JPH0128044B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to normal heparin.
The present invention relates to a process for obtaining low molecular weight heparin starting from a substance with significantly advantageous pharmacological and therapeutic properties.

Heparin is well known and is a stable monovalent or divalent salt of the unstable heparic acid. It is a polymer of disaccharide units formed by hexuronic acid (D-glucuronic acid and L-iduronic acid) and glucosamine 60- and N-sulfate linked by α1-4 glycosidic bonds.

its anti-clotting properties
has been known for many years since it was discovered and isolated from tissue in 1917.

Heparin exists in various forms in many tissues and cells, more or less loosely bound to protein moieties. In the skin, a partially different species of heparin exists in the lungs in a polymeric form, which is sensitive to the action of ascorbic acid or certain enzymes thought to be present in the intestinal mucosa. Ascorbic acid or intestinal mucosa
After treatment with homogenates) these macromolecular forms of lung or skin heparin can be reduced to the same molecular size as heparin that can be isolated from the intestinal mucosa.

Heparin isolated from intestinal mucosa having an average molecular weight (W) of 15,000 Daltons or heparin from other sources reduced to this W by some of the conventional methods as described above is the smallest natural molecule size of heparin. give. According to the invention, such type of heparin is defined as normal heparin. In fact, there are no enzymes in the body that can further degrade heparin from the intestinal mucosa into lower molecular weight fractions, and no chemistry is capable of depolymerizing normal heparin molecules without losing all of their biological activity. No method was developed. Flavobacterium heparinum
Certain microbial enzymes from
Only enzymes, such as heparinase, can completely dissociate heparin molecules into denatured disaccharide units. Although this method has been applied to structural studies, it has proven inadequate for obtaining new biologically active molecules.

One object of the invention is therefore to provide a process which makes it possible, starting from normal heparin, to obtain a low molecular weight heparin fraction with increased pharmacological activity.

To achieve this objective, the present invention involves the following steps: (a) acidifying normal heparin to obtain heparic acid; (b) acidifying the heparic acid by heating in the presence of peroxide in an autoclave. (c) sulphation of the heparamine to obtain the corresponding low molecular weight heparin.

The reaction scheme according to the method proposed by the present invention is described below.

7 if < and 26.7, which is the value corresponding to 15000 D of heparin.
<<22.

It is calculated as follows: =15000/56226.7 where 567 is the disaccharide unit (chemical formula
C ₁₂ H ₁₅ O ₁₆ NS ₂ Na ₃ ).

According to the above reaction formula, the formula represents the sodium salt of normal heparin; for simplicity, the formula is D-
Only glucuronic acid is shown and the portion of the molecule containing L-iduronic acid is not shown. Similarly, other monovalent or divalent cations can be present in place of the sodium cation.

The heparin is acidified according to step (a) above to obtain the free acid, ie, heparinic acid of formula.

Heparic acid of the formula is depolymerized in step (b) to lose most of the N-sulfate groups and produce heparamine of the formula.

Finally, the heparamine of formula is treated according to step (c) to give the final product according to the invention, namely LMW (low molecular weight), by rearrangement of the N-sulfate group.
Heparin of the formula defined as heparin or RD (reconstituted depolymerized heparin) is obtained.

According to a more particular description of the process of the invention, the heparic acid is obtained from normal heparin by simple acidification or treatment, preferably with a cationic exchange resin.

The heparic acid then proceeds through a depolymerization step (b), which is preferably carried out in the presence of a peroxide, such as H ₂ O ₂ by heating at a pressure of 1 to 2 atmospheres. Performed in an autoclave, the autoclaving process can be stopped at preselected times (eg, 15, 30, 60, 120, 240 minutes). In this method, =
Starting from 15000D normal heparin, it is possible to obtain the final product, ie L or RD heparin, with various average molecular weights () ranging from about 12000 to 4000D. The yield of the depolymerization is the same in all examples (approximately 65% by weight). The decrease in is followed by titration of terminal reducing groups (Somoji's method) or by decrease in specific viscosity.

The product of the depolymerization is a low molecular weight heparin, the molecular weight of which is lower than that of the corresponding starting heparin, which is reduced by about 1/3 following autoclaving for various preselected times.

In the depolymerization step, as mentioned above, most of the sulfate groups bonded to nitrogen are lost,
Finally, the heparamine cools the reaction mixture and
After the pH rises, active sulfuric groups (active sulfuric
group), for example by reaction of a nitrogenous base with a sulfotrioxide (e.g. pyridine sulfotrioxide, trimethylamine sulfotrioxide, etc.) or with chlorosulfonic acid and a nitrogenous base in the presence of an alkali carbonate. The final product is obtained according to step (c) by known methods such as the reaction of The yield of the final product is the same in all cases, regardless of the autoclaving time, in terms of weight relative to the starting amount of heparin.

Some examples will be described for a better understanding of the invention, but are not intended to limit the invention.

Example 1 An amount of 20 g of sodium or calcium heparin (anticoagulant activity = 150 IU/mg) is dissolved in 100 ml of deionized water and Amberlite in H ⁺ form
Place in a column containing 100 ml of IRC120 (Amberlite IRC120).

Wash the column with 100 ml of deionized water and collect the liquid. The pH of the solution was 1-1.5. 20 ml of a saturated solution of hydrogen peroxide (36%) were added and the liquid was heated in an autoclave for 15 minutes at 1 atm. After cooling to room temperature, the pH was raised to 7.2 with 2N NaOH and 16 ml of pyridine and 16 g of pyridine sulfotosoxide were added under stirring.

The reaction is continued for 12 hours with the addition of some pyridine to maintain the pH between 5 and 6. The pH is then raised to 8 with NaOH and the solution is concentrated in vacuo to 100 ml to remove excess pyridine. Salts were removed in a mixed bed ion exchange column, the solution was sterile filtered and it was lyophilized.

Yield: 85-100 IU/mg anticoagulant activity and original 2/
65% as a white powder with a temperature of 3 (Somogyi method).

Example 2 Sodium or calcium heparin, 150IU/
mg, 20 g was dissolved in 100 ml of water for injection and decationized with a cation exchange resin in H ⁺ form.

Peracetic acid (40% solution) in the solution (PH=1-1.5)
20 ml were added and the whole was then autoclaved for 60 minutes at 1 atm.

After cooling and raising the pH to 7.2, add 20 g of trimethylamine sulfotrioxide +
20g of Na ₂ CO ₃ was added. The reaction mixture was stirred for a number of hours at a temperature not exceeding 60°C and then
Retardion 11A8 column (Dowex
Retardion11A8column) was passed. The pH of the solution was adjusted to 6, the solution was cooled, and 3 volumes of ethanol were added. The white precipitate was collected, washed with ethanol or methanol or acetone, and dried in vacuo.

The final product has an anticoagulant activity of 35-50 IU/mg and is 1/3 of the original (Somegey method).

Example 3 By operating as described in Example 1 using the following operating conditions, autoclaving at 2 atmospheres for 30 minutes resulted in an anticoagulant activity of 35-50 IU/mg and approximately 1/3 of the original amount. A final product with is obtained.

The product has a chemical composition (sulfate, hexosamine, hexuronic acid content) that is identical to that of purified heparin and certain physicochemical properties (specific
(optical rotation)] but give different values in the titration of the terminal reducing group (Somezie method), different electrophoretic mobilities and shifted maximum absorption wavelengths in complexes formed with basic dyes, such as toluidine blue.

LMW heparin (RD
heparin) exhibits very interesting pharmacological and therapeutic properties compared to normal heparin.

The most important of these are the Anti-Xa test/APTT, where Anti-Xa test = Yin's test, APTT = Activated partial thromboplastin time.
anti-thrombotic activity measured by Partial thromboplastin time.
activities) and total anti-coagulant activity (total anti-
clotting activities) in vitro or in vivo (in laboratory animals and humans).

Different RD heparins show that the specific anti-clotting activity decreases with a decrease in , whereas the value of the ratio of antithrombotic activity/total anti-clotting activity increases with a decrease in .

In some cases, normal heparin with = 15000 has a total anticoagulant activity = 150 IU/mg and anti-
Xa/APTT ratio = 1, whereas the RD heparin of the present invention has a total anticoagulant activity <150 IU/mg.
and anti-Xa/APTT>1. In particular, the ratio is almost double for the compound compared to the compound, the antithrombotic activity of the compound is twice that of normal heparin, in other words, in terms of anticoagulant activity, the administration of the compound Half the amount or less is required to produce the same antithrombotic effect produced by a whole dose of normal heparin.

These data were confirmed in experimental animals by measuring the protective effect of intravenously administered compounds and against thrombin and thromboplastin-induced thrombi formation.

Therefore, in the family of RD heparins obtainable by the method of the present invention, in terms of specific anticoagulant activity and antithrombotic/anticoagulant ratio for specific use in the field of protection against thrombosis. The most suitable product can be chosen.

For example, patients with clot-forming conditions with very short clotting times require antithrombotic agents with significantly higher anticoagulant activity, whereas patients undergoing surgical intervention are less likely to experience bleeding and Antithrombotic agents with low anticoagulant activity are required when there is a risk of thrombosis after surgery.

Furthermore, after subcutaneous administration to humans, the compounds remain in the blood longer than the compounds. Another test, namely recalcification time
time), activated partial thromboplastin time (APTT), thrombin time, anti-Xa activity (Anti-
Xa activity) is used to test how long a compound remains in the circulation.

Additionally, the compounds exhibit oral absorption in some animal species (rats, mice, dogs), whereas the compounds are not absorbed orally.

Claims (1)

[Scope of Claims] 1 The following properties: Hexosamine after hydrolysis; uronic acid after hydrolysis same as for heparin; same sulfate group as for heparin; same optical rotation as for heparin; same average molecular weight () as for heparin. ; 12000-4000D total anticoagulant activity; <150 IU/mg anti-Xa/APTT; >1. 2. A method for obtaining low molecular weight heparin with enhanced pharmacological properties, comprising the steps of: (a) acidifying normal heparin to obtain heparic acid; (b) heating in the presence of peroxide. (c) sulfating the heparamine to obtain the corresponding low molecular weight heparin. 3. The method according to claim 2, wherein the depolymerization is carried out in an autoclave. 4. Claim 2, characterized in that the sulfation is carried out by treatment of a nitrogen-containing organic base with sulfotrioxide in the presence of a base.
The method described in section. 5. Process according to claim 4, characterized in that said base is a free nitrogen-containing base corresponding to said sulfotrioxide. 6. The method according to claim 4, wherein the base is an alkali carbonate. 7. The method according to claim 2, characterized in that the sulfating is carried out by treatment with chlorosulfonic acid and a nitrogen-containing organic base. 8 The following properties: Hexosamine after hydrolysis: Same as for heparin, Uronic acid after hydrolysis: Same as for heparin, Sulfate group: Same as for heparin, Optical rotation: Same as for heparin, Average molecular weight (): 12000 -4000D A pharmaceutical composition for the treatment of thrombosis, comprising as an active ingredient a low molecular weight heparin with a total anticoagulant activity of <150 IU/mg anti-Xa/APTT; >1.