HK1059273B - Heparin-derived polysaccharide mixtures, preparation method and pharmaceutical compositions containing same - Google Patents

Description

heparin-derived polysaccharide mixtures, process for their preparation and pharmaceutical compositions containing them

The present invention relates to mixtures of polysaccharides derived from heparin, to a process for their preparation and to pharmaceutical compositions containing them.

Heparins are mixtures of mucopolysaccharides of animal origin, used in particular because of their anticoagulant and antithrombotic properties.

However, heparin has a number of drawbacks that limit its use. In particular, its high anticoagulant activity (anti-IIa activity) may cause bleeding.

Low molecular weight heparins obtained by alkaline depolymerization of heparin esters have been proposed (EP 40144); however, these products also have a high IIa-resistance activity.

The invention relates to heparin-derived polysaccharide mixtures which, compared with heparin, produce activity in a more selective manner with respect to activated factor x (factor Xa) and activated factor II (factor IIa).

More specifically, the invention relates to sulfated polysaccharide mixtures having the general structure of polysaccharides constituted by heparin and having the following characteristics:

their average molecular weight is 1500-3000 daltons, the anti-Xa activity is 100-150 IU/mg, the anti-IIa activity is 0-10 IU/mg, the anti-Xa activity/anti-IIa activity ratio is higher than 10,

the constituent polysaccharides of these mixtures contain 2 to 26 sugar building blocks with unsaturated 4, 5-glucuronic acid 2-O-sulfate building blocks at one end,

these polysaccharide mixtures are in the form of alkali metal or alkaline earth metal salts.

As the alkali metal or alkaline earth metal salt, sodium, potassium, calcium and magnesium salts are preferable.

The average molecular weight is determined by high pressure liquid chromatography using two columns in series, such as columns sold under the tradenames TSK G3000XL and TSK G2000 XL. The detection is performed using a refractometer. The eluent used was lithium nitrate at a flow rate of 0.6 ml/min. Standards were prepared by separating enoxaparin (AVENTIS) using agarose-polyacrylamide gel (IBF) chromatography and used to calibrate this system. Such preparation is carried out according to the techniques described in Barrowcliffe et al, Thromb. Res., 12, 27-36(1977-78), or D.A. Lane et al, Thromb. Res., 12, 257-271 (1977-78). The results were calculated using the software GPC6(Perkin Elmer).

anti-Xa activity was measured using the amidolytic method using the first international standard for low molecular weight heparin as a standard and using chromogenic substrates as described by Teien and co-workers, Thromb. Res., 10, 399-.

The anti-IIa activity was measured using the first international standard for low molecular weight heparins as standard, using the technique described in Anderson L.O. and co-workers, Thromb. Res., 15, 531-541 (1979).

Preferably, the anti-Xa activity of the above mixture is 125-150 IU/mg.

Very particularly, the anti-Xa activity of the above mixture was 140 IU/mg. The average molecular weight is 2000-3000 dalton.

Preferably, the mixture of the invention has an anti-IIa activity of 0-5 IU/mg.

More particularly, these mixtures have a ratio of Xa-activity/IIa-activity resistance of higher than 25.

The oligosaccharide mixture of the invention can be prepared by the following method: the heparin benzyl ester quaternary ammonium salt is depolymerized in an organic medium by means of a strong organic base having a pka higher than 20 or sodium imidazolide, and the depolymerized heparin benzyl ester quaternary ammonium salt is then converted into the sodium salt, this ester being saponified and optionally purified.

The heparin benzyl ester quaternary ammonium salt is preferably a benzethonium salt, a cetylpyridinium salt or a cetyltrimethylammonium salt.

The depolymerization is generally carried out in an inert organic solvent, for example a chlorinated solvent like dichloromethane, tetrahydrofuran, anisole, at a temperature of-20 ℃ to 40 ℃.

As strong organic bases having a pKa above 20, it is possible to use the bases of 1, 5, 7-triazabicyclo [4.4.0] dec-5-ene, 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphospho-cyclohexatriene, guanidine and phosphazenes.

The guanidine base is preferably of the formula

Alkali

In the formula R₁Is hydrogen or alkyl, R₂、R₃、R₄And R₅Identical or different, each represents an alkyl group.

More particularly, R₁Is hydrogen, R₂、R₃、R₄And R₅Is methyl.

Strong organic bases belonging to the class of phosphazenes are for example defined in accordance with R.Schwesinger and co-workers, "Angew. chem.int.ed. Engl., (1167) -1169(1987), R.Schwesinger and co-workers," Angew. chem., (105), 1420 (1993).

Among the phosphazene bases, preference is given to using bases of the formula:

in the formula R₁-R₇The radicals, which are identical or different, represent alkyl radicals.

In the above formula, the alkyl group contains C_1-6Straight chain or branched chain.

Advantageously, the molar ratio of the strong organic base or sodium imidazolide/heparin benzyl ester quaternary ammonium salt with a pka higher than 20 is between 0.2 and 5, preferably between 1 and 4.

More particularly, the esterification rate of the quaternary ammonium salt of heparin benzyl ester is 50-100%, preferably 70-90%. This esterification rate corresponds to the molar percentage of uronic acid esterification of heparin.

In general, the depolymerised heparin benzyl ester quaternary ammonium salt is converted to the sodium salt by treating the reaction medium with an alcoholic solution of sodium acetate, preferably with a 10% (w/v) solution of sodium acetate in methanol, at a temperature of 15-25 ℃. The weight equivalent of the acetate added is preferably 3 times higher than the mass of the quaternary ammonium salt of heparin benzyl ester which takes part in the depolymerization reaction.

In general, the saponification is carried out at a temperature of from 0 to 20 ℃ and preferably from 0 to 10 ℃ in an aqueous medium by means of an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, lithium hydroxide. Generally, 1 to 5 molar equivalents of alkali metal hydroxide are used. Preferably, the saponification is carried out in the presence of 2-3 molar equivalents of alkali metal hydroxide.

The final product may optionally be purified using any known depolymehzation heparin purification method, in particular according to the method described in patent EP 0037319. Preferably, the purification is carried out with the aid of hydrogen peroxide in an aqueous medium at a temperature of from 10 to 50 ℃. Preferably, this operation is carried out at 20-40 ℃.

The heparin benzyl ester quaternary ammonium salt can be prepared according to the following reaction flow:

a) heparin in the form of its sodium salt is converted with the aid of benzethonium chloride in order to obtain benzethonium heparin,

b) esterifying the benzethonium salt obtained above with benzyl chloride and treating with an alcoholic sodium acetate solution to obtain heparin benzyl ester sodium salt,

c) heparin benzyl ester sodium salt is converted (transmutation) to a quaternary ammonium salt, preferably a benzethonium salt, a cetylpyridinium salt or a cetyltrimethylammonium salt.

The reaction of step a) is carried out by allowing an excess of benzethonium chloride to act on heparin in the form of the sodium salt in an aqueous medium at a temperature of about 15-25 ℃. Advantageously, the molar ratio benzethonium chloride/heparin in the form of the sodium salt is between 2 and 3, more particularly 2.5.

The starting heparin used in the form of the sodium salt is preferably porcine heparin. This heparin may be previously purified according to the method described in patent FR 2663639, in order to reduce its chondroitin sulfate B content.

Preferably, the esterification reaction of step b) is carried out at a temperature of 25-45 ℃, preferably at 30-40 ℃, in a chlorine-containing organic solvent (e.g. chloroform, dichloromethane). The ester is then recovered as the sodium salt by precipitation using a solution of 10% (w/v) sodium acetate in an alcohol such as methanol. Typically, 1 to 1.2 volumes of alcohol are used per volume of reaction medium. The amount of benzyl chloride and the reaction time are adjusted so that the esterification rate reaches 50 to 100%, preferably 70 to 90%. Preferably, 0.5 to 1.5 parts by weight of benzyl chloride are used per 1 part by weight of heparin benzethonium salt. Also preferably, the reaction time is 10-35 hours.

The trans-salification reaction of step c) is carried out in an aqueous medium at a temperature of 10-25 ℃ by means of a quaternary ammonium chloride, preferably by means of benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride. Advantageously, the molar ratio of quaternary ammonium chloride/sodium salt of heparin benzyl ester is between 2 and 3.

The inventive mixtures in the form of the sodium salts can be converted into other alkali metal or alkaline earth metal salts. Optionally converting one salt into another salt using the process described in patent US 4168377.

The mixtures of the invention are non-toxic and can therefore be used as medicaments.

The mixtures of the invention can be used as antithrombotic agents. In particular, they are effective in the prevention of venous thrombosis, arterial thrombotic disorders, especially in the case of myocardial infarction. They are also effective in the prevention and treatment of smooth muscle cell proliferation, angiogenesis and as neuroprotective agents in atherosclerosis and arteriosclerosis.

The invention also relates to pharmaceutical compositions containing as active ingredient a mixture of polysaccharides according to the invention, optionally in combination with one or more inert excipients.

These pharmaceutical compositions are, for example, solutions which can be injected subcutaneously or intravenously. They are also effective via the pulmonary (inhalation) route.

The dosage is varied according to the age, weight and health of the patient. For adults, such doses are typically 20-100 mg daily administered intramuscularly or subcutaneously.

The following examples illustrate the invention.

Example A: preparation of benzethonium salt of heparin benzyl ester

Heparin benzethonium

To a solution of 10 g of heparin in the form of the sodium salt in 100 ml of water at a temperature of about 20 c, a solution of 25 g of benzethonium chloride in 125 ml of water is added. This product was filtered, washed with water and dried.

Heparin benzyl ester (sodium salt)

To a solution of 20 g heparin benzethonium in 80 ml dichloromethane, 16 ml benzyl chloride was added. This solution was heated at a temperature of 30 ℃ for 12 hours. At this point 108 ml of a 10% sodium acetate in methanol solution was added, filtered, washed with methanol and dried. This gives 7.6 g of heparin benzyl ester in the form of the sodium salt, whose esterification rate is 77%.

Heparin benzyl ester (benzethonium salt)

Into a 2 l Erlenmeyer flask A were added 36 g (0.0549 mol) heparin benzyl ester (sodium salt) and 540 ml distilled water. After homogenization at a temperature of about 20 ℃, a pale yellow solution is obtained. A solution of 64.45 g (0.1438 mol) benzethonium chloride and 450 ml of water was prepared in a 1 l Erlenmeyer flask B with magnetic stirring. The solution of Erlenmeyer flask B was poured into the solution of Erlenmeyer flask A with stirring over about 35 minutes. A large amount of white precipitate was observed to form. Erlenmeyer flask B was rinsed with 200 ml of distilled water and the rinse was added to Erlenmeyer flask A. At this point the stirring was stopped and the suspension was left for 12 hours. The clear portion of the supernatant was withdrawn and separated. To the settled (sludge-like) precipitate, 560 ml of water were added and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (560 ml). This washing operation was repeated twice with about 560 ml of distilled water for the settled precipitate. In the latter washing operation, this precipitate is made into a suspension and filtered through sintered glass 3. The filter cake was then washed 4 times with 200 ml portions of distilled water. The wet white solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 87.5 g of heparin benzyl ester, benzethonium salt are obtained. The yield achieved was 94.9%.

Example 1

Depolymerization and conversion to form sodium salt:

in a 50 ml Erlenmeyer flask A, 28 ml of dichloromethane were added. 4 g (0.00238 mol) of heparin benzyl ester obtained as described in example A (esterification rate: 77%, benzethonium salt) were slowly charged with stirring. After complete dissolution, 1.32 g (0.00948 moles) of 1, 5, 7-triazabicyclo [4.4.0] dec-5-ene were added. Stirring was carried out at a temperature of about 20 ℃ for 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. An almost translucent gelatinous yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the supernatant was withdrawn and separated (62 ml). To the precipitated precipitate (yellow mud), 50 ml of methanol was added and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (49 ml). To the settled precipitate, 50 ml of methanol was added and stirred for 20 minutes. The precipitate in suspension is then filtered through sintered glass 4. The resulting golden cake was washed twice more with 25 ml of methanol each time. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 1.21 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. The yield achieved was 77.2%.

Saponification:

in a 25 ml Erlenmeyer flask, 1.21 g (0.0018 mol) of previously obtained depolymerized heparin (benzyl ester, sodium salt) and 11 ml of water were added. Under magnetic stirring, 0.18 ml (0.0018 mol) of 30% sodium hydroxide lye is added. Upon completion of the addition, the mixture was cooled to 4 ℃ and stirred for 2 hours. 1.43 g NaCl was added and the solution was neutralized by the addition of 1 mol/l HCl (14 ml). This mixture was transferred to a 100 ml Erlenmeyer flask and 52 ml methanol was added. A yellow precipitate was observed to form. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 20 ℃ for 12 hours. The supernatant was then withdrawn and separated again (44 ml). To the settled (yellow mud) precipitate was added 25 ml of methanol and stirred for 20 minutes. This precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (21 ml). To the precipitated precipitate, 25 ml of methanol was added and stirred for 20 minutes. This precipitate in suspension is filtered through sintered glass 3. The resulting pale yellow cake was washed twice with 10 ml of methanol each time. The warm solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 0.66 g of crude depolymerized heparin (sodium salt) was obtained. The yield achieved was 60%.

And (3) purification:

in a 10 ml Erlenmeyer flask, 0.66 g of previously obtained depolymerized heparin and 5.9 ml of distilled water were added. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was raised to 9-10 by adding 0.1 mol/l sodium hydroxide, and 33. mu.l of 30% aqueous hydrogen peroxide was added. After stirring for about 2 hours, 0.65 g of sodium chloride was added. This mixture was then neutralized by the addition of 0.1 mol/l HCl. This solution was then filtered and poured into a 25 ml Erlenmeyer flask. 23.3 ml of methanol were poured in. The formation of a white precipitate was observed. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 20 ℃ for 12 hours. The supernatant was then withdrawn and separated (5 ml). To the settled (sludge-like) precipitate was added 5 ml of methanol and stirred for 20 minutes. This precipitate was then allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (5 ml). To the settled precipitate was added 5 ml of methanol and stirred for 20 minutes. This precipitate in suspension is then filtered through sintered glass 3. The resulting white filter cake was washed twice with 5 ml each time of methanol. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 0.51 g of the purified polysaccharide mixture (sodium salt) is obtained. The yield achieved was 77.2%.

The properties of this mixture are as follows:

average molecular weight: 1600 dalton

anti-Xa activity: 94 IU/mg

anti-IIa Activity: < 0.1 IU/mg

The ratio of Xa-resistant activity to IIa-resistant activity is > 100.

Example 2

Depolymerization and conversion to form sodium salt:

in a 100 ml Erlenmeyer flask A, 70 ml of dichloromethane were added. 10 g (0.00595 mol) of heparin benzyl ester obtained as described in example A (esterification rate: 77%, benzethonium salt) were slowly charged with stirring. After complete dissolution, 1.7 ml (0.00587 mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethyl-perhydro-1, 3, 2-diazaphospho-cyclohexatriene (phosphorine) are added. The reaction was allowed to proceed at a temperature of about 20 ℃ for about 3 hours and 30 minutes. During this time, a solution of 30 g of sodium acetate in 300 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. An almost-translucent gelatinous yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the supernatant was withdrawn and separated (204 ml). To the settled (yellow mud) precipitate was added 125 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (162 ml). To the precipitated precipitate, 125 ml of methanol was added and stirred for 20 minutes. The gel precipitate in suspension is then filtered through sintered glass 3. The resulting yellow gel cake was washed twice more with 63 ml portions of methanol. The gel-like solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 3.34 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. The yield achieved was 85.3%.

Saponification:

1.67 g of previously obtained depolymerized heparin (benzyl ester, sodium salt) was saponified according to the saponification method described in example 1. 0.94 g of a pale yellow powder is obtained. The yield of crude depolymerized heparin (sodium salt) was 61%.

And (3) purification:

according to the purification method described in example 1, 0.94 g of the previously obtained crude depolymerized heparin (sodium salt) was purified. 0.71 g of a white powder is obtained. The yield was 75.5%.

The resulting purified polysaccharide mixture (sodium salt) had the following properties:

average molecular weight: 2500 Dalton

anti-Xa activity: 146.6 IU/mg

anti-IIa Activity: 2.15 IU/mg

anti-Xa activity/anti-IIa activity ratio: 68.

example 3

Depolymerization and conversion to form sodium salt:

in a 50 ml Erlenmeyer flask A, 28 ml of dichloromethane were added. 4 g (0.00238 mol) of heparin benzyl ester obtained according to example A (esterification rate: 77%, benzethonium salt) are slowly charged with stirring. After complete dissolution and cooling to 2 ℃, 0.333 g (0.00239 moles) of 1, 5, 7-triazabicyclo [4.4.0] dec-5-ene were added. The reaction was allowed to proceed at a temperature of about 20 ℃ for about 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. A yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the supernatant was withdrawn and separated (90 ml). To the settled (yellow mud) precipitate was added 50 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (61 ml). To the settled precipitate was added 50 ml of methanol and stirred for 20 minutes. The precipitate in suspension is then filtered through sintered glass 4. The resulting filter cake was washed twice more with 25 ml of methanol each time. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 1.19 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. This solid was dark yellow. The yield achieved was 75.9%.

Saponification:

1.19 g of the previously obtained depolymerized heparin (benzyl ester, sodium salt) was saponified according to the saponification method described in example 1. 0.78 g of a pale yellow powder is obtained. The yield of crude depolymerized heparin (sodium salt) was 71.5%.

And (3) purification:

according to the purification method described in example 1, 0.78 g of the previously obtained crude depolymerized heparin (sodium salt) was purified. 0.58 g of a white powder is obtained. The yield was 72.5%.

The resulting purified polysaccharide mixture (sodium salt) had the following properties:

average molecular weight: 2700 Dalton

anti-Xa activity: 100.1 IU/mg

anti-IIa Activity: 3.3 IU/mg

anti-Xa activity/anti-IIa activity ratio: 27.3.

example 4

Depolymerization and conversion to form sodium salt:

in a 50 ml Erlenmeyer flask A, 28 ml of dichloromethane were added. 4 g (0.00238 mol) of heparin benzyl ester obtained according to example A (esterification rate: 77%, benzethonium salt) are slowly charged with stirring. After complete dissolution and cooling to 2 ℃, 0.6 ml (0.00222 mol) of 2-tert-butylimino-tris (dimethylamino) phosphorane is added. The reaction was allowed to proceed at a temperature of about 0 ℃ for about 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. An almost translucent gelatinous yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the supernatant was withdrawn and separated (108 ml). To the settled (yellowish mud) precipitate was added 50 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (60 ml). To the precipitated precipitate, 50 ml of methanol was added and stirred for 20 minutes. The yellowish white precipitate in suspension is then filtered off with sintered glass 4. The resulting filter cake was washed twice more with 25 ml of methanol each time. The solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 1.22 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. The yield achieved was 77.8%.

Saponification:

1.22 g of previously obtained depolymerized heparin (benzyl ester, sodium salt) was saponified according to the saponification method described in example 1. 0.69 g of a very pale yellow powder is obtained. The yield of crude depolymerized heparin (sodium salt) was 61.6%.

And (3) purification:

according to the purification method described in example 1, 0.69 g of the previously obtained crude depolymerized heparin (sodium salt) was purified. 0.67 g of a white powder was obtained. The yield was 97.1%.

The resulting purified polysaccharide mixture (sodium salt) had the following properties:

average molecular weight: 2900 Dalton

anti-Xa activity: 145.2 IU/mg

anti-IIa Activity: 4.5 IU/mg

anti-Xa activity/anti-IIa activity ratio: 32.6.

example 5

Depolymerization and conversion to form sodium salt:

in a 50 ml round bottom flask A, 28 ml dichloromethane was added. 4 g (0.00238 mol) of heparin benzyl ester (esterification rate: 77%, benzethonium salt) were slowly charged under stirring. After complete dissolution and temperature up to 40 ℃, 0.95 g (0.00949 moles) of sodium imidazolide was added. The reaction was allowed to proceed under reflux of dichloromethane for about 4 hours. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. An almost translucent gelatinous yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the orange supernatant was withdrawn and separated (88 ml). To the settled (orange sludge) precipitate was added 50 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (51 ml). To the settled precipitate was added 50 ml of methanol and stirred for 20 minutes. The orange precipitate in suspension is then filtered with sintered glass 4. The resulting filter cake was washed twice more with 25 ml of methanol each time. The solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 1.34 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. The yield achieved was 76.6%.

Saponification:

1.2 g of the previously obtained depolymerized heparin (benzyl ester, sodium salt) was saponified according to the saponification method described in example 1. 0.63 g of a grayish brown powder was obtained. The crude depolymerized heparin (sodium salt) yield was 52.5%.

And (3) purification:

according to the purification method described in example 1, 0.63 g of the previously obtained crude depolymerized heparin (sodium salt) was purified. 0.42 g of pale grayish brown powder was obtained. The yield was 66.7%.

The resulting purified polysaccharide mixture (sodium salt) had the following properties:

average molecular weight: 2250 daltons

anti-Xa activity: 134.5 IU/mg

anti-IIa Activity: 1.5 IU/mg

anti-Xa activity/anti-IIa activity ratio: 90.5.

example 6

Depolymerization and conversion to form sodium salt:

in a 50 ml Erlenmeyer flask A, 28 ml of dichloromethane were added. 4 g (0.00238 mol) of heparin benzyl ester obtained as described in example A (esterification rate: 77%, benzethonium salt) are slowly charged with stirring. After complete dissolution, 1.33 g (0.00956 moles) of 1, 5, 7-triazabicyclo [4.4.0] dec-5-ene were added. Stirring was carried out at a temperature of about 20 ℃ for 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. The reaction mixture in Erlenmeyer flask A was poured into sodium acetate in methanol with magnetic stirring. An almost translucent gelatinous yellow precipitate appeared. At this point the stirring was stopped and the suspension was allowed to decant for one hour. The clear portion of the supernatant was withdrawn and separated (56 ml). To the settled (yellow mud) precipitate was added 60 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional 30 minutes. The supernatant was withdrawn and separated (70 ml). To the settled precipitate was added 50 ml of methanol and stirred for 15 minutes. The precipitate in suspension is then filtered through sintered glass 4. The resulting golden cake was washed twice with 50 ml of methanol each time. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 0.92 g of depolymerized heparin (benzyl ester, sodium salt) was obtained. The yield achieved was 64%.

Saponification:

in a 25 ml Erlenmeyer flask, 0.92 g (0.0014 mol) of the previously obtained depolymerized heparin (benzyl ester, sodium salt) and 17.5 ml of water were added. Under magnetic stirring, 0.38 ml (0.00379 mol) of 30% sodium hydroxide lye is added. After the addition, the mixture was kept at a temperature of about 20 ℃ and stirred for 5 hours. 1.8 g NaCl was added and the solution was neutralized by the addition of concentrated HCl (the final volume of the solution was about 18 ml). This mixture was poured into a 100 ml Erlenmeyer flask and 46 ml methanol was added. A yellow precipitate was observed to form. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 20 ℃ for 12 hours. The supernatant was then withdrawn and separated (52 ml). To the settled (yellow mud) precipitate was added 25 ml of methanol and stirred for 20 minutes. The precipitate was allowed to settle for an additional about 1 hour. The supernatant was withdrawn and separated (27 ml). To the settled precipitate was added 25 ml of methanol and stirred for about 1 hour. The precipitate in suspension is then filtered through sintered glass 4. The resulting filter cake was washed twice more with 10 ml portions of methanol. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 3 hours, 0.42 g of crude depolymerized heparin (sodium salt) was obtained. The yield achieved was 45.6%.

And (3) purification:

in a 10 ml Erlenmeyer flask, 0.42 g of the previously obtained crude depolymerized heparin and 3.8 ml of distilled water were added. This mixture was warmed to 38 ℃ with magnetic stirring. The pH was raised to 9-10 by adding 0.1 mol/l sodium hydroxide and 25. mu.l of 30% aqueous hydrogen peroxide was added. After stirring for about 2 hours 30 minutes, 0.5 g of sodium chloride was added. This mixture was then neutralized by the addition of 0.1 mol/l HCl. This solution was then filtered and poured into a 25 ml Erlenmeyer flask. 11.3 ml of methanol were poured in. The formation of a white precipitate was observed. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 20 ℃ for 12 hours. The supernatant was then withdrawn and separated (9.8 ml). To the settled (mud) precipitate was added 5 ml of methanol and stirred for 15 minutes. This precipitate was then allowed to settle for an additional 3 hours. The supernatant was withdrawn and separated (6.2 ml). To the settled precipitate was added 5 ml of methanol and stirred for 20 minutes. This precipitate in suspension is then filtered through sintered glass 3. The resulting white filter cake was washed twice with 5 ml each time of methanol. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for about 2 hours and 20 minutes, 0.39 g of pure depolymerized heparin (sodium salt) is obtained. The yield achieved was 92.8%.

The properties of the depolymerized heparin thus obtained are as follows:

average molecular weight: 1950 Dalton

anti-Xa activity: 115.6 IU/mg

anti-IIa Activity: < 2 IU/mg

The anti-Xa activity/anti-IIa activity ratio > 57.

Example 7

Depolymerization and conversion to form sodium salt:

in a 400 ml reactor A, 140 ml of dichloromethane were added. 20 g (0.0119 mol) of heparin benzyl ester obtained as described in example A (esterification rate: 77%, benzethonium salt) are slowly charged with stirring. After complete dissolution, the water content of this reaction medium was determined by the Karl Fisher method. The value obtained was 0.1% water. 3.5 ml (0.0121 mol) of 2-tert-butylimino-2-diethylamino-1, 3-dimethyl-perhydro-1, 3, 2-diazaphosphocyclohexatriene are then added. The reaction was allowed to continue at a temperature of about 25 ℃ for about 24 hours. During this time, a solution of 30 g of sodium acetate in 300 ml of methanol was prepared at 4 ℃ in Erlenmeyer flask B. Half of the reaction mixture in reactor a was poured into sodium acetate methanol solution with magnetic stirring. An almost translucent gelatinous yellow precipitate appeared. Stirring was maintained for one hour and the suspension was allowed to settle at 4 ℃ for about 12 hours. The clear portion of the supernatant was withdrawn and separated (220 ml). To the settled (yellow mud) precipitate was added 220 ml of methanol and stirred for 50 minutes. The precipitate was allowed to settle for an additional about 40 minutes. The supernatant was withdrawn and separated (204 ml). To the precipitated precipitate was added 204 ml of methanol, and the mixture was stirred for 40 minutes. The gel-like precipitate in suspension is then filtered through sintered glass 3. The resulting yellow gel cake was washed twice with 100 ml portions of methanol. The gel solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for about 12 hours, 2.6 g of depolymerized heparin (benzyl ester, sodium salt) were obtained. The yield achieved is 70.6% (calculated on the basis of the half of the reaction medium which has been worked up).

Saponification:

2.6 g of the previously obtained depolymerized heparin (benzyl ester, sodium salt) was saponified according to the saponification method described in example 1. 1.48 g of a pale yellow powder are obtained. The crude depolymerized heparin (sodium salt) yield was 62.9%.

And (3) purification:

in a 50 ml Erlenmeyer flask, 1.48 g of the previously obtained crude depolymerized heparin and 15 ml of distilled water were added. This mixture was warmed to 40 ℃ under magnetic stirring. The pH was raised to 9-10 by the addition of 0.1 mol/l sodium hydroxide. This solution was filtered through a filter membrane with a porosity of 1 μm. Then 76 microliters of 30% aqueous hydrogen peroxide was added. After stirring for about 2 hours, 1.5 grams of sodium chloride was added. This mixture was then neutralized by the addition of 1 mol/l HCl. This solution was then filtered through a filter membrane with a porosity of 1 μm. Then 38 ml of methanol was poured in. The formation of a white precipitate was observed. At this point the stirring was stopped and the suspension was allowed to settle at a temperature of about 20 ℃ for 1 hour. The supernatant was then withdrawn and separated (37 ml). To this precipitate was added 37 ml of methanol, and stirred for 45 minutes. This precipitate was then allowed to settle for an additional 45 minutes. The supernatant was withdrawn and separated (34 ml). To the settled precipitate was added 34 ml of methanol, and stirred for 15 minutes. This precipitate in suspension is then filtered through sintered glass 3. The resulting white filter cake was washed twice with 25 ml each time of methanol. The wet solid was dehydrated and dried under reduced pressure (2.7 kPa) at a temperature of about 60 ℃. After drying for 12 hours, 1.29 g of pure depolymerized heparin (sodium salt) are obtained. The yield achieved was 87.2%.

The characteristics of the purified depolymerized heparin (sodium salt) obtained were as follows:

average molecular weight: 2250 daltons

anti-Xa activity: 149.6 IU/mg

anti-IIa Activity: 0.85 IU/mg

anti-Xa activity/anti-IIa activity ratio: 176.

Claims (17)

1. Sulfated polysaccharide mixtures having the general structure of polysaccharides consisting of heparin and having the following characteristics:

their average molecular weight is 1500-3000 daltons, the anti-Xa activity is 100-150 IU/mg, the anti-IIa activity is 0-10 IU/mg, the anti-Xa activity/anti-II-a activity ratio is higher than 10,

the constituent polysaccharides of these mixtures contain 2 to 26 sugar building blocks with unsaturated 4, 5-glucuronic acid 2-O-sulfate building blocks at one end,

these polysaccharide mixtures are in the form of alkali metal or alkaline earth metal salts.

2. The mixture of claim 1, wherein the anti-Xa activity is 125-150 IU/mg.

3. The mixture of claim 1, characterized in that the anti-Xa activity is 140-150 IU/mg and the average molecular weight is 2000-3000 daltons.

4. A mixture according to any one of claims 1 to 3 in the form of sodium, potassium, calcium or magnesium salts.

5. The mixture according to any one of claims 1 to 3, which has an anti-IIa activity of from 0 to 5 IU/mg.

6. The mixture of claims 1-3, which has an anti-Xa activity/anti-IIa activity ratio of greater than 25.

7. A process for the preparation of the mixture according to claim 1, characterized in that the depolymerization of heparin benzyl ester quat in an organic medium with the aid of a strong organic base having a pKa higher than 20 or sodium imidazolide, the conversion of the depolymerized heparin benzyl ester quat into the sodium salt, the saponification of this ester and the optional purification of this product,

wherein the strong organic base having a pKa above 20 is selected from the group consisting of bases of 1, 5, 7-triazabicyclo [4.4.0] dec-5-ene, 2-tert-butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2-diazaphosphocyclohexatriene, guanidine and phosphazenes,

wherein the guanidine base is a base of the formula:

in the formula R₁Is hydrogen or alkyl, R₂、R₃、R₄And R₅The same or different, each represents an alkyl group having C_1～6A straight-chain or branched-chain,

wherein the base of the phosphazene is a base of the formula:

in the formula R₁-R₇The radicals, which are identical or different, represent a compound having C_1～6Linear or branched alkyl.

8. The process of claim 7, wherein the guanidine base is a base of the formula:

wherein R is₁Is hydrogen, R₂、R₃、R₄And R₅Is methyl.

9. The method of claim 7, wherein the heparin benzyl ester quaternary ammonium salt is a benzethonium salt, a cetyl pyridinium salt, or a cetyl trimethylammonium salt.

10. The process according to any one of claims 7 to 9, wherein the molar ratio of the strong organic base having a pka higher than 20 or of the sodium imidazolide/heparin benzyl ester quaternary ammonium salt is between 0.2 and 5.

11. The method of any one of claims 7-9, wherein the esterification rate of the heparin benzyl ester quaternary ammonium salt is 50-100%.

12. The process of any one of claims 7 to 9, wherein the depolymerized heparin benzyl ester quaternary ammonium salt is converted to the sodium salt by treating the reaction medium with an alcoholic solution of sodium acetate.

13. The method of any one of claims 7-9, wherein the saponification is performed using an alkali metal hydroxide.

14. The method of any one of claims 7-9, wherein the purification is performed using hydrogen peroxide.

15. Use of a mixture as defined in any one of claims 1 to 6 as a medicament.

16. Use of a mixture as defined in any one of claims 1 to 6 as an antithrombotic agent.

17. Pharmaceutical composition containing as active ingredient a mixture according to any one of claims 1 to 6.