SI8110736A8 - Process for obtaining mucopolysaccharides as heparine derivatives - Google Patents

Description

PROCEDURE FOR OBTAINING MUCOPOLISACCHARIDE DERIVATIVES

HEPARINA

The field of the technique of the invention

The present invention relates to a process for the production of mucopolysaccharides having biological properties which in particular enable them to control in some remarkable specific manner certain stages of blood coagulation. The invention falls within Class C 08B 37/10 and A 61K 31/725 of the International Patent Classification.

The present invention particularly relates to a method for producing mucopolysaccharides (hereinafter referred to as MPS abbreviation) having activity more selective than heparin, namely, against active factor X, or factor Xa, blood, and with strong antithrombotic activity without risk of bleeding for the patient .

2.

2. Technical problem

The presence of access routes to products of the aforementioned type, their creation and the attainment of increased yields, has led to this invention in particular by studying chemically depolymerized heparin.

It should be noted that the term heparin is used in the description and requirements in a broad sense, to individually designate a preparation of commercially available pharmaceutical grade heparin or crude heparin such as that obtained by extraction "starting from biological agents, especially mammalian tissues ·

3 · Technique groan

Heparin is known to adhere to its anticoagulant activity and enhance the inhibitory effect of antithrombin AT (AT Ul), which is a plasma protein, as opposed to cascades of enzyme reactions that occur during coagulation,

Heparin is known to be able to simultaneously attenuate the large swarm of coagulation factors that affect its formation in the maintenance of various forms of hypercoagulability, with its activity not specific but general. "

3.

If this anticoagulant activity is determined, then every time the executive returns the reversed equilibrium of the coagulation-fibrinolysis system to the treated patients, due to the general nature of its effect. "It appears that administration (because of a dangers of hypercoagulation, for example, the occurrence of post-surgical (post-surgical) thrombosis), too high doses of anticoagulation drug, or nontarget selectivity of the drug, may eventually cause severe bleeding. "

The inventors have been examining methods to obtain mucopolysaccharide arrays, their heparin derivatives, which are more suitable than heparin arrays in terms of their biological properties. "

A thorough study of the variable conditions for depolymerization of heparin and mixtures obtained from depot 1 of the rice name led these inventors to conclude that it was possible, under certain mild conditions, to partially depolymerize the string! of heparin to a degree corresponding to the production of mucopolyaccharide (MPS) arrays having valuable antitrarobic properties and a very high yield (almost quantitative) of active molecules. These mucopolysaccharides may inhibit factor Xa according to a degree of selectivity greater than heparin when their total anticoagulation activity is lower than heparin ”this is due to the favorable yield of their g Yin-Wessler titer (activity) and their USP titer.

Yin-Veeeler's activity is known to be a more specific reflection of the ability of active fractions to potently inhibit Xa blood by assisting at III in an appropriate assay and USP ti tar (activity) is a reflection of the power of active fractions to inhibit total blood or blood coagulation plasma.

Yin-Veseler's tar is a measure of the method described by these authors in J. Lab. Ciin, Med. 81, 298-300 (1976) and the USP is a measure according to the method described by Phaimacopea pf tke United States of America p, 229-230 (see also the second USP-NH appendix, p. 62 and the fourth USP addition. p «90, entitled Drug Substances and Dosage fonas).

The present invention is therefore intended to provide a new process for the production (in higher yields and with convenient performance) of products which yield a more favorable yield of heparin than their Yin-Vesaler and USP ti (activity), and which can be used as drug active substances.

4 »Description of the solved problem

The method according to the invention is that heparin having molecular weight sequences of the order of 2,000 to 50,000 is subjected to a mild chemical agent, which can depolymerize or fragment the heparin sequences, in particular the action of nitric acid, whereby this reaction is carried out in these formulations. those conditions to enable the vapor and vapor deposition of the starting heparl substance to be obtained and to obtain a mixture in which a large portion of the product has Yin-Wessler and USP tl greater than 2, especially at least 3, preferably greater than 6. containing terminal reductive groups of the 2,5-anhydro-D-mannose structure t

When ae achieves the desired degree of depolymerization, the products are separated, which can be precipitated with an alcoholic solvent and then collected * «·

The realization of these prod ucts allows to obtain suitably mucopolys of saccharides having USP thy tar weaker than the starting heparin while their Yin – Vessler thy tar is larger than the heparin, and in practically quantitative yield ·

According to the supplementary transcript, which is carried out in order to make available the mixtures of saccharide mucopoly having terminal groups already stable, the resulting pre-mixture is subjected to a treatment that allows the aldehyde group to be converted to a more stable functional group, especially to an acid or alcohol group.

According to the general method, the process of the present invention is carried out in a mixture limited to most of the heparin derivatives soluble in the hydro-alcoholic medium (water-alcohol) having a tare of 55-6l ° C3L, claiming water-ethanol insolubility in the middle which has an increased content of alcohol and insoluble in pure alcohol. They represent Yin-Vessler cereals and USPs in a ratio equal to at least 2, especially at least 3, by a year greater than 6, close to about 1O.

These arrays of saccharide mucopolys contain thermal units having a base structure of 2,5-anhydro-D-mannose whose primary alcoholic function in position 6 may be substituted or not by the group -SO ^ * · This structure results in

7.

from the action of azo taste acid to the level of the basic unit

N-Eulphate-glucosate heparadine «

The terminal unit (discussed) corresponds to the general following formula t

in which j Rg denotes a hydrogen atom Hi group SO ^ and denotes a functional group selected from an aldehyde "al-school or carboxyl group or their derivative groups" especially ace tal, amide "e tara" ether or corresponding salts "

The process according to the invention is conveniently carried out "as already emphasized without applying fractions" and in products having Yin-Veesler / uSP orders of magnitude 10 or more "with Yin-Vessler activity above about 200 Ul / mg, preferably above about 250 Ul / mg.

8.

These products consist mainly of mole types - kulaks weighing 2,000 to 6,000 daltons, corresponding to structures of 8 to 30 saccharide units.

According to a convenient method of carrying out the process of the present invention, heparin having a molecular weight of from about 2,000 to about 50,000 is conveniently used as a starting material.

It can be affected by heparin with classic pharma- ceutical properties, and may be given by injection, or crude heparin obtained as a product of active principle extraction operations starting from tissues or organs of mammals, especially mucus from Hi gut, for example, pigs or bovine animals . The hedgehog may consist of products that are normally extracted during purification of heparin and obtain heparin of such quality that it can be injected with injection and increased specific activities.

Bi heparin is subjected to the action of a chemical agent which, under mild conditions, can be used to partially depolymerize heparin and lead to active biological arrays as mentioned above.

Speci Jalne is used as nitric acid

9.

IINOg «This acid acts on the level of units of N-eulfatglucosamine heparin and accompanies them to groups of structures

2,5 — Anhydro — D-mannose “Suitably nitric acid is produced in situ by the addition of controlled amounts of an acid to the mineral salt of azo taste, especially to alkaline or zesaioalkalic nitric acid ·

According to one convenient method of the present invention, sodium nitrite NaNO is especially used. · To obtain azo taste acid in situ, controlled amounts of an acid containing a physiologically acceptable anion such as acetic acid U and preferably HCl are added.

The action of nitric acid on Jieparin is conveniently carried out in aqueous and aqueous medium ·

The realization of these transcripts is of particular importance from the point of view of the biological applications under consideration.

The aqueous medium in which the depolymerisation is carried out provides the effect of an acceptable physiological environment, which avoids all problems related to the solvent elimination, which is detrimental in this respect. "On the other hand, the salt that is pruned in this medium during the production of nitric acid is a gel. dissolved in water, nairae say natri jumohiorld, whose presence does not appear to be detrimental *

The various parameters, which operate during the course of a chemical reaction, especially the finished gossip is a reagent, crows, temperature 1 pH, are adjusted so that the desired products are debuted at the end of the experimental conditions.

From this point of view it is said how many different pairs of tare are in a strict relationship at all,

Obviously, calculating one Ml of these factors may entail adjusting one or more other factors. "

Studies of these experimental conditions by these applicants have shown that it is advantageous to use reagents in amounts leading to a final concentration of heparin of a suitably size of 1 to 10 g, preferably 1.5 to 5 g, particularly about 2 g per 100 ml of reaction In the middle, the final concentration of sodium yu-three may vary from about 0.02 M to 0.1 M and is primarily of the order of 0.05 H. "Chloro hydrochloric acid is used in an amount that is

π.

sufficient to obtain "pH of reaction medium of order of magnitude ed 2 to 3, preferably ed 2,2 to 2,7 * primarily 2,5"

It operates at a temperature of from 0 to 1 ° C, preferably about 4 ° C, and is aged to react for the period of time allowed to obtain the desired degree of depolymerisation. As an indication, an incubation of 10 minutes when run at 4 ° C seems sufficient.

It is interrupted during the degree of depolymerization,

Increasing the pH of the medium is conveniently adjusted for this purpose *

The addition of an alkaline reagent, for example, to a soda solution in an amount sufficient to obtain a pH value that is at least neutral or weakly alkaline, allows the desired depolymerization reaction to be interrupted *

All raucopolysaccharides which are precipitated with an alcohol solvent are separated *

The use of absolute ethanol, about 5 volumes, provides the desired separation *

The precipitate is also collected depending on the use of chipper and sushi «

12.

In order to achieve the above transcripts, muco * polysaccharides having these three Yw / USPs at the bottom of the order of magnitude 10/1 are isolated, with those activities YU above about 200 Ul / ag, starting from heparins having these YW / USP ratios order size 1.

According to the supplementary regulation, the aldehyde functions of terminal reductive groups are converted into more stable functional groups such as alcohol or acid groups, leading to arrays of terminal mucopolyaccharides for most of the 2.5-anhydro-D-mannitol or 2,5-anhydro structures JMsenonski Iti line,

In order to convert 2,5-anhydro-D-eanose terminal groups into 2,5-enhydro-D-raanitol groups, the previously precipitated precipitated products are subjected to the action of a reductive agent, working under conditions that enable at least part of the desired transformation to be achieved.

Reductive agents are mediated by the agents commonly used to transmit aldehyde groups into alcohol groups. Of these agents, it seems appropriate to apply a metal boride anhydride.

The reaction of ed is conveniently carried out in an aqueous medium in the presence of sodium borohydride or potassium hydrochloride over a period of several hours. "

η.

Smoking at room temperature, suitable with stirring, it seems sufficient to leave the mixture to react for a period of about 4 hours. An increase in the pH of the reaction medium is observed. · This pH value can reach the order of magnitude of IO when using NaBIi ^ - I'll hear the release of soda "(sodium hydroxide) ·

Upon the destruction of the unreacted borohydride, the pli is reduced by the addition of acid. "

In a special case »it seems appropriate to tear the pH down to 4 by adding» »for example» acetic acid *

The pH is then adjusted back to a value close to neutrality, especially of the order of magnitude 7 »5 by adding an alk! no g agent "for example" soda "from the reaction medium regenerates that product which can be precipitated with alcohol and regenerates a rewarded precipitate containing the required products in which the terminal structure of 2,5-anhydro-D-mannose was converted to structure 2" 5 —Henhydro — D-taenitol “

Said deposition can be done by adding an absolute

1 * »ethyl alcohol, suitable in 5 volumes ·

In order to isolate the beautifully reduced products, centrifugation is conveniently applied and a centrifugation precipitate is collected, which then, if desired, is washed and dried,

A study of the saccharide mucopolys thus obtained has shown that they have Ttf / USP titers with a size ratio of 10 or more, with YK activity titers above about 200 'IU / mg, preferably above 256 Ul / mg.

According to one embodiment of the method of this invention, the 2,5-anhydro-D-mannose groups are converted to the 2,5-anhydro-IMaanonic acid groups ·

Reacts, under the conditions necessary to achieve the desired transformation, products containing terminal groups of 2,5-anhydro-D-mannose with an oxidizing agent, selected from agents commonly used to transform aldehyde groups into carboxylic acid groups, in particular from pereangants ·

The reaction is conveniently carried out in an aqueous medium at pH above neutral.

15 «

Oxidation of the aldehyde groups into the alkaline groups results in a decrease in the value of the pile, which is conveniently distributed continuously during the reaction, smoking at room temperature, the desired oxidation being achieved within about 15 hours. growth - boil.

Then the taiog was washed and dried for a year.

It was concluded that the values (indicators) of molecular weights given above (and the following, especially in the lower examples) are derived from the measures of retention solutions of the carrier having a certain studied substance, subject to the permeability of the gel through the column. gels, under similarly determined elution conditions, whereby the logarithms of these molecular weight indicators are in the same "retention ratio" with the above® measured® retention times (retention times), such as molecular weights of 4,000, 6,500, 16,000 and 11,000, ethanol sodium sulphonate ethanol, especially commercial products obtained from CHRGMPACK (0raay-les-Ulis, France), versus their retention times measured in the system and under conditions identical to gel leakage.

16.

Depending on the degree of purity to the needle, the treated products, which may be in the form of salts, are physiologically infused with metals such as sodium, and then they may not be converted into salts or salts or salts containing other salts. physiologically acceptable metal such as calcium, using all the processes that can be converted to heparin salts. The process described in French patent no. 73 135θο pre dated 13 April 1973 · on behalf of the public. It recalls that, in the drying process, starting from (for example) sodium heparin, from contacting it with a salt of a different physiologically acceptable metal, for example, with calcium chloride, or with some solution thereof, then to separate metallic yen unrelated to heparin (aa, for example, by alcohol precipitation or dialysis), and, if the measure and yield of substitution are unsatisfactory, re-contact, salts or salts thereof, between the Soviets and heparin salts obtained after of the first contact, with the new dosage of the second salt, in particular the calcium chloride, of the thorn as the yield of the final substitution is desired.

Other features and advantages of the present invention derive from the description of the exemplary embodiments of the invention.

17 «

Example 1

Partial de-polymerization and production of oligosaccharides having terminal reductive groups ea structure «

2,5-anhydro-u-mannose, nalmeth

whereby the primary. the hydroxyl moiety at position 6 may be admixed with another substituent primarily by the group sg ₃ ""

Suitably, the starting bepetrin is subjected to mild HNOg action and acts as below.

Dilute ee 60 g of commercial heparin with a YV / USP yield of about 1 and a USP titer of 160 Ul / mg in 3 1 of distilled water at + 4 ° C »

Sodium nitrite NaNOg is added in an amount sufficient to give a 0.05 M solution, say 10.35 g,

18.

then the pH was adjusted to 2.5 with pure hydrochloric acid and stirred at + 4 ° C for 10 minutes. The pH of the batina was adjusted to 7> 5 with 5 N sodium hydroxide solution.

Addition of 5 volumes of pure ethanol (above 15 · 500 ml) precipitates the reaction. Centrifugation collects cheeks »! sediment. This was washed with ethanol and dried at 60 ° C under high vacuum. 6o g of product is obtained which has the following characteristics!

USP titer 17 µl / mg

YW titer 24θ ul / mg titer AP TT * 12 uI-Hng (* English language term activated partial thromboplastin time * equivalent to time - no cephalin kaolin / Coen J «et al, L 'Rento st ase ezpansion sc ± entifique, * 197O, pp. 169-170 /).

In the second test, it was proceeded as described above except that the second batch of heparin was used as a deposition substance, gossip is 165 Ul / mg in USP units and having a YV / USP ratio of order of magnitude 1.

Dissolve 3 g in 150 ml of distilled water at temperature

19.

from + 4 ° C, a total of 517 of that NaNO _{n is} added to the reaction center.

After pi erado as described above, 2.8 g of product were obtained with a titre USP of 24 µl / rag and a titre in Y & aktivnosti activity of 250 µl / rag,

According to another test, which was performed according to the above procedure, under the above conditions, 50 e of heparin were titrated with USP 158 Ul / rag bottom and he: Y ¥ / USP about 1 "

Dissolve in 2500 ml of extruded water and add 3.625 g of NeN0 ₂ at + 4 ° C. <Upon completion of the processing, 46 g of product is collected which has the following characteristics:

USP ti tar 13 ul / mg ti tar activity aktivnosti from ¥ 270 ul / mg ul / rag · APTT titer

20.

Example 2

Partial depolinorisation of lieparin 1 to produce oligo aaccharides forming terminal groups with structures

2,5-anhydro-D-mani tola, namely s

CH ₂ OH

OH

About what the primary alcohol group in position 6 may be substituted as mentioned above,

Dissolve 47 g of the product obtained in Example 1 in 1200 ml of distilled water at room temperature. Strong stirring is added to 7 g of potassium borohydride KBH ^. Stirring is continued for 2 hours at room temperature with acetic acid in the reaction mixture. lowers pH to 4.0 and destroys KDII ^ not consumed «

The reaction mixture was then stirred for 30 minutes, then adjusted to pH 7.5 with 5 N sodium hydroxide solution.

5 volumes of alcohol are added to the reaction medium. "

The formed precipitate was collected, dried in air, washed with pure ethanol and dried in vacuo at 6 ° C.

Collect 46.5 g of product that has the following characteristics!

OSP titer 17 ul / mg ti tar in Y&W activity 250 ul / rag ti tar APTT 11 ul / mg.

Partial depolymerization of heparin and preparation of ligands of saccharides having terminal groups from the structure

2,5-anhydro-O-raanoic acid, nalme:

COOH

22.

wherein the primary liidrcksi group at position 6 may be substituted as described above.

Dilute 4 ϋ g of the product obtained by following the procedure described in Example 1 in 400 ml of distilled water at room temperature.

The pH value was adjusted to 5 with 5 k of soda solution and then added 2 g of potassium solute dissolved in 40 ml of water.

The reaction mixture was stirred vigorously for 15 hours. During this tea, the mixture was continuously adjusted to 8.5 per 5 g of soda solution.

At the end of this period of 15 hours, 0.2 preconceived (yo ml) alcohol is added to reduce the clinical effects of which did not respond *

The reaction amoeba will be left to stand for 1 hour. Ethanol precipitation precipitates the reaction products, and then they are washed and dried. 55 g of product are thus obtained which has the following characteristics:

USP ti tar 12 ul / mg ti tar in cdetivity Y R V 27 o ul / mg 8 ul / mg.

the APTT titer

The products obtained according to this invention can be used in the form of all. therapeutic salts applied, (especially salts of physiologically acceptable metals such as salts of sodium, calcium, magnesium, or salts).

in the present case, according to one of the methods described in the patent application «no. 73 13530 surrendered it on 13 April 1973 in the name of these applicants.

Pharmacological examination of the obtained products *, which are conveniently purified according to classical "techniques", for example, by dialysis, chromatography, or by alkalinity by alcohol, shows that they have a more selective effect, especially with respect to factor ia inhibition, than reference heparin. among other things, these products have the advantage that they do not cause blood platelet aggregation in the duckweed - in which heparin causes such a reaction »

These products are atoxic »

Administration to mice at a significant dose of 3200 mg / kg did not facilitate determination

The present invention particularly relates to the preparation of muco-polysaccharides of the type described above, which have activity

24.

at least 2θο bl / rag (Yin-Wossler) and a Υίί / oSP ratio above 6. On the other hand, it refers to the production of farraaceutslili preparations having similar activities, bon pyro-driven substances, and in combination with farmaceutslciu excipients. This invention particularly relates to the preparation of conextractable solutions of these fractions, which are stable and suitable for injection, and to be used in therapy for the control of blood coagulation, and in particular a suitable way of preventing postoperative thrombosis or embolism, whereby these solutions are contain from 1,000 to ICO,000 bi (Υλη-Jesaier) / acre mucopoisaluride, preferably from 3,000 to 30,000, -m example, 25,000 U / ml, although these solutions are intended for subcutaneous injection or contain, for example, from 300 to 10.00G, for example, 5,000 U / ml of nuconolysaccharides, when these are intended for infusional injection or perfusion.

The cucopolysaccharides obtained by the present invention are conveniently in the form of a sludge of at least one physiologically infused ji c metal, such as sodium and / or Italy.

Conveniently, these pharmaceutical relationships are applied when assisted by syringes and are not used before the appropriate time of use.

25.

The compositions obtained according to the present invention are particularly adapted to control (preventive or curative) blood coagulation in human 111 live, especially when a separate host is at risk of hypercoagulability, especially if this is a result of perturbation of the exterior phase mentioned above. for example, as a result of thromboplastin staining in the body, for example, tissue thromboplastin (surgical interventions, atheromatic processes, tumor bleeding, disorders of coagulation mechanisms by bacterial or enzymatic activators, etc.). of the present invention will be given below, as an example, the penology of their administration to man "it includes, for example, administering to a patient from 1,000 to 25,000 Ul subcutaneous eubcutaneous routes, two to three times daily, according to the level of hypercoagulability or thrombotic condition batch is from 1,000 to 25,000 Ul per 24 hours intrav elnozaim, and discontinuous administration at regular intervals, or continuous administration with perfusion, or at least 1,000 to 25,000 Ul (m. times per week) by intramuscular route (tl t trenches are expressed in Ul Yin-Wessler units). The doses given above may be adjusted for each of the ducks according to the results of the previously performed blood tests, according to the nature of the disease the patient is suffering from, in general according to his or her state of health which must be well known.

The present invention also relates to the use of muco-polysaccharides obtained according to the invention having a composition of biological lactatives that can be used in the laboratory, in particular as a reference for the study of other products in which their anticoagulant activity is glued "especially at the level of inhibition factor Xa,

Claims (2)

PATENT REQUIREMENTS 1. A process for the preparation of mucopolysaccharides of heparin derivatives formed from unit-exchanged units / D-glucosamine_7 - uronic acid, or vice versa, with terminal groups 2,5-anhydromanose of the formula in which R ^ represents the group -CHO, COOH or -CH ^ OH, and R ₂ represents a hydrogen atom or a group -SO ^ ^- , wherein these mucopolysaccharides have increased anti-Xa (Yin-Wessler) activity , and a Yin and Wessler titer to USP titer of at least 2 by depolymerization of heparin by a chemical agent, wherein the heparin having molecular weight sequences of the order of 2,000 to 50,000 is subjected to the action of nitric acid created in situ by adding controlled amounts of acid to the mineral nitric acid salts in aqueous medium, at a temperature of about 0 ° C to ambient temperature, which depolymerization is interrupted when portions of heparin from the depolymerization mixture are formed by a large portion of arrays having a molecular weight of 2,000 to 8,000, titers YW and USP relative to higher of 2, a 28. especially of 3, and containing terminal reductive groups with the structure of 2,5-anhydro-D-mannose ch ₂ oh II CHOs which isolate mucopolysaccharides precipitated by an organic solvent and collect, and, if desired, the resulting mixture of mucopolysaccharides with terminal groups of formula I, wherein R1 has the meaning -CHO, are subjected to reduction with the aid of potassium borohydride, to obtain a product whose terminal the groups of formula I have the meaning -CH ^ OH for the radical R ,,, or else, which results in a mixture of mucopolysaccharides with the terminal groups of formula I, wherein Rhyme means -CHO, is oxidized by an oxidizing agent such as potassium permanganate, and thus obtained a compound of formula I in which the radical R 1 has the meaning COOH. A process according to claim 1, characterized in that, as the mineral salt of nitric acid, alkaline or earth alkaline salt is used. 3) The method of claim 2, wherein the mineral salt of nitric acid is sodium nitrate and that nitric acid is formed in situ by the addition of sodium nitrate to an acid having physiologically acceptable anions, such as acetic acid and preferably hydrochloric acid. A method according to any one of claims 1 to 3, wherein the final concentration of heparin is preferably from 1 to 10 g, preferably from 1.5 to 5 g, with especially close to 2 g per 100 ml of the reaction medium, wherein the final the concentration of sodium nitrate may vary between 0.02 H and 0.1 H and preferably be about 0.05 H, and the nitric acid is formed in situ by the addition of hydrochloric acid, in a controlled amount sufficient to achieve a pH of the order of 2 to 3, preferably from 2.2 to 2.7 and primarily 2.55. The method according to any one of claims 1 to 4, characterized in that it is operated at a temperature of from 0 to 10 ° C, preferably at 4 ° C, for a period of time sufficient to achieve the desired degree of depolymerization, wherein this time period is about 10 minutes when operated at 4 ° C.