A1 (en) * | 1994-08-29 | 1996-03-01 | Debiopharm Sa | HEPARIN - BASED ANTITHROMBOTIC AND NON - HEMORRHAGIC COMPOSITIONS, PROCESS FOR THEIR PREPARATION AND THERAPEUTIC APPLICATIONS. |
| DE19646901A1 (en) * | 1996-11-13 | 1998-05-14 | Helmut Prof Dr Heusinger | Process for the production of degradation products of polymeric glycosaminoglycans by means of ultrasound |
| SK286024B6 (en) * | 1996-11-27 | 2008-01-07 | Aventis Pharmaceuticals Inc. | Pharmaceutical composition and use of the composition for medicine preparation |
| BR0012202A (en) * | 1999-06-30 | 2002-04-02 | Hamilton Civic Hospitals Res | Compositions of heparin of average molecular weight (mmwh), method of treating a thrombotic condition, method of preventing the formation of a thrombus, method of inhibiting the formation of thrombi, pharmaceutical composition, method of treating deep vein thrombosis, method of prevention of pulmonary embolism, method of preparation and uses of a composition of mmwh |
| AU2001291549A1 (en) * | 2000-09-08 | 2002-03-22 | Hamilton Civic Hospitals Research Development Inc. | Antithrombotic compositions |
| WO2002076475A2 (en) * | 2001-03-23 | 2002-10-03 | Bioparken As | Glycosaminoglycan anticoagulants derived from fish |
| US20040171819A1 (en) | 2002-10-10 | 2004-09-02 | Aventis Pharma S.A. | Mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them |
| JP2006096668A (en) * | 2002-11-08 | 2006-04-13 | Ono Pharmaceut Co Ltd | Medicine comprising combination of elastase inhibitor with enzyme inhibitor of blood coagulation system and/or fibrinolysis system |
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|---|---|---|---|---|
| US3451996A (en) * | 1968-02-12 | 1969-06-24 | Thompson Farms Co | Method for the preparation of heparin |
| US3766167A (en) * | 1971-03-26 | 1973-10-16 | Research Corp | Orally active anticoagulant |
| DE2652272C2 (en) * | 1976-11-12 | 1979-02-15 | Schering Ag, 1000 Berlin Und 4619 Bergkamen | Process for the production of heparin |
| IT1075117B (en) * | 1977-02-14 | 1985-04-22 | Fedeli Gianfranco | ARTERIAL POLYSACCHARIDIC COMPLEX, PROCESS FOR ITS PREPARATION AND USE IN HUMAN THERAPY |
| US4301153A (en) | 1977-03-21 | 1981-11-17 | Riker Laboratories, Inc. | Heparin preparation |
| DE2800943C2 (en) * | 1978-01-06 | 1984-09-27 | Schering AG, 1000 Berlin und 4709 Bergkamen | Use of intestinal brine to obtain pure heparin |
| US4175182A (en) | 1978-07-03 | 1979-11-20 | Research Corporation | Separation of high-activity heparin by affinity chromatography on supported protamine |
| CA1136620A (en) | 1979-01-08 | 1982-11-30 | Ulf P.F. Lindahl | Heparin fragments having selective anticoagulation activity |
| DE2903701C2 (en) | 1979-01-31 | 1980-10-16 | Boehringer Mannheim Gmbh, 6800 Mannheim | Control reagent for the determination of the heparin activity |
| US4351938A (en) | 1980-05-19 | 1982-09-28 | Riker Laboratories, Inc. | Anticoagulant substance |
-
1982
- 1982-05-07 AT AT82200559T patent/ATE15142T1/en not_active IP Right Cessation
- 1982-05-07 DE DE8282200559T patent/DE3265781D1/en not_active Expired
- 1982-05-07 EP EP82200559A patent/EP0066908B1/en not_active Expired
- 1982-05-10 IE IE1118/82A patent/IE53255B1/en not_active IP Right Cessation
- 1982-05-11 ZA ZA823248A patent/ZA823248B/en unknown
- 1982-05-12 US US06/377,581 patent/US4438108A/en not_active Expired - Lifetime
- 1982-05-19 PT PT74928A patent/PT74928B/en not_active IP Right Cessation
- 1982-05-19 DK DK227282A patent/DK157061C/en not_active IP Right Cessation
- 1982-05-20 HU HU821620A patent/HU190687B/en unknown
- 1982-05-20 FI FI821804A patent/FI75491C/en not_active IP Right Cessation
- 1982-05-20 CA CA000403376A patent/CA1186646A/en not_active Expired
- 1982-05-20 NZ NZ200688A patent/NZ200688A/en unknown
- 1982-05-20 JP JP57085653A patent/JPS57197221A/en active Granted
- 1982-05-20 GR GR68206A patent/GR76794B/el unknown
- 1982-05-21 AU AU83717/82A patent/AU550317B2/en not_active Ceased
- 1982-05-21 ES ES512464A patent/ES512464A0/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| HU190687B (en) | 1986-10-28 |
| DK157061B (en) | 1989-11-06 |
| DE3265781D1 (en) | 1985-10-03 |
| ZA823248B (en) | 1983-03-30 |
| JPH0216733B2 (en) | 1990-04-18 |
| AU550317B2 (en) | 1986-03-20 |
| FI75491B (en) | 1988-03-31 |
| FI75491C (en) | 1988-07-11 |
| ES8307098A1 (en) | 1983-07-01 |
| DK227282A (en) | 1982-11-22 |
| ES512464A0 (en) | 1983-07-01 |
| US4438108A (en) | 1984-03-20 |
| DK157061C (en) | 1990-04-09 |
| FI821804A0 (en) | 1982-05-20 |
| ATE15142T1 (en) | 1985-09-15 |
| IE53255B1 (en) | 1988-09-28 |
| PT74928B (en) | 1983-12-07 |
| PT74928A (en) | 1982-06-01 |
| NZ200688A (en) | 1985-10-11 |
| IE821118L (en) | 1982-11-21 |
| EP0066908A1 (en) | 1982-12-15 |
| GR76794B (en) | 1984-09-04 |
| JPS57197221A (en) | 1982-12-03 |
| AU8371782A (en) | 1982-11-25 |
| EP0066908B1 (en) | 1985-08-28 |
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