HRP920795A2 - Heparosan-n, o-sulfates, the process for obtaining them and pharmaceutical preparations containing the same - Google Patents

Description

The object of the present invention is new heparosan-N, sulfates, preparations of heparosan-N,O-sulfates that contain these new heparosan-N,O-sulfates and pharmaceutical preparations that contain heparosan-N,O-sulfates as an active principle.

Glycosaminoglycans are known to be suitable products for extraction from animal tissues. Some of these glycosaminoglycans have very interesting anticoagulant and antithrombotic properties. Typical products of this family are heparin, its fragments and their derivatives, such as heparan sulfate and dermatan sulfate, which, however, have a disadvantage because they are very expensive due to their origin.

More precisely, it is known that dermatan sulfate is a family of polymers with a variable degree of polymerization that consists of repeating units of the uronic acid group (iduronyl or glucuronyl) and the acetylsulfato-4-galactosaminyl group (H.W. Stuhlsatz "The Methodology of Connective Tissue Research", 1976, 137-146) . Natural dermatan sulfate has a molecular weight between 20,000 and 40,000 Da. This product is particularly interesting as an anticoagulant and antithrombotic (F. Fernadez et al, British Journal Haematology (1986, 64, 309-317).

On the other hand, it is known (I. Björk et U. Lindhal, "Molecular and Cellular Biochemistry", (1982), Dr. W. Junk Publishers-Pays-Bas) that blood coagulation is a physiologically complex phenomenon, the mechanism of which can be schematized and summarized as follows:

[image]

Some stimuli, such as contact activation and tissue factors, involve the sequential activation of a series of coagulation factors present in the blood plasma, these are indicated in advance by Roman numerals on the diagram, and the presence of an arrow indicates the activated form (arrow present) or unactivated (arrow absent) of a given coagulation factor .

Whatever the nature of the stimulus, the final stages are identical: Factor Xa activates factor II (also called prothrombin), which in its activated form (factor IIa, also called thrombin) causes partial proteolysis of soluble fibrinogen with the release of insoluble fibrin, which forms the main blood clot.

Under normal physiological conditions, regulatory proteins such as antithrombin III (ATIII) and heparosan cofactor II (HCII) are equally present in plasma.

Antithrombin III exhibits inhibitory activity on the coagulation factor system marked with an asterisk (*) in the above scheme. This inhibition is strongly enhanced in the presence of heparin or oligosaccharides of heparin-type synthesis (D. H. Atha et al, Biochemistry, 1985, 24, 6723-6729).

Cofactor II of heparin shows inhibitory activity only on factor IIa (thrombin), which catalyzes the last stage of coagulation. This activity is greatly enhanced in the presence of heparin or dermatan sulfate (D. M. Tollefsen, J.Biol.Chem. (1983), 258, 6713-6716).

Inhibition of factor Xa or factor IIa constitutes a privileged way to obtain anticoagulant and antithrombotic activity because these two factors intervene in the last two stages of coagulation, which are independently exclusive stimulants.

To obtain inhibition of factor IIa itself, one possibility is particularly interesting, and consists in using the specificity of cofactor II heparin and testing the enhancement of its inhibitory activity. Dermatan sulfate is a well-known product that has a very strong amplifying activity of this type.

It is equally known that the structure of the heparin main chain is formed in two stages. At first, heparin is biosynthesized starting from a precursor proteoglycan, the polysaccharide part of which is composed of a family of proteoglycan polymers, the polysaccharide part of which is composed of a family of polymers of a variable degree of polymerization formed from repeating units of beta-D-glucoronyl-1,4-alpha-N -acetyl-D-glucosamyl (1,4) (disaccharide units) molecular weight 379 Da. This polysaccharide moiety is commonly called N-acetylheparosan (J. Navia, Anal.Biochem., (1983) 135, 134-140). This first stage of biosynthesis is the only moment where one can really speak of a "disaccharide motif" because the second stage of biosynthesis modifies this simple skeleton in a desirable way ("L'Héparine, fabrication, structure, properties, analyses", J.P. Duclos (1984) p. 81-83, Masson Ed., France).

Indeed, natural heparin, the result of biosynthesis is a polysaccharide composed of molecules of glucoric acid and iduronic acid (uric acid), possibly sulfated in position 2, bound to glucosamine molecules, possibly sulfated in position 6 and sulfated or acetylated to an amine in position 2.

The structure of heparin can be satisfactorily represented by the following formula (I):

[image]

where A represents H and SO3-, B represents SO3- and COCH3 and n is a number between 20 and 30, corresponding to a molecular weight of 12000 ̧18000 Da (EP-A-O 116 801).

The terms "H and SO3-" as used for the substituents A and B, respectively, indicate that in the 20-30 disaccharide units forward, A is in some cases hydrogen and in other SO3- groups and analogously, B is in most cases SO3-, and in other cases it is an acetyl group.

Also, the bond indicates that the COO- group in 20-30 disaccharide units forward has in some cases configuration (ii)

[image]

(it is about D-glucuronic acid) and in most n cases, configuration (iii)

[image]

(it is about L-iduronic acid).

Thus, heparin and heparin fragments as obtained by different depolymerization procedures are macromolecules that also contain glucuronic acid units as well as iduronic acid units.

Certain depolymerization procedures allow obtaining heparin fragments with a molecular weight between 2000 and 9000 Da and a degree of sulfation above at least 20% of the starting heparlin. Such "supersulfate" heparins are described in patent application EP-A-0 116 801, and have for immersion units, the two structures (ii) and (iii) mentioned above.

Some Escherichia coli bacteria are also known to produce a capsular polysaccharide, commonly called the K5 antigen, which is a family of polymers composed of repeating units of beta-D-glucuronyl-1,4-alpha-N-acetyl-D-glucosamyl-(1,4 ) (W. F. Vann et al, Eur. J. Biochem (1981) 116, 359-364).

This polysaccharide, of the same chemical nature as the polysaccharide part of proteoglycin heparin, will be called here N-acetyl-heparosan. This product has a molecular weight between 105 and 2106 and a level of "uronic acid" units, a structure very regular for a unique D-glucuronic acid compound (W.F. Vann et al, Eur. J. Biochem (1980), 116, 359-364 and patent application EP-A-O 333 243).

Patent application EP-A-0 333 243 describes the polysaccharide K5 O-sulfate as well as certain compounds of fragments, respectively of 4, 6 or 8 "sugar" units, both O-sulfates. These products have antiangiogenic and antitumor activity with a suitable relationship of these activities to anticoagulant properties. This document also describes fragments of N-acetylheparosan compounds respectively of 4, 6, 8 or 10 "sugar" units.

Patent application EP-A-0 333 243 also describes the preparation of a pentasaccharide of the structure N-acetylheparosan-O-sulphate, completely by chemical synthesis.

It has now been found that heparosan-N,O-sulfates of variable molecular weight between 1500 and 15000 Da have anticoagulant activity, against heparin cofactor II and very high anti-Xa activity.

Heparosan-N-O-sulfates, the object of the present invention, differ from other products already described in the literature by their original structure, mainly by their degree of sulfation (sulfation also on the amine group of glucosamine) and their pharmacological properties. Among other pharmacological properties, they have anticoagulant activity against heparin cofactor II (HCII) stronger than dermatan sulfate and have very interesting pharmacokinetic characteristics. Furthermore, the products of the invention obtained by chemical chemosynthesis have the pharmacological activities of glycosaminoglycans usually used therapeutically, mainly heparin, and can be used to regulate coagulation and can more specifically find their application as antiplatelets.

The subject invention for the object has heparosan-N,O-sulfates composed of chains or a mixture of chains of molecular weight between 1500 and 15000 Da, characterized by the repeating disaccharide structure of formula I:

[image]

where,

E represents in 0-80% of the disaccharide units of the mentioned heparosan-N,O-sulfate, an acetyl group and in the remaining disaccharide units a sulfate group and possibly a hydrogen atom.

G represents a hydrogen atom and a sulfate group,

and pharmaceutically acceptable salts of said heparosan-N,O-sulfates.

The degree of sulfation of heparosan-N,O-sulfate expressed as sulfate/carboxyl yield is 1.5-3.0.

The invention also relates to a preparation of heparosan-N,O-sulfate which contains at least 70% by mass of the heparosan-N,O-sulfate described above and the object of the present invention, preferably containing at least 90 mass. % heparosan-N,O-sulfate of the subject invention.

Heparosan-N,O-sulfates object of the present invention can be composed of equal polysaccharide chains of well-defined molecular mass, this molecular mass can be in the interval 500 ̧15000 Da. They can also be composed of a mixture of chains of variable molecular weights that can be between 1500 and 15000 Da. The dispersion of the molecular masses of these chains can be even greater. In fact, heparosan-N,O-sulfates, the object of the present invention, can be composed of chains between which the difference in molecular masses can be a maximum of about 13500 Da, or in contrast to a chain between which the difference in molecular masses is about 300 Da, which corresponds to a unit of immersion structure (D -glucuronic acid or its derivatives) or glucosamine structures. It is also evident that for the building function of each heparosan-N,O-sulfate, the molecular weight of the chains having, say, a lower molecular weight, say a higher molecular weight, can only correspond to a value between 1500 and 15000 Da.

The expression "G represents a hydrogen atom and a sulfate group" used above indicates that G in the disaccharide unit represents a hydrogen atom in some positions and a sulfate group in others. In the same way, E represents in some disaccharide units an acetyl group and in other of these units a sulfate group or possibly an atom of the heparosan-N,O-sulfate disaccharide unit, they are not all identical.

Formula I is a repeating disaccharide structure formed from a glucosamine unit and a D-glucuronic acid unit. The mentioned units can be inverted, more precisely, if it is considered that the disaccharide structure of formula I is repeated n times, that the non-reducing unit of the chains can be indifferent, for example, a glucosamine unit, such as is represented in formula I with a hydroxy group in position 4, this unit of glucosamine it can be sulfated or not, for example D-glucuronic acid containing possibly a double bond in the C4-C5 position and sulfated or not. The reductive unit can be indifferent, for example D-glucuronic acid as shown in formula I, substituted with hydrogen on the enomer oxygen, for example glucosamine, for example the 2,5-anhydromanno structure, as obtained in saltpeter depolymerization (2,5-anhydro- D-mannose) possibly followed by oxidation (2,5-anhydro-D-mannonic acid) or reduction (2,5-anhydro-D-mannitol).

Preferred products are those whose two sides, reductive and non-reductive heparosan-N,O-sulfate chains of the objects of the subject invention are uronic units sulfated or not, glucosamine sulfated or not N-acetyl glucosamine sulfated or not or the structural units are 2,5-anhydromanne.

Preferred heparosan-N,O-sulfates consist of chains whose two reductive and non-reductive sides are sulfated or not immersion units, sulfated or not glucosamine and N-acetyl glucosamine sulfated or not.

The subject invention also has for its object a procedure for obtaining a preparation containing 70-100% heparosan-N,O-sulfate, characterized by the fact that it implies a sequence of the following stages:

- stage a: cultivation of Esherichia coli species (K5) for the production of N-acetylheparosan

- stage b: isolation and purification of the formed N-acetylheparosan to obtain a compound containing 70-100% molecular weight between 1500 and 15000 Da characterized by a repeating disaccharide structure of formula II:

[image]

- stage c: partial deacetylation of this N-acetylheparosan to obtain a compound containing 70-100% of heparosan composed of chains or a mixture of chains with a molecular weight between 1500 and 15000 Da, characterized by a repeating disaccharide structure of formula III:

[image]

in which R' represents, 0-80% of the disaccharide units, an acetyl group and in the rest a hydrogen atom,

- stage d: let's say partial N,O-sulfation of this heparosan compound,

for example partial N,O-sulfation of this heparosan compound followed by a step of complete N-sulfation,

say complete or partial N-sulfation followed by a step of complete or partial O-sulfation.

and possibly includes one or more stages of fractionation of molecular masses carried out at the end of stages, b, and d.

To obtain the compound containing 70-100% heparosan-N,O-sulfate, the object of the present invention and according to the method of the invention, it is preferably used as Escherichia coli type (K5), Escherichia coli type SEBR 3282. This type is derived from the type Bi 8337- 41 (010:K5H4) ATTCC 23506 (described mainly by D.S. Gupta et al FEMS Microbiology Letters (1982) 14, 75-78 and W. Vann Eur.J.Biochem. (1981) 116, 359-364).

The strain Escherichia coli SEBR 3282 responds positively to the typing test with the specific phage K5 according to the method of B. Kaiser et al (J.Clin.Microbiol. (1984) 19, 2, 264-266). It is therefore a species of Escherichia coli (K5). This species has been deposited with CNCM Institut Pasteur, Paris, France under no. I.1012. A mutant, say spontaneously produced by this species, can also be used, as well as other suitable strains of Escherichia coli (K5), eg Bi 626-42 (012:K5:NM) ATCC23508.

The culture medium used is preferably a medium rich in nitrogenous matter, for example, a medium rich in yeast extract and casein hydrolyzate, a slightly expensive agent that carries a significant amount of amino acids.

Cultivation of the Escherichia coli species (K5) preferably continues for at least two hours after stopping the biomass growth.

Isolation and purification of N-acetylparosan in order to obtain a compound containing 70-100% of N-acetylparosan composed of a mixture of chains with a molecular weight between 1500 and 15000 Da, characterized by the structure of a repeating disaccharide of formula II, is realized by a process that includes at least one stage of precipitation and ion exchange chromatography. This step is carried out using preferably a Q-Sepharose column or an equivalent column. Precipitation is carried out with a suitable organic solvent, and mainly alcohol, preferably ethanol. According to this process, N-acetylheparosan can be in the form of a salt, preferably in the form of a sodium salt.

By way of example, a preferred isolation and purification procedure can be schematized as follows:

- stage a1: precipitation with ethanol,

- stage b1: dialysis,

- stage c1: precipitation with ethanol, then dehydration and drying,

- stage d1: purification by amino-exchange chromatography,

- stage e1: ethanol precipitation of the eluate obtained in stage d1, dehydration, drying and grinding.

With regard to stages a1, b1, c1, the order in which they are performed is of little importance. One of the steps a1 or c1 can be omitted.

Step e1, ethanol precipitation is not necessary. N-acetylheparosan can be isolated by other methods, for example by evaporation under vacuum of the eluate obtained in step d1.

Isolation and purification of N-acetylheparosan to obtain a compound containing 70-100% of N-acetylheparosan composed of a mixture of chains of molecular weight between 1500 and 15000 Da can also be carried out in the following way:

- stage a'1: dialysis,

- stage b'1: purification in an acidic environment, elimination of insoluble impurities in aqueous solutions pH=3.5 and pH=1.8,

- stage c'1: precipitation with ethanol, then dehydration and drying,

- stage d'1: alkaline hydrolysis and dialysis,

- stage e'1: purification by anion-exchange chromatography,

- stage f'1: purification by exclusion chromatography.

This process of isolation and purification is also a preferred process of the invention.

Alkaline hydrolysis is performed with NaOH solution, at a temperature between 30-80oC.

When applying the isolation and purification procedure, the starting product can be used, for example the suspension obtained at the end of the cultivation and in this case prior filtration is required, for example the product that has already been subjected to preliminary purification performed according to the procedure that includes the following stages:

- stage a''1: centrifugation of the resulting suspension at the end of cultivation,

- stage b''1: contacting the supernatant with an alkaline solution,

- stage c''1: pre-filtering,

- stage d''1: concentration on the membrane of a certain threshold opening size, which allows separation as a function of molecular mass,

- stage e''1: dialysis.

Stage e''1 is not necessary.

As an alkaline solution, 0.1M NaOH can be used.

In a preferred embodiment, a preliminary purification of the product obtained at the end of cultivation is performed, according to the procedure described above.

Cultivation of Escherichia coli species (K5) and the isolation and purification procedures as well as the pre-purification step mentioned above, allow obtaining a compound of N-acetylheparosan containing 70-100% of N-acetylheparosan made from a mixture of chains of molecular weight between 1500 and 15000 Da, characterized by a repeating disaccharide by the structure of formula II:

[image]

In fact, thanks to the isolation and purification procedures described above and the preliminary purification step, it is possible to obtain N-acetylheparosan compounds containing at least 80-100% N-acetylheparosan composed of a mixture of molecular weight chains between 1500 and 15000 Da.

N-acetylheparosans composed of a mixture of chains with molecular weights between 1,500 and 15,000 are new products and are also the subject of the present invention.

Both reductive and non-reductive sides of the novel N-acetylheparosans such as are obtained according to the process described above and using Escherichia coli strain SEBR 3282 or other suitable species as indicated above, are uronic or N-acetylglucosamine units.

In most chains, the non-reducing side is the immersion unit of formula (a)

[image]

The eventual enrichment of the obtained compound in N-acetylheparosan composed of a mixture of chains of molecular weight between 1500 and 15000 Da of the repeating disaccharide structure of formula II, can be carried out in various degrees and mainly until the isolation of the mentioned N-acetylheparosan. Enrichment is performed using classical molecular mass fractionation techniques such as gel permeation chromatography and ultrafiltration (A.A. Homer, Biochem.J., (1989), 262, 953-958; G. Pyler et al, J. Biol. Chem. (1984 ), 259, 20, 12368-12376). The ethanol fractionation process can also be used (patent application EP-A-0 287 477). This last fractionation procedure is particularly appreciated among other known techniques.

The N-acetylheparosans described above are used as intermediates for obtaining heparosan-N,O-sulfate, the object of the present invention, but also for obtaining other derivatives.

To obtain heparosan-N,O-sulfate, the object of the present invention, other known N-acetylheparosans can also be used, such as, for example, N-acetylheparosans built mainly from polysaccharide chains that are, in addition, disaccharide units, such as described in patent application EP-A-0 333 243, and mainly composed of chains containing at least 10 monosaccharide units (Grupa et al, FEMS Microbiology Letters (1983), 16, 13-17) or high molecular weight N-acetylheparosan which can be immediately depolymerized according to the procedures of use for obtaining heparin of low molecular mass.

In fact, heparosan-N,O-sulfates, the object of the present invention, can be obtained by other methods, starting from the known psosaccharides (K5). In this case, a depolymerization step is required.

This depolymerization can be carried out before deacetylation of high molecular weight N-acetylheparosan, after deacetylation, after N-sulfation or even after N,O-sulfation.

As previously mentioned, the depolymerization can be performed according to the procedures described in the literature for obtaining low molecular weight heparin, e.g., by periodate depolymerization, free radicals, beta-elimination or the action of nitric acid (patent applications or patents EP-O 040 144, EP- O 037 319, EP-O 121 067). These procedures are provided by way of example and are not limiting. All other glycosaminoglycan depolymerization procedures can be used.

As heparosan-N,O-sulfates are obtained by depolymerization starting from N-acetylheparosan of high molecular mass (previously deacetylated and possibly N-sulfated) by subjecting to the action of nitric acid, the reductive unit of heparosan-N,O-sulfate, the object of the present invention, can have the structure 2,5-anhydromannose and mainly 2,5-anhydro-D-mannose. Depolymerization by the action of nitric acid can be performed after the N-deacetylation or N-sulfation stage. When this step is followed by oxidation or reduction, heparosan-N,O-sulfates of the reductive unit are obtained, the structures corresponding to 2,5-anhydro-D-mannonic acid or 2,5-anhydro-D-mannitol.

Partial deacetylation of compounds containing 70-100% N-acetylheparosan, which is carried out to obtain compounds containing 70-100% and also 80-100% heparosan composed of a mixture of chains of molecular weight between 1500 and 15000 Da, characterized by a repeating disaccharide structure of formula III , given in advance, is realized by treatment with a deacylating agent.

Phosphorus pentasulfide, triethyloxonium fluoroborate, sodium carbonate or hydrazine can be mentioned as deacylating agents, these last two agents being particularly well known. You can also use strong mineral acids, such as HCl, H2SO4, etc. The duration of the reaction depends on the selected operating conditions and mainly on the temperature and concentration of the deacetylating agent in the reaction medium.

Enrichment of heparosan compounds in heparosan made from chains or a mixture of chains with a molecular weight between 1500 and 15000 Da, characterized by a repeating disaccharide structure of formula III, is realized by implementing the classic molecular weight fractionation techniques mentioned above (gel permeation chromatography, ultrafiltration and ethanol fractionation). In this case, compounds containing 90-100% by mass of heparosan composed of a mixture of chains of molecular weight between 1500 and 15000 Da of the repeating disaccharide structure of formula III are obtained.

To obtain heparosans made from a mixture of molecular chains between 1500 and 15000 Da, it is also possible to use N-acetylheparosans of higher molecular weights. Heparosans of high molecular weight obtained after deacylation can be depolymerized by the methods already described for obtaining low molecular weight and already mentioned in this claim. The depolymerization products can then be subjected to fractionation to obtain the desired heparosans of the invention.

Heparosans composed of a mixture of chains of molecular weight between 1500 and 15000 Da, characterized by a repeating structure of formula III:

[image]

in which R' represents in 0-80% of the disaccharide units, an acetyl group and in the remaining hydrogen atoms are new products and therefore also part of the invention.

Preferred heparosans of the invention are heparosans made from a mixture of chains of molecular weight between 1500 and 15000 Da, characterized by a repeating disaccharide structure of formula III in which the acetyl group (R') is present in a percentage below or equal to 60%.

Heparosan compounds containing at least 70% by weight of heparosan as described above also form part of the present invention.

These heparosans and heparosan compounds are intermediates applicable for obtaining heparosan-N,O-sulfate of the object of the present invention, but they can also be used for obtaining other products, for example products that undergo epimerization, due to obtaining D-glucuronic acid.

Before the partial N,O-sulfation step, heparosans can be converted to an organic base or quaternary ammonia salt. For the formation of the quaternary ammonium salt of heparosan, tetrabutylammonium is preferably used.

The stage of partial N,O-sulfation realized according to the procedure described in French patent no. 2 584 728 of 10.11.1987 (which corresponds to the application FR-85.10787) is carried out in an aprotypical polar solvent, such as dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide or acetonitrile or a mixture of these solvents, using, for example, a sulfur anhydride complex (sulfur trioxide: SO3) with an organic base, such as trimethylamine, triethylamine or pyridine. It can also be performed with chlorosulfonic acid in a pyridine solvent. Sulfur trioxide-pyridine complex is preferably used.

It is also possible to use other sulphating agents, mainly those given in E.E. Gilbert in Chemical Review, (1962), 62, 549-589. The N,O-sulfation reaction is usually carried out at a temperature between 0 and 100°C, preferably between 10 and 50°C for between six and 14 hours.

During the preparation process, at the end of the partial N,O-sulfation reaction, the heparosan-N,O-sulfate compound containing 70-100% heparosan-N,O-sulfate, the object of the present invention, is precipitated with the addition of sodium chloride until a 0.33M solution is obtained NaCl, and then an adequate amount of ethanol. The formed precipitate is taken in a 0.5 M NaCl solution. This solution is then neutralized. After adding an adequate amount of ethanol, the formed precipitate is taken with ultrapurified water and dialyzed against it, lyophilised and dried.

The stage of N,O-sulfation as well as the stages of purification of the heparosan-N,O-sulfate compound described in the previous paragraph can be repeated several times. The purification procedure is given as an example and is not exclusive of equivalent procedures.

This step of N,O-sulphation is preferably followed by a step of complete sulphation, usually carried out in an aqueous solvent, of a suitably basic pH, with a sulphating agent such as a complex of sulfur trioxide with an organic base, eg trimethylamine.

At the end of the complete sulfation stage, the heparosan-N,O-sulfate compound is precipitated after the addition of sodium chloride until a 0.5M solution of NaCl and ethanol is obtained. The formed precipitate is then re-dissolved in 0.5M NaCl solvent and re-precipitated with ethanol, taken up in ultrapure water, dialyzed against it, lyophilised and dried.

Enrichment of the compound heparosan-N,O-sulfate in heparosan-N,O-sulfate is carried out by the classic molecular mass fractionation techniques already mentioned,

It is interesting to perform this enrichment.

Heparosan-N,O-sulfates, the object of the present invention and compounds of heparosan-N,O-sulfate containing at least 70% of the new heparosan-N,O-sulfate, the object of the present invention are conveniently obtained by a process that includes the final N-sulfation reaction. E then represents, for repeating structures in formula (I), an acetyl or sulfate group.

It is also possible to obtain heparosan-N,O-sulfates, the object of the invention, by performing first N-sulfation followed by O-sulfation. This variant of the procedure is the application of the preferred method.

N-sulfation is realized using a sulfur trioxide complex with an organic alkali such as trimethylamine, triethylamine or pyridine. It can also be performed with chlorosulfonic acid in solution in pyridine. A sulfur trioxide complex with trimethylamine is suitably used and the N-sulfation reaction is carried out at a temperature between 20 and 80oC in an introduced alkaline environment. At the end of the N-sulfation reaction, the product thus obtained is precipitated by adding an appropriate amount of ethanol. The resulting precipitate is taken with ultrapure water, dialyzed against it, lyophilized and dried. The purification procedure is given as an example and is not exclusive of equivalent procedures. The stages of purification can be repeated several times.

According to the process of the invention, before the O-sulfation step, heparosan-N-sulfate is preferably converted into an organic alkali or quaternary ammonium salt. For the formation of quaternary ammonium salts, tetrabutylammonium is preferably used.

The O-sulfation reaction is carried out in formamide or another chemically equivalent solvent, using, for example, a complex of sulfur trioxide with an organic alkali, such as trimethylamine, triethylamine or pyridine. Sulfur trioxide-pyridine complex is preferably used. The O-sulfation reaction is usually carried out at a temperature between 10 and 50 oC.

Heparosan-N,O-sulfate is then precipitated by adding sodium chloride to the reaction medium until a 0.33M NaCl solution is obtained, followed by an appropriate amount of ethanol, as in the case of N,O-sulfation. Then the heparosan-N,O-sulfate compound is purified. the various steps are already described in detail, forward (ethanol precipitation, dialysis, etc.).

According to the invention, heparosan-N,O-sulfate preferably comprises at least 90 wt. % of molecular weight chains below 11000 Da. It is clear that this heparosan-N,O-sulfate contains at least 0.2 µmole/mg amino group NH2

The acetyl group is preferably present in a percentage less than or equal to 60% and it is clear that the degree of sulfation, expressed as a sulfate/carboxyl ratio, is, for example, between 1.5 and 3.0.

Preferred products of the invention are heparosan-N,O-sulfates made from chains of an average molecular weight of about 4000 to 7000 Da and a degree of sulfation, expressed as a sulfate/carboxyl ratio between 1.7 and 3, as well as heparosan-N,O-sulfates, N-deacetylated up to about 80% (the percentage of the acetyl group is present about 20%) and made up to at least 70 wt. % of chains with a molecular weight between 5000 and 7000 Da and a degree of sulfation of 1.8-2.5 or more heparosan-N,O-sulfates made of at least 70 wt. % of chains with molecular weights between 10,000 and 12,000 Da, N-acetylated up to about 80% (the percentage of the present acetyl group is about 20%) and a sulfation degree of 1.8-2.5 or more heparosan-N,O-sulfates, N-deacetylated up to about 40% (the percentage of the present acetyl group is about 60%) and made of at least 70 wt. % of chains with molecular weights between 6000 and 8000 Da and degree of sulfation 2.0-2.8.

Preferred heparosan-N,O-sulfates of the invention are more specifically:

- Heparosan-N,O-sulfates, N-acetylated up to about 80% (the percentage of the present acetyl group is about 20%) composed of at least 80 wt. % of molecular masses between 2300 and 7200 Da and degrees of sulfation from 1.8 to 2.5;

- Heparosan-N,O-sulfates, N-acetylated up to about 80% (the percentage of the present acetyl group is about 20%) composed of at least 80 wt. % of molecular masses between 3300 and 7700 Da and degree of sulfation from 1.8 to 2.5;

- Heparosan-N,O-sulfates, N-acetylated up to about 80% (the percentage of the present acetyl group is about 20%) composed of at least 80 wt. % of molecular masses between 6900 and 13500 Da and degree of sulfation from 1.8 to 2.5;

- Heparosan-N,O-sulfates, N-acetylated up to about 40% (the percentage of the present acetyl group is about 60%) composed of at least 80 wt. % molecular masses between 4000 and 10300 Da and degree of sulfation from 2.0 to 2.8.

Heparosan-N,O-sulfates are understood to mean all pharmaceutically acceptable salts. These salts are obtained by classical procedures described mainly for the preparation of heparin salts (US patent 4,168,377).

The procedure for obtaining heparosan-N,O-sulfate described above as well as the purification procedures allow obtaining heparosan-N,O-sulfate in the form of sodium salt. Starting from these salts, the applicable procedures used to obtain different salts of heparin or unsalified heparin can be obtained (L'Heparin, fabricatio structure, properties, analysis, J.P. Duclos (1984) p. 81-83, Masson Ed. France) for example other salts heparosan-N,O-sulfate, say heparosan-N,O-sulfates unsalified.

Heparosan-N,O-sulfates, the object of the subject invention, have interesting pharmacological and biochemical properties and quite surprisingly with the teachings of the prior art.

Mainly contrary to the sulfated products of antigen K5 described in patent EP-A-0 33 243, which have anti-angiogenic and anti-tumor activity with a suitable ratio of these activities to the ratio of anticoagulant properties, the heparosan-N,O-sulfates of the present invention have a good activity of regulating coagulation. This activity is superior to that of dermatin sulfate in various coagulation parameters and can be better compared to that of heparin.

More specifically, the dependence of the anti-IIa, ATIII or HCII activity of representative products of the invention was determined according to the procedures described by D. Dupouy et al in Thrombosis and Haemostasis (1988), 60, 2, 236-239, for heparin cofactor II (HCII) and M.L. Larsen et al in Thrombosis Research (1978), 13, 2, 285-288 for antithrombin (ATIII).

In both cases, the test consists in measuring the in vitro inhibitory effect of the studied product on purified thrombin (factor IIa) in the presence of HCII and ATIII purified in the dose of the amidolytic activity of thrombin towards the chromogenic substrate. As it has a stronger anti IIa CHII dependence, dermatan sulfate obtained according to the procedure described by H.W. Stuhisatz et al in "The Methodology of Connective Tissue Research" (1976) 137-146, is used as a reference product in the test to measure this activity, the results are expressed in mg of dermatin sulfate (DS) equivalent to the activity of 1mg of the studied product (mg DS eq. /mg).

The anti-Xa activity (Yin-Wessler value) of said representative products of the invention was measured by the method described by E.T. Yin et al in J. Lab. Clin. Honey. (1973), 81, 2, 298-310, while their global anticoagulant activity was measured according to the APTT test described by R.R. Proctor et al in Am. J. Clin. Path. (1961), 36, 212-219.

All treated products showed anti-IIa HCII dependence activity clearly better than that of dermatin sulfate. The dependence of the activity of anti-IIa ATIII and as Yin-Wessler, lower than those for heparins are shown to be better than those for dermatin-dermatin sulfate and can reach up to 60% of that for heparin. Their value in APTT is about 2 and about 20 times better than that of dermatin sulfate and can reach up to 60% of that of heparin.

Heparosan-N,O-sulfates of the invention therefore have a specific effect and extremely interesting anticoagulant activity. On the other hand, the small molecular masses of these products give them very interesting pharmacokinetic properties.

Heparosan-N,O-sulfates of the present invention are very slightly toxic; their toxicity is quite compatible with their use as medicines.

The invention also relates to pharmaceutical preparations that include as an active principle heparosan-N,O-sulfate, the object of the present invention, one of their salts or heparosan-N,O-sulfate compounds containing at least 70 wt. % of this heparosan-N,O-sulfate or one of its salts together with one or more suitable pharmaceutical carriers.

These pharmaceutical preparations are mainly used to treat, for the purpose of prevention or treatment, the difficulties of blood vessels such as atherosclerosis and arteriosclerosis and hypercoagulability conditions observed, for example, after surgical operations, tumor development or coagulation irregularities caused by the activities of bacteria, viruses or enzymes.

The dosage can vary widely as a function of the patient's age, weight and health status, the nature and severity of the addiction, and the method of administration.

This dosage involves taking one or more doses of about 1 mg to 1 g per day, preferably about 5 mg to 500 mg per day, eg 200 mg per day intravenously or subcutaneously at regular intervals or one dose per day of 200 to 1000 mg orally .

These doses can of course be adjusted for each patient in function of the described results and analysis of the type of previous preferences.

The invention is illustrated by the examples given below:

N-ACETYLHEPAROSANS

Example 1

Obtaining N-acetylheparosan mainly of low molecular weight (Procedure I)

1) Cultivation of the bacterial species Escherichia coli (K5) with the species Escherichia coli SEBR 3282 (deposited at the CNCM Institute Pasteur, Paris, France, under No. I-1013) and incubated with agitation for 2 hours at 37oC.

The resulting preculture is then transferred to an 18.5-liter fermenter containing 11 liters of agent A, the composition specified in Table I, below, and is incubated for four hours at 37oC and pH=7.4, at a partial oxygen pressure maintained at 40 mmHg by regulation air injection (up to 20 lit/min) and with mixing. Then glucose is added by continuously introducing a sterile solution containing 600 g/l of glucose at a rate of 250 ml/h for eight hours.

Cultivation continues under the same conditions of temperature, pH and partial pressure of oxygen for 10 hours after stopping the addition of glucose. Control of the DO (ó=600 nm) of the culture medium allows confirmation that there is no increase in biomass during the last twelve hours of cultivation.

The culture broth mixture is then cooled to 25oC, then filtered through a membrane with a porosity of 0.22 µm. About 12 liters of filtrate containing N-acetylheparosan is thus obtained.

Table I

Composition and preparation of means A and means B

RESOURCE A

Medium A is obtained by mixing the three sterile solutions given below:

Solution no. 1

Dissolve in 700 ml of ultra-pure water in the following order:

Complexon: N/Tris-(hydroxymethyl)methyl)methyl/glycine

(Tricin commercialized by Fluka®) 360 mg

FeSO4·7H20 280 mg

CaCl2·2H2O 6.7 mg

MgCl2 ·6H20 1280 mg

K2SO4 500 mg

KCl 5000 mg

Casein hydrolyzate (main source of amino acids) Hy Case SF

(commercialized by Sheffield®) 25000 mg

Yeast extract (commercialized by Difco® 18000 mg

Solution of oligo elements (see table II) 1 ml

Antifoaming agent Struktol J 673 (commercialized by

Schill et Seilacher®) a few drops of Pipette Pasteur

The pH is adjusted to 7.4 with KOH solution (d=1.38) and made up to

850 ml of ultra-pure water. The middle is autoclaved for 45 min

at 120o C.

Solution no. 2

In about 40 ml of ultrapure water, dissolve 5 g of K2HPO4, after which it is adjusted to 50 ml with the same solvent. The resulting solution is filtered through a 0.2 µm porosity filter.

Solution no. 3

Dissolve 20.7 g of glucose in an adequate amount of ultrapure water and adjust the volume to 100 ml with the same solution. It is autoclaved at 110o C for 30 minutes.

ASSET B

Preparation of agent B is identical to that of agent A with the difference that 20 g of buffer pH=7.2 (3-morpholinopropanesulfonic acid) is added after the addition of antifoam.

Table II

Obtaining a solution of oligo elements used in obtaining agent A and agent B

Dissolve in 800 ml of ultra-purified water in the following order:

H3BO3 500 mg

Na2MoO4 ·2H20 1930 mg

CoCl2 ·6H20 11850 mg

CuSO4 ·5H20 25 mg

ZnSO4 ·7H20 2000 mg

AlCl3 ·7H20 2410 mg

Add 100 ml of hydrochloric acid with a density of 1.19 and make up to 1000 ml with ultra-purified water.

2) Isolation and purification of N-acetylheparosan, mostly of low molecular weight:

Stage a - Ethanol precipitation

About 48 l of 95% ethanol was added to the filtrate and the mixture was allowed to settle and decanted at 4o C for eight hours. The supernatant is eliminated by suction, then it is centrifuged and the centrifugation residue is taken up in about 1 liter of ultrapurified water.

Stage b - Dialysis

The solution obtained in step a is dialyzed for 24 hours previously placed in a NOJAX 40 hose carrying a cellulose-based membrane with a porosity of 24 Å against ultra-purified water (1 volume of solution/6 volumes of water, repeated after 2, 8 and 16 hours). This operation allows the elimination of small molecules present in the culture medium, such as salts, sugars, amino acids, oligonucleotides and oligopeptides.

Stage c - Precipitation, dehydration and drying

0.5 M NaCl and 4 volumes of ethanol are added to one volume of the dialyzed solution. Allow to form a precipitate for 5 minutes at room temperature. It is centrifuged at 5000 g for 20 min. The centrifugation residue is taken up in ethanol, the resulting suspension is stirred and left to recover for one hour at room temperature. The centrifugation and suspension operations are repeated. Centrifuge again at 5000 g for 20 min. After centrifugation, the precipitate obtained is dried in an oven under vacuum at 40°C for 24 hours.

Stage d - Shredding

The dried sediment from centrifugation is pulverized in a mortar under anhydrous conditions.

Stage e - Anion exchange chromatography

The crushed centrifugation sediment is taken into a buffer called buffer D, composed of Tris-HCl 20 mM pH=7.5 for 100 ml/g. The resulting solution is chromatographed on a strong anion exchange column containing a mesh agarose matrix with quaternary ammonium groups ("Q-Sepharose fast flow", Pharmacija®) previously equilibrated with buffer D, with 50 ml of gel for 1 g of powder. The gel is washed with a sufficient amount of buffer D to return to the alkaline line of UV detection at 214 nm, and then with a pH=3.5 solution composed of: =.5M NaCl and 25 nM piperazine. The eluate is neutralized with a 5N NaOH solution.

Stage f - Precipitation, dehydration, drying and shredding

The operations described in steps c and d are repeated, forward, without the addition of sodium chloride.

The N-acetylheparosan obtained as a result of step f was named lot A.

One variant of the purification procedure consists in sequential execution of stages a, c, b, d, e and f. The N-acetylheparosan thus obtained was called lot B.

3) Characterization of N-acetylheparosan obtained as a result of different stages of purification:

Nuclear magnetic resonance (NMR) spectrum

Proton and carbon 13C NMR spectra were compared with those of N-acetylheparosan described by W.E. Vann (Eur. J. Biochem. (1982), 116, 59-364).

Study of the spectra obtained with N-acetylheparosan, lot A and lot B, confirm the chemical identity of the product N-acetylheparosan described by W. E. Vann. It consists of polymer chains made of repeating structures beta-D-glucoronyl-1,4-alpha-N-acetyl-D-glucosaminyl-(1,4).

Determination of molecular mass distribution by exclusion chromatography

Molecular mass distribution was determined by HPLC exclusion under the following conditions:

- A column composed of silica balls with a diameter of 10 µm and a porosity of 250 Å

- Eluent: aqueous solution of 0.5 M sodium sulfate

- Flow: 1 ml/min

- UV detection at ó=205 nm

Calibration was performed using oligosaccharide derivatives of heparin, with the following molecular weights: 1324, 2436, 3411, 3996, 4535, 4995, 5365, 6150, 6671, 7542, 8655, 10088, 11561, 12950, 14809, 17374 and 226 Da.

Considering these gamma standards, only molecular masses between 934 and 56703 Da were taken into account. It is accepted that the detected optical density is proportional to the amount of N-acetylheparosan. However, the precision of the procedure decreases exponentially for high molecular masses and mostly greater than 20000 Da.

The exclusion chromatography elution profile for lot A is given in Fig. 1. When examining Fig. 1, it is noted that the distribution is polydisperse and has a huge peak at about 4700 Da. A mass fraction equal to at least 70% of lot A has a mass between 1700 and 8000 Da.

A very similar chromatogram is obtained when lot B is analyzed. The main peak of the distribution is at about 5000 Da. A mass fraction equal to at least 70% for lot B has a molecular weight between 1500 and 8000 Da.

Monitoring the distribution of molecular masses by polyacrylamide gel electrophoresis

These samples are analyzed so that the migration end includes bromophenol blue in electrophoresis in Tris-borate buffer in a 15% polyacrylamide gel obtained by polymerization of a mixture of 29/l acrylamide and N,N'-methylene-bis-acrylamide. Migration is performed under a current of 40 mA for about four hours on a gel 18 cm long until the end of migration marker exits. The gel is then stained in alkaline blue and then in silver, according to the technique of S. Pelkonen et al (J. Bact. (1985), 170, 6, 2646) specifically in polysaccharide acids.

This electrophoresis analysis is performed on the partially purified product obtained after the implementation of stage a and the purified product lot A or lot B from the final stage, in order to confirm the absence of a significant modification of the molecular weight distribution of N-acetylheparosan during purification.

Content of uronic acids

The amount of uronic acid per unit mass of the purified product (lot A or lot B) obtained from the final stage was determined colorimetrically according to the procedure described by T. Bitter (Analytical Biochemistry, (1962), 4, 330-334). This dosage procedure is based on the reaction of glycosaminoglycans with carbazole in an acidic warm environment, which causes a pink coloration proportional to the amount of released uronic acid.

For lot A, the partially purified product obtained in the realization of stage d and the purified product obtained in the realization of the final stage f, respectively, the content of uronic acid is 1.3 and 2.1 µmol/mg.

For lot B, the purified product from the realization of the final stage and the content of uronic acid is 2.1 µmol/mg.

Spectrophotometry in the visible and ultra-violet region.

The purified product (lot A) is dissolved in ultra-purified water and the obtained solution (C= 1 mg/ml) is placed in a cuvette with a 1 cm optical path. The absorption spectrum is registered between 200 and 500 nm.

The obtained spectrum confirms, especially on the basis of absorption at 256 nm, that lot A contains at least 1% AND.

Total protein content

The "protein assay" equipment commercialized by BIORAD was used to determine total proteins. The dosing procedure is based on the fact that the position of the maximum absorbance signal of Coomassie g-250 acid solution shifts brilliantly blue from 465 to 595 nm, when proteins are fixed in it (Reisner et al. Anal. Buochem (1975), 64, 509).

The total protein content of lot A is below 1.5%.

Content of free amino groups (NH2)

This determination is performed according to the procedure described by Zensaku Yosizawa et al. in Biochemica et Biphysica Acta (1967), 141, 358-365.

The NH2 content parameter (expressed in µmol/mg) is an indicator of the amount of beta-D-glucuronyl-1,4-alpha-N-acetyl-D-glucosaminyl-(1,4) deacetylated units and contaminants containing a free amino group.

Lot A and lot B each have an NH2 content of 0.05 µmol/mg. The NH2/glucuronic acid ratio = 0.05/2.1 is lower than 2.5%. There is therefore (in moles) for 100 units of beta-D-glucuronyl-1,4-alpha-N-acetyl deacetylated type.

Example 2

Obtaining N-acetylheparosan mainly of low molecular weight (process II)

1) Cultivation of the bacterial species Escherichia coli (K5) and separation of the filtrate containing N-acetylheparosan.

Cultivation of Escherichia coli SEBR 3282 and separation of the filtrate containing N-acetylheparosan is realized according to the procedure described in example 1.

2) Isolation and purification of N-acetylheparosan, mostly of low molecular weight

Stage a - Dialysis

375 ml of filtrate is subjected to dialysis according to the procedure described in example 1, /2). Isolation and purification of N-acetylheparosan, mainly of low molecular weight, Stage b/. After dialysis, about 1020 ml of purified solution is obtained.

Stage b - Purification in an acidic environment

An adequate amount of 5N Hcl solution is added to the dialyzed solution to obtain pH=3.5. By centrifugation, the formed precipitate is eliminated, then the solution is acidified with the same acid (5M HCl) to obtain ph = 1.8. A sediment can be formed which eliminates centrifugation. The solution is then neutralized using a 5M NaOH solution.

Stage c - Precipitation, dehydration and drying

An adequate amount of sodium chloride is added to the neutralized solution to obtain a 0.5M NaCl solution, then 4 volumes of ethanol are added. Allow to form a precipitate for 5 minutes at ambient temperature. Centrifuge at 5000 g for 20 minutes. The centrifugation residue is taken up in ethanol and the resulting suspension is stirred and allowed to recover for one hour at room temperature. Centrifugation operations are repeated and suspensions are created. Centrifuge again at 5000 g for 20 minutes. The resulting centrifugation residue is dried in a dryer under vacuum at 40oC for 24 hours.

Stage d - Alkaline hydrolysis and dialysis

The product obtained in the previous stage, after drying, dissolves up to 2.5% in 0.25N NaOH solution. The solution thus obtained is kept at 50oC for two hours. It is then neutralized using a 5N HCl solution and the solution containing the polysaccharide is then subjected to dialysis according to the procedure described in example 1,/2). Isolation and purification of N-acetylheparosan, mainly of low molecular weight, Stage b/. After dialysis, about 990 ml of solution is obtained.

Stage e - Anion exchange chromatography

Adequate amounts of piperazine, EDTA, and triton X-100 (Prolabo®) are added to the dialyzed solution to provide concentrations of 25 mM piperazine, 2 mM EDTA, and 0.2% triton X-100, respectively. The pH is then adjusted to 3.5 using 5N HCl solution. This solution was applied to a 400 ml Q-Sepharose Fast Flow column equilibrated with piperazine buffer containing 25 mM piperazine and 2 mM EDTA and 0.2 mM triton X-100 (pH=3.5). It is washed with piperazine buffer and eluted with 0.5 M NaCl solution and then precipitated with 4 volumes of ethanol. It is dried under vacuum at 40oC. About 9.85 g of N-acetylheparosan is thus obtained.

Stage f - Exclusion Chromatography

4 g of the product obtained in the previous stage is dissolved in 60 ml of a buffer solution composed of tris-HCl 20 mM pH=7.5 and NaCl 1 M, then passed through a 200 ml octyl Sepharose® column previously equilibrated with the same buffer. 4 volumes of ethanol are added to the fraction not retained. The resulting precipitate is washed and dried at 40oC under vacuum.

Thus, 3.90 g of N-acetylheparosan is obtained.

3) Characterization of N-acetylheparosan obtained in various stages of purification

The spectrum of nuclear magnetic resonance NMR

Studies of proton and carbon 13C NMR spectra obtained with this N-acetylheparosan confirm the chemical identity of the N-acetylheparosan product described by W. F. Vann (Eur. J. Biochem. (1981) 116, 59-364).

Determination of molecular mass distribution by exclusion chromatography

The molecular mass distribution was determined by HPLC exclusion according to the procedure used to determine the molecular mass distribution of N-acetylheparosan described in example 1. The mass fraction of at least 86% of the chains that make up the lot obtained in example 2 has a molecular mass between 1500 and 15000 Da.

Content of uronic acids

The N-acetylheparosan obtained in step e has a uronic acid content of 1.94 µmol/mg.

Spectrophotometry in the visible and ultraviolet regions

The obtained spectrum confirms that the obtained N-acetylheparosan contains at least 1% AND.

Total protein content

The total protein content of this lot of N-acetylheparosan is below 1%.

Content of free amino groups (NH2)

The NH2 content is below 0.1 µmol/mg.

Example 3

Obtaining N-acetylheparosan mainly of low molecular weight (process III)

1) Cultivation of Escherichia coli SEBR 3282 is carried out according to the procedure described in example 1. About 12 liters of culture containing N-acetylheparosan is obtained.

2) Preliminary purification

Stage a - Centrifugation

At the end of cultivation, the resulting suspension (12 liters) is centrifuged at 8000 rpm (say between 11000 and 14000 g) for 20 min.

Stage b - Contact with an alkaline solution

After centrifugation, the precipitate is eliminated and the supernatant is contacted with 0.1 M NaCH solution for about one hour.

Stage c - Filtering

The solution obtained in the previous stage is subjected to filtration on a 3M ® series 300 polypropylene filter.

Stage d - Concentrating on a membrane of a certain cut-off

The filtrate obtained in step c is concentrated on an Amicon® hollow fiber pellet of cutoff 10000 Da or equivalent. Thus, a solution enriched in low molecular weight N-acetylheparosan is obtained.

Stage e - Dialysis

A solution enriched in low molecular weight N-acetylheparosan is dialyzed against ultra-purified water, always on the Amicon® system, with a very large dilution factor, above 10,000.

3) Isolation and purification of N-acetylheparosan, mostly of low molecular weight

It is done as indicated in example (1/2) Isolation and purification of N-acetylheparosan mainly of low molecular weight, Stage c - Stage f) and N-acetylheparosan with characteristics similar to lot A or as indicated in example (2) is obtained /2) isolation and purification of N-acetylheparosan, mostly of low molecular weight, Stage a - Stage f.

HEPAROSAN-N,O-SULFATE

Example 4

Chemical modifications of N-acetylheparosan mainly of low molecular weight obtained in example 1

1) Chemical modifications lot B

Stage a - Partial deacetylation

Dissolve 500 mg of lot B in 10 ml of 1M NaOH solution. The solution is brought to 50 oC and left to react at this temperature for eight hours with stirring. The solution is then neutralized with a 2M HCl solution, dialyzed against ultrapurified water and then lyophilised.

Stage b - Formation of tetrabutylammonium salt

The previous lyophilizate is taken in 20 ml of ultra-purified water. The resulting solution is applied to an ion-exchange column based on mesh polystyrene with divinylbenzene (Dowex 50 W 8 Dow Dhemical®), previously equilibrated in an acidic environment in order to regenerate the acidic form of the product. The solution is then mixed with 0.4 ml of 40% tetrabutylammonium solution. Liphilized.

Stage c - Partial N,O-sulfation

Dissolve 421 mg of the salt obtained in the previous step in 35 ml of dimethylformamide and add 3.71 g of sulfur trioxidepyridine complex (commercialized by Aldrich® under ref. S755-6). Allow to react with stirring for six hours at room temperature. NaCl is added to one volume of the reaction medium to obtain a concentration of 0.33 M NaCl in the solution, and then 2 volumes of ethanol. Leave to form a precipitate. Centrifuge and eliminate the supernatant. The centrifugation sediment is taken into a 0.5 M NaCl solution, which is neutralized. Then 2 volumes of ethanol are added. Allow to form a precipitate, centrifuge and collect the centrifugation precipitate with ultrapurified water. It is dialyzed against ultrapurified water and lyophilized.

The operations described in the preceding paragraph are repeated together.

The obtained lyphilizate has the following characteristics:

* uronic acid content: 1.11 µmol/mg

* content of free NH2: 0.01 µmol/mg

* sulfation content: 2.64 per disaccharide unit.

The content of uronic acid and NH2 were measured as described in example 1.

Sulfation content, also called the sulfate/carboxyl ratio, is the average number of sulfate groups per carboxyl group, measured by the conductometric method of sulfate group content of a carboxyl group as described by B. Casu et al, in Carbohydrate Research, (1975) 39, 168-176 .

2) Chemical modifications of lot A

Lot A is divided into fractional aliquots, called lot A1, lot A2 and lot A3.

Stage a - Partial deacetylation and gel filtration

Lot A1, A2 and lot A3 are treated as described in step a, for lot B with the difference that only lot A1 is dialysed and lyophilised. Lot A1, lot A2 and lot A3 are then fractionated by gel filtration (also called gel permeation chromatography or exclusion chromatography) under the following conditions:

Ball carrier cross section 25-75 µm based on reticulated N,N'-methylene-bis-acrylamide (Sephakril S 300 HR commercialized by Pharmacia®). Used eluent: 0.5 M NaCl solution.

Mixtures of corresponding fractions were made in Kav from 0.46 to 1 for heparosan originating from lot A1, from 0.43 to 1 for heparosan originating from lot A2 and from 0.43 to 0.64 for heparosan originating from lot A3. These fractions were then called "heparosan gel filters".

Kav is a coefficient commonly used in size exclusion chromatography and allows for fractional repeatability in size exclusion chromatography. It is defined by the formula:

Ve - Vt

Kav = ---------

Vo - Vt

where Ve = eluate volume of the considered fraction

Vo = exclusion volume

Vt = total gel volume

The molecular mass distribution of heparosans originating from log A1 immediately after partial deacylation as well as for gel-filtered heparosans lot A1, lot A2 and lot A3 was determined by exclusion chromatography according to the technique described in example 1.

The obtained elution profiles before and after gel filtration for the heparosan obtained from lot A1 are shown respectively in Fig. 2 and 3. Some results of said elution profiles are given in Table III, below, in which PMo represents the molecular weight so that a mass fraction of 1% of the product has a molecular mass greater than PMo, PM1 represents a molecular mass such that a mass fraction of 10% of the product has a molecular mass above PM1, PM3 represents a molecular mass such that an average of 10% of the product has a molecular mass of PM3 and PM2 represents a molecular mass that corresponds to the absorption maximum.

Table III

Distribution of molecular weights of heparosan originating from lot A1 (which is not gel filtered) and gel filtered heparosan originating from lot A1, A2 and lot A3

[image]

By comparing figures 2 and 3, it can be concluded that the filtering gel allows the elimination of a small fraction of the high molecular mass of heparosan.

This result is clearly shown in Table III, where a strong decrease in PMo is noted in the next gel filtration operation. Heparosan originating from each lot contains at least 90 wt. % of chains with a molecular weight below 7000 Da.

Heparosan gel filtered originating from lot A1 and lot A2 were then treated as described in example 1/2) Isolation and purification of N-acetylheparosan mainly of low molecular weight, Step c/.

Heparosan gel filtered from lot A3 does not settle but is dialyzed against ultra-purified water.

The content of uronic acid and free amino groups is measured as described in Example 1.

The obtained results are given in Table IV, below. The residual acetyl group content is estimated by considering that there are as many glucoronyl groups as glucosaminyl groups.

The content of deacetylation is calculated as the ratio of the content of free amino groups to the content of uronic acid.

Nuclear magnetic resonance spectrum

The study of the proton nuclear magnetic resonance spectrum obtained on gel-filtered heparosan from lot A3 allows the conclusion that the product has the expected structure.

The deacetylation content calculated by integration was found to be 44%. This value is close to the one calculated using the ratio of the content of free amino groups to the content of uronic acid (41%).

Table IV

Characteristics of gel-filtered heparosans originating from lot A1, lot A2 and lot A3

[image]

Stage b - Partial n,O-sulfation

Gel filtered heparosans originating from lots A1, A2 and A3 deacetylated in the manner previously described for lot B (stage c - partial N,O-sulfation), with the difference that the operations described in the first paragraph are not repeated.

For heparosan originating from lot A3, after the first precipitation, the precipitate is taken in ultrapurified water, dialyzed against ultrapurified water and left in solution.

The characteristics of the N,O-sulfated products are given in Table V, below

Table V

Characteristics of heparosan-N,O-sulfate originating from lots A1, A2 and A3 from the reaction of partial N,O-sulfation

[image]

It is noted that after the N,O-sulfation reaction, the content of residual NH2 groups (0.10 µmol/mg) is higher than for purified N-acetylheparosan (0.05 µmol/mg). Sulfation is therefore not complete on the nitrogen atom.

Stage c - Complete sulphation

It is mixed in a volume of 20 ml of ultra-purified water per gram of N,O-sulfate product used, 1 part by mass of N,O-sulfate product, 1 part by mass of sodium bicarbonate, 1 part by mass of sulfur trioxide-trimethylamine complex and left to react at 55oC with stirring for 20 hours. Then the reaction mixture is diluted (dilution factor 10), the conductivity of the resulting solution is adjusted to that of a 0.5 M NaCl solution. Then precipitation is carried out by adding 2 volumes of ethanol, after centrifugation, the precipitates are taken in 0.5 M NaCl solution and the precipitation is repeated by adding 2 volumes of ethanol. After taking in ultra-purified water and dialysis against ultra-purified water, the products are lyophilized and dried at 40oC under vacuum.

Nuclear magnetic resonance spectrum

Study of the NMR13C spectrum of heparosan-N,O-sulfate originating from lot A3, shows that for this compound the alcohol in position 6 of the glucosaminyl group is completely in the form of a sulfate ester.

Certain characteristics of the obtained heparosan-N,O-sulfates determined according to the methods described above are given in Table VI, below:

Table VI

Characteristics of the obtained products originating from complete N-sulfation lot A1, A2 and A3.

[image]

It is found that the content of the residual NH2 group is small (0.02 µmol/mg) and lower than that of purified N-acetylheparosan (0.05 µmol/mg for lot A and lot B) as well as that of heparosan-N,O- sulfate originating from partial N,O-sulfation (specified in Table V). This shows the spontaneous nature of the N-sulfation reaction.

The molecular weight distribution of the products obtained after N-sulfation was analyzed by the technique described in example 1.

The obtained elution profile for the product originating from lot A1 is shown in Fig. 4. Very similar elution profiles are obtained for the products originating from lot A2 and A3.

Certain results of the mentioned elution profiles are given in Table VII. The definitions of PMo, PM1 and PM3 are identical as indicated for Table III.

Table VII

Distribution of molecular masses of products obtained in complete N-sulfation for lot A1, A2 and A3.

[image]

Heparosan-N,O-sulfate of each lot contains at least 90% of the mass of chains with molecular masses smaller than 10,000 Da.

In fact, lots A1, A2 and A3 contain 80% of chains with molecular weights between 2600 and 9000 Da, 3400 and 10000 Da and 1800 and 7600 Da, respectively.

Example 5

Chemical modifications of N-acetylheparosan mainly of low molecular weight originating from example 2. Obtaining heparosan-N,O-sulfate, 80% of N-deacetylated derivatives.

N-acetylheparosan was used to obtain, according to the procedure described in example 2. The uronic acid content of the product is 2.12 µmol/mg.

Determination of molecular mass distribution is performed by exclusion chromatography, according to the procedure described in example 1. One mass fraction of at least 87.5% has chains of molecular masses between 1500 and 15000 Da. The maximum peak of the distribution is at around 4900 Da. This lot of N-acetylheparosan is called lot C.

Stage a - Partial deacetylation

Dissolve 2.5 g of lot C in 50 ml of 2N NaOH solution. The solution is brought to 50 oC and left to react at this temperature for eight hours with stirring. The pH is adjusted to 8 using 2N HCl solution, dialyzed against ultrapurified water, then lyophilized.

1.96 g of product is thus obtained.

N-deacetylation content

The content of free amino groups (NH2) indicates that N-acetylheparosan is N-deacetylated 80%.

Stage b - N-sulfation

The previous lyophilisate is taken in 70 ml of water and 2.5 g of Na2CO3 and 2.5 g of sulfur trioxide-trimethylamine complex are added. Leave to react for 20 hours at 55 oC.

The conductivity of the reaction solution is adjusted to that of a 0.5 M NaCl solution by adding demineralized water. Precipitate with 4 volumes of ethanol. It is centrifuged.

The centrifugation sediment is taken with ultra-purified water, this solution is centrifuged according to the already described procedure (example 1) and lyophilized. About 2 g of product is obtained.

Stage c - Formation of tetrabutylammonium salt

The lyophilizate obtained in the previous stage is converted into tetrabutylammonium salt according to the procedure described in example 4/1). Chemical modifications lot B, stage b/.

2.7 g of salt is obtained.

Stage d - O-sulfation

Dissolve 2.680 g of the salt obtained in the previous step in 196 ml of formamide and add 11.27 g of the sulfur trioxide-pyridine complex. Leave to react with stirring for six hours at 80oC. 1 volume of 2M NaCl solution per 5 volumes of reaction solution is added and the pH is adjusted to 7 using NaOH solution. Reprecipitate with 2 volumes of ethanol, centrifuge and redissolve the precipitate in 0.5 M NaCl solution. Then 2 volumes of ethanol are added. Leave to form a precipitate, centrifuge and take the centrifugation precipitate with 80 ml of ultra-purified water. Add 20 ml of 2 M NaCl solution and then 4 volumes of ethanol. Leave to form a precipitate and centrifuge.

Stage e - Gel filtration

The product obtained in stage d is taken with ultra-purified water and then fractionated by gel filtration according to the conditions described in example 4/2) Chemical modifications lot A, stage a/. The molecular weights of the chains that make up heparosan-N,O-sulfate are determined by exclusion chromatography, according to the procedure described in example 1. Part of the fractions containing chains of molecular masses between 1500 and 12000 Da/solution C(A) and the other part, the fractions, are regrouped which contain heparosan-N,O-sulfate composed of chains of molecular masses from 2000 to 30000 Da /solution C(M)/.

4 volumes of ethanol are added to the solution C(A). Allow to form a precipitate, centrifuge and collect the centrifugation precipitate with ultrapurified water. It is dialyzed against ultra-purified water and lyophilized.

1.8 g of product is obtained.

Heparosan-N,O-sulfate thus obtained was named lot C1.

Certain characteristics of this heparosan-N,O-sulfate, determined according to the procedures previously described, are given in Table VIII.

Table VIII

Characteristics of the product corresponding to lot C1

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The molecular weight distribution of the product was evaluated using the technique described in the example. Certain results of said elution profile are given in Table IX.

Table IX

Molecular mass distribution of the product corresponding to lot C1

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The definitions of PMo, PM1, PM2 and PM3 are identical to those indicated for Table III.

Heparosan-N,O-sulfate lot C1 contains 95% mass chains that range between 1500 and 15000 Da.

The spectrum of nuclear magnetic resonance NMR

NMR spectra of protons and carbon 13C were obtained with heparosan-N,O-sulfate lot C1 (spectra were obtained with AMX 500, solvent D2O).

Study of NMR spectra of carbon 13C protons confirms the expected structure of the product. It is heparosan-N,O-sulfate.

Studying the ratio of the intensity of sugar protons to acetyl protons of the proton spectrum leads to a deacetylation content of 84%. This value is close to the one calculated using the ratio of the content of free amino groups to the content of uronic acid (80%). The carbon 13C NMR spectrum confirms mainly that glucosamine is N-sulfated almost completely. Glucuronic acid is not sulfated in position 2 and 3.

Stage f - Gel filtration

4 volumes of ethanol are added to the C(M) solution. Leave to form a precipitate, centrifuge and take the precipitate in ultra-purified water, dialyze and lyophilize.

The lyophilisate is then dissolved in a 0.5 M NaCl solution and fractionated by gel filtration according to the conditions defined in stage e.

A regrouped group of fractions containing a chain of molecular masses between 1300 and 21000 Da /solution C(B)/ and another part of masses between 14000 and 37000 Da /solution C(C)/.

These two solutions are treated as indicated above for solution C(A) and obtained after lyophilization with solution C(B), lot C2 and with solution C(C), lot C3.

Table X indicates certain results of said elution profiles for the products lot C2 and lot C3.

Table X

Distribution of molecular masses of products corresponding to lot C2 and C3

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The definitions of PMo, PM1, PM2 and PM3 are identical as indicated for Table III.

Heparosan-N,O-sulfate corresponding to lot C2 contains about 99 wt. % of chains whose molecular weight is between 1500 and 15000 Da, and heparosan-N,O-sulfate corresponding to lot C3 contains about 73 wt. % of chains whose molecular weight is between 1500 and 15000 Da.

Example 6

Chemical modifications of N-acetylheparosan mainly of low molecular weight from example 2. Obtaining heparosan-N,O-sulfate, a 40% deacetylated derivative whose sulfation content is 2.6.

N-acetylheparosan used as starting material is obtained according to the procedure described in example 2. The uronic acid content of this product is 1.96 µmol/mg. This lot is called lot D.

Stage a - Partial deacetylation

Dissolve 3.7 g of lot D in 74 ml of 1 M NaOH and treat according to the procedure described in example 5 (Step a). Deacetylation is performed under nitrogen.

After lyophilization, 2.1 g of product is obtained.

Deacetylation content

The content of free amino groups (NH2) of the product obtained after lyophilization indicates that N-acetylheparosan is N-deacetylated 40%.

Stage b - N-sulfation

The product obtained in the previous step is treated with 3.7 g of sulfur trioxide-trimethylamine complex, in the presence of 3.7 g of Na2CO3 and according to the procedure described in example 5 (Step b).

About 3 g of heparosan-N-sulfate is obtained.

Stage c - Formation of tetrabutylammonium salt

About 2 g of heparosan-N-sulfate obtained in the previous step is formed according to the procedure described in example 5 (Step c) into tetrabutyl ammonium salt.

2.99 g of lyphilizate is obtained.

Stage d - O-sulfation

2.98 g of the salt obtained in the previous step are reacted with 14 g of the sulfur trioxide-trimethylamine complex, according to the procedure described in example 5 (Step d), then precipitation and purification are carried out in the same way as described in example 5 (Step d).

2.8 g of product is obtained.

Stage e - Gel filtration

The product obtained in the previous stage is fractionated by gel filtration using the method and material described in example 4, /2) Chemical modifications lot A, Stage a/.

The molecular weight distribution of heparosan-N,O-sulfate fractions is determined by exclusion chromatography according to the technique described in example 1. The fractions that make up heparosan-N,O-sulfates, most of whose chains have a molecular weight between 1500 and 15000 Da, are isolated.

These fractions are then concentrated and dialyzed against ultrapurified water. Then precipitation is performed by adding 5 volumes of ethanol, centrifuged, the precipitate is dissolved in 0.5 M NaCl solution and the precipitation operation is repeated.

The purified product thus obtained is subjected to a second fractionation using the protocol mentioned at the beginning of this stage. The molecular weight distribution of the fractions is determined by exclusion chromatography according to the already described procedure. Fractions containing products with molecular masses between 4000 and 8000 Da are collected.

The fractions are dialyzed and the product is then precipitated by the addition of ethanol. The precipitate is collected and dissolved in 0.5 M NaCl solution. The solution thus obtained is dialyzed against ultra-purified water and then lyophilized.

About 1 g of heparosan-N,O-sulfate called lot D1 is obtained.

The characteristics and molecular weight distribution of this heparosan-N,O-sulfate are given respectively in Table XI and Table XII.

Table XI

Characteristics of the product corresponding to lot D1

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Table XII

Molecular weight distribution of the product corresponding to lot D1

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Heparosan-N,O-sulfate lot D1 contains 80 wt. % of chains between 4047 and 10305 Yes and about 98.5 wt. % of chains having a molecular weight between 1500 and 14600 Da. Figure 5 presents the elution profile for this heparosan-N,O-sulfate.

Nuclear magnetic resonance (NMR) spectrum

NMR spectra of protons and carbon 13C were obtained for heparosan-N,O-sulfate lot D1 (spectra realized on AMX, solvent D2O).

Study of proton and carbon 13C NMR spectra confirms the expected structure of the product. It is heparosan-N,O-sulfate.

The carbon 13C NMR spectrum confirms mainly that each amino group of glucosamine is not in free form. Glucosamine is fully sulfated at the C6 position and, in contrast, all hydroxy groups of glucuronic acid are not sulfated.

Example 7

Chemical modifications of N-acetylheparosan, mainly of low molecular weight, originating from example 2. Obtaining heparosan-N,O-sulfate, 40% deacetylated derivative with degree of sulfation 3.

The lot of N-acetylheparosan obtained according to the procedure described in example 2 is used as the starting material. This lot is called lot E and has a uronic acid content of 2 µmol/mg.

The molecular weight distribution of this N-acetylheparosan was evaluated by the technique described in example 1. This N-acetylheparosan contains about 75 wt.% of chains between 1500 and 15000 Da and the average mass of these chains is about 10900 Da. The molecular weight of most chains is 5135 Da.

Stage a - Partial deacetylation

N-Acetylheparosan was 40% N-deacetylated according to the procedure mentioned in Example 6 (Step a). To perform this deacetylation, 2.5 g of starting material is used, which is dissolved in 50 ml of 1 M NaOH. At the end of the reaction, the pH of the medium is adjusted to 6 with HCl and then concentrated.

N-deacetylation content

The content of free amino groups (NH2) indicates that N-acetylheparosan was 40% N-deacetylated.

Stage b - Gel filtration

A Sephacryl S 300 HR column (5 cm x 100 cm) equilibrated with 0.5 M NaCl is used.

The distribution of molecular masses of heparosan contained in different fractions is determined by exclusion chromatography. Fractions containing chains of molecular masses from 6000 to 20000 Da are regrouped, concentrated in a rotary evaporator, 4 volumes of ethanol are precipitated and the formed precipitate is dried.

Thus, 1 g of product is obtained.

Stage c - Formation of tetrabutylammonium salt and partial N,O-sulfation

For the formation of the tetrabutylammonium salt, 300 mg of the product obtained in the previous stage is used and the procedure described in example 4/1) Chemical modifications of lot B, Stage b/ is applied. 490 mg of salt is obtained, which is dissolved in 30 ml of formamide. Then partial N,O-sulfation is performed according to the procedure described in example 4/1) Chemical modifications lot B, Stage c - first paragraph/.

Starting from 490 mg of the salt that reacts with 2.25 g of the sulfur trioxide pyridine complex, 406 mg of heparosan-N,O-sulfate is obtained.

Stage d - Complete N-sulfation and gel filtration

372 mg of the product obtained in the previous step is dissolved in 15 ml of 5% Na2CO3 solution and treated with 372 mg of sulfur trioxide-trimethylamine complex, according to the procedure described in example 4/2) Chemical modifications Lot A, Step c.

The product obtained after complete N-sulphation is then fractionated by gel filtration using a Sephacryl S 300 column (2.5 cm x 100 cm) and applying the procedure already described.

Molecular weight fractions from 6000 to 20000 To be regrouped, concentrated on a rotary evaporator, extensively dialyzed against cold ultrapurified water and lyophilized.

Thus, 260 mg of heparosan-N,O-sulfate called lot E1 is obtained.

The characteristics of this product are given in table XIII.

Table IX indicates the distribution of molecular weights of the chains that make up lot E1.

This heparosan-N,O-sulfate contains about 75 wt. % of chains with molecular weights between 1500 and 15000 Da and about 65 wt. % chains of molecular masses between 7700 and 15000 Da.

Table XIII

Characteristics of the product corresponding to lot E1

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Table XIV

Molecular weight distribution of the product corresponding to lot E1

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The definitions of PMo, PM1, PM2 and PM3 are identical to those indicated in Table II.

Example 8

Obtaining 2 lots of heparosan-N,O-sulfate, 80% N-deacetylated derivative with sulfation content of 2.25 and 2.4.

The starting material used, lot F, is a lot of N-acetylheparosan obtained according to the procedure described in example 2.

Determination of the distribution of the molecular chains that make up the compound is realized by exclusion chromatography according to the procedure described in example 1.

Examination of the profile allows the conclusion that lot F contains 92 wt. % of chains whose molecular weight is between 1500 and 15000 Da.

The main peak is at around 4800 Da. One mass fraction of lot F of at least 80% has a molecular weight between 2400 and 10000 Da.

The uronic acid content of this product is 2.4 µmol/mg.

Stage a - Partial deacetylation

Proceed as described in Example 5 (Step a) using 2.5 g lot F and 50 ml 2 M NaOH.

1.6 g of 80% deacetylated product is obtained.

The percentage of N-deacetylation is determined by the content of free amino groups.

Stage b and c - N-sulfation and formation of tetrabutylammonium salt

These two stages are the same as stages b and c described in example 5.

4.7 g of tetrabutylammonium salt is obtained.

Stage d - O-sulfation

Dissolve 2.9 g of the salt obtained above in 290 ml of formamide and add 17.4 g of the sulfur trioxide-pyridine complex.

It is done as described in step d of example 5.

The precipitate obtained after the second centrifugation is dissolved in ultrapurified water, then dialyzed against ultrapurified water and lyophilized.

Thus, 3.68 g of product is obtained.

Stage e - Gel filtration

The product obtained in the previous step is introduced into a 0.5 M NaCl solution and then applied to a Sephacryl S 300 HR column equilibrated with 0.5 M NaCl. The effluent is collected using a fraction collector.

Fractions with a molecular weight between 1400 and 10000 Da are regrouped and concentrated on a rotary evaporator. They are precipitated using 4 volumes of ethanol, centrifuged, the precipitate is taken in ultra-purified water, the resulting solution is dialyzed, widely opposite to ultra-purified water, lyophilized and dried.

Thus, 1.6 g of heparosan-N,O-sulfate called lot F1 is obtained.

Fractions corresponding to molecular masses between 5,000 and 35,000 are collected, concentrated on a rotary evaporator and 4 volumes of ethanol are added to the resulting solution, centrifuged, the precipitate is taken up in water, the resulting solution is dialyzed against ultra-purified water and lyophilized. The lyophilizate is subjected to fractionation again according to the procedure identical to the beginning of this stage.

The molecular mass distribution of the different fractions is determined by exclusion chromatography according to the procedure described in example 1. The fractions containing heparosan-N,O-sulfates with a molecular mass between 2000 and 26000 Da are regrouped. They are precipitated with 4 volumes of ethanol, centrifuged and the precipitate is redissolved in distilled water, dialyzed and lyophilized. Thus, 0.6 g of heparosan-N,O-sulfate called lot F2 is obtained.

The characteristics of lot F1 and F2 are given in table XV. The results of the mentioned elution profiles are given in Table XVI.

Table XV

Characteristics of heparosan-N,O-sulfate for lot F1 and F2

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Table XVI

Distribution of molecular masses of the corresponding products Lot F2 and F1

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The definitions of PMo, PM1, PM2 and PM3 are identical to those given in Table III.

Heparosan-N,O-sulfate lot F1 contains about 99 wt. % of chains with molecular weights between 1500 and 15000 Yes, that lot F2 also contains about 84.6 wt. % of chains with molecular weights of 1500 and 15000 Da and about 70 wt. % chains of molecular masses from 6900 to 13500 Da.

The spectrum of nuclear magnetic resonance NMR

NMR spectra of protons and carbon 13C are obtained from heparosan-N,O-sulfate lot F1 (spectra realized on AMX 500 solvent D2O).

Study of the 13C spectrum confirms that the glucosamine motif is N-sulfated. Glucuronic acid in the majority of disaccharide structures is not O-sulfated in position 2 and 3.

OBTAINING A

Obtaining N-acetylheparosan mainly of high molecular weight (Procedure I)

1) Cultivation of the Escherichia coli species (K5) and separation of the filtrate containing N-acetylheparosan

400 ml of medium D, composition specified in Table XVII, below, is inoculated with Escherichia coli SEBR 3282 and incubated with stirring for two hours at 37oC.

The resulting pre-culture is then transferred to an 18.5-liter fermenter containing 11 liters of medium C, the composition of which is also specified in Table XVII, below, and is incubated for 6.5 hours at 37oC and pH=7.2, with a partial oxygen pressure maintained to 40 mmHg by regulating the air injection (up to 20 lit/min) and from mixing. Glycerol is then added by continuously introducing a sterile solution containing 500 g/l of glycerol at 18 g/h for 16-17 hours.

Cultivation continues under the same conditions of temperature, pH and oxygen partial pressure until the glycerol is almost completely consumed. Monitoring the DO (= 600 nm) of the culture suspension after the end of glycerol addition, shows a stationary state or slight lysis until the cultivation stops after 28-30 hours of aging in the fermenter.

The soup mixture is then cooled to 25 oC, then filtered through a membrane with a porosity of 0.22 µm. Thus, 12 liters of filtrate containing N-acetylheparosan, mostly of high molecular weight, are obtained.

Table XVII

Composition and preparation of medium C and medium D

Middle C

Dissolve in 900 ml of ultra-purified water in the following order:

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The pH is adjusted to 7.2 with concentrated potash with a density of 1.38 and adjusted to 1000 ml with ultrapurified water. Filter the sterilant on a 0.2 µm membrane.

Glycerol solution

Dissolve 50 g of glycerol in an adequate amount of ultra-purified water and adjust the volume to 1000 ml with the same solvent. Filtration of the sterilant is performed on a 0.2 µm membrane.

The antifoam used during fermentation is Struktol J 673 (SChill et Seilacher®).

Middle D

The preparation of medium D is the same as for medium C, with the difference that buffer (pH = 7.2) and 3-morpholinepropanesulfonic acid are added after the addition of antifoam.

2) Isolation and purification of N-acetylheparosan, mostly of high molecular weight

N-acetylheparosan of mostly high molecular weight is isolated and purified according to the procedure described in example 2 /2) Isolation and purification of N-acetylheparosan of mostly low molecular weight, Stage a - Stage f/.

3) Characterization of the obtained N-acetylheparosan

Determination of molecular mass distribution by exclusion chromatography

According to the ethanols currently used, an approximate distribution of molecular masses was made.

The N-acetylheparosan obtained for this preparation is a compound of chains with molecular weights between 20,000 and 500,000 Da and an average molecular weight of about 100,000-200,000 Da.

Content of uronic acids

The uronic acid content of the purified product from the final stage is 2.2 µmol/mg.

Spectrophotometry in the ultraviolet and visible range.

The resulting spectrum confirms that the lot contains at least 0.5% ADN.

Total protein content

The total protein content is below 0.5%.

Content of free amino groups NH2

The NH2 content is below 0.1 µmol/mg.

OBTAINING B

Obtaining N-acetylheparosan mainly of high molecular mass (Procedure II)

1) Cultivation of Escherichia coli (K5)

Cultivation of Escherichia coli SEBR 3282 is carried out according to the procedure described in Preparation A. About 12 liters of culture containing N-acetylheparosan of mostly high molecular weight is obtained.

2) Preliminary purification

It is done as described in Example 3 /2) Preliminary purification using in stage D a fiche of hollow fibers Amicon® cut start 30000 Da or equivalent.

3) Isolation and purification of N-acetylheparosan, mostly of high molecular weight

It is done as indicated in example 3 /3) Isolation and purification of N-acetylheparosan, mostly of low molecular weight

OBTAINING C

Chemical modifications of N-acetylheparosan mainly of high molecular weight originating from Obtaining A

As a starting material for various chemical modifications, N-acetylheparosan called lot G1 obtained according to the procedure described in Preparation A is used.

The uronic acid content of this product is 2.41 µmol/mg.

Stage a - Partial deacetylation

Dissolve 1.9 g of lot G1 in 38.5 ml of NaOH. The solution is brought to 50oC and left to react for eight hours under nitrogen.

Then the pH is adjusted to 8.25 by adding 2 M HCl. It is dialyzed against ultra-purified water and lyophilized.

Thus, 1.6 g of product is obtained.

Deacetylation content

The content of free amino groups (NH2) shows that N-acetylheparosan was 80% n-deacetylated.

Stage b - N-sulfation

Dissolve 1.3 g of the product obtained in the previous stage in 57 ml of ultrapurified water, add 1.9 g of Na2CO3 and 1.9 g of the sulfur trioxide-trimethylamine complex and keep it at 55oC for 20 hours. The conductivity of the solution is then adjusted to that of a 0.5 M NaCl solution by the addition of demineralized water and precipitated with 4 volumes of ethanol. It is centrifuged and the precipitate is taken with a 0.5 M NaCl solution, precipitated with 4 volumes of ethanol and centrifuged.

The precipitate is dissolved in ultrapurified water and dialyzed with ultrapurified water according to the procedure described in Example 1, then lyophilized.

1.809 g of heparosan-N-sulfate is thus obtained.

Stage c - Formation of tetrabutylammonium salt

Dissolve 800 g of the product obtained in the previous step in 100 ml of water. This solution is applied to a Dowex 50X8 ion exchange column and is carried out as described in example 4 /1) Chemical modifications of lot B, Stage b/.

After lyophilization, about 1.3 g of salt is obtained.

Stage d - O-sulfation

Dissolve the previously obtained salt in 80 ml of formamide, add 5.6 g of the sulfur trioxide-pyridine complex. It is left to react for six hours at 30oC, then 16 ml of 2 M NaCl is added. It is brought to pH=7 and precipitated with two volumes of ethanol. The precipitate is taken up with 0.5 M NaCl solution, reprecipitated with 2 volumes of ethanol, dialyzed against ultrapurified water and concentrated on a rotary evaporator.

Stage e - Gel filtration

The concentrated solution obtained in the previous step is fractionated by gel filtration using a Sephacryl S 300 HR column and 0.5 M NaCl solution as eluent.

Fractions corresponding to molecular masses between 10,000 and 500,000 Da are isolated. 2 volumes of ethanol are added and then concentrated. Then the conductivity is adjusted to that of a 0.5 M NaCl solution by adding water.

Fractionation by gel filtration is repeated and fractions containing heparosan-N,O-sulfate composed of chains of molecular masses of an average of 100,000 to 200,000 Da are collected, as well as fractions containing heparosan-N,O-sulfate made of chains of molecular masses of about 50,000 and from about 12,000 Yes.

To each of these three fractions, 5 volumes of ethanol are added, they are subjected to dialysis against ultra-purified water and after dialysis the solutions obtained are lyophilized.

Thus, 3 lots of heparosan-N,O-sulfate are obtained:

- lot G1 is heparosan-N,O-sulfate composed of chains with an average molecular weight of 100,000 to 100,000 Da. 0.14 g of this lot is isolated.

- lot G2 is heparosan-N,O-sulfate composed of chains with an average molecular weight of about 50,000 Da. 0.250 g of this heparosan-N,O-sulfate is isolated.

- lot G3 is heparosan-N,O-sulfate composed of chains with an average molecular weight of 12,000 Da. About 0.100 g of the last lot is isolated.

The characteristics of these three lots of heparosan-N,O-sulfate described in this example are given in Table XVIII.

Table XVIII

Characteristics of heparosan-N,O-sulfate for lot G1, G2, G3

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Claims (30)

1. N,O-sulfated heparosans characterized by the fact that they contain chains or mixtures of chains with a molecular weight between 1500 and 15000 Da, with a repeating disaccharide structure of formula I: [image] where E represents an acetyl group in 0 to 80% of the disaccharide units of the mentioned N,O-sulfated heparosans, a sulfate group and optionally a hydrogen atom in the remaining disaccharide units, G represents a hydrogen atom and a sulfate group, the degree of sulfation expressed as the ratio of sulfate to carbonyl, which is 1.5 and 3.0, and pharmaceutically acceptable salts of the mentioned N,O-sulfated heparosans. 2. N,O-sulfated heparosans according to claim 1, characterized in that E represents an acetyl group and a sulfate group. 3. N,O-sulfated heparosan according to claim 1, characterized in that it contains chains or mixtures of chains having a molecular weight between 1500 and 15000 Da, with a repeating disaccharide structure of the formula I: [image] where E represents an acetyl group in 0 to 80% of the disaccharide units of the aforementioned N,O-sulfated heparosan, a sulfate group, and optionally a hydrogen atom in the remaining disaccharide units, G represents a hydrogen atom and a sulfate group, the degree of sulfation expressed as the ratio of sulfate to carboxyl, which is 1.5 and 3.0, the two ends of the reductive and non-reductive chains of the mentioned N,O-sulfated heparosans are immersion units, sulfated or not, glucosamines sulfated or not, N-acetyl glucosamines, sulfated or not, and pharmaceutically acceptable salts of said N,O-sulfated heparosan. 4. N,O-sulfated heparosan according to one of claims 1 to 3, characterized in that E represents an acetyl group in 0 to 60% of the dihydrin units of said N,O-sulfated heparosan. 5. N,O-sulfated heparosan according to one of claims 1 to 4, characterized in that it contains at least 90 mass percent of chains with a molecular weight of less than 11,000 Da. 6. N,O-sulfated heparosan according to claim 1, characterized in that it contains less than 0.2 µmol/mg amino group (NH2). 7. N,O-sulfated heparosan according to one of claims 1 to 4, characterized in that it has an average molecular weight of 4000 Da to 7000 Da and a degree of sulfation expressed as a ratio of sulfate to carboxyl between 1.7 and 3. 8. N,O- sulfated heparosan according to claim 1, characterized in that it contains at least 70 wt. % of chains with a molecular weight between 5000 and 7000 Da and the degree of sulfation expressed as the ratio of sulfate to carboxyl, which is 1.8 and 2.5, and E represents the acetyl group in 0 to 20% of the disaccharide units of the mentioned N,O-sulfated heparosan. 9. N,O- sulfated heparosan according to claim 1, characterized in that it contains at least 70 wt. % of chains with a molecular weight between 10,000 and 12,000 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl between 1.8 and 2.5, and E represents an acetyl group in 0 to 20% of the disaccharide units of the mentioned N,O-sulfated heparosan. 10. N,O- sulfated heparosan according to claim 1, characterized in that it contains at least 70 wt. % of chains with a molecular weight between 6000 and 8000 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl between 2.0 and 2.8, and E represents an acetyl group in 0 to 20% of the disaccharide units of the mentioned N,O-sulfated heparosan. 11. N,O-sulfated heparosan according to claim 1, characterized by the fact that it contains at least 80 mass percent of chains with a molecular weight between 2300 and 7200 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl is between 1.8 and 2.5, and E represents an acetyl group in 0 to 20% of the disaccharide units of said N,O-sulfated heparosan. 12. N,O- sulfated heparosan according to claim 1, indicated by the fact that it contains at least 80 mass percent of chains with a molecular weight between 3300 and 7700 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl is between 1.8 and 2.5, and E represents an acetyl group in 0 to 20% of the disaccharide units of said N,O-sulfated heparosan. 13. N,O- sulfated heparosan according to claim 1, characterized by the fact that it contains at least 70 mass percent of chains with a molecular weight between 6900 and 13500 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl is between 1.8 and 2.5, and E represents an acetyl group in 0 to 20% of the disaccharide units of said N,O-sulfated heparosan. 14. N,O- sulfated heparosan according to claim 1, characterized in that it contains at least 80 mass percent of chains with a molecular weight between 4000 and 10300 Da, the degree of sulfation expressed as a ratio of sulfate to carboxyl is between 2.0 and 2.8, and E represents an acetyl group in 0 to 60% of the disaccharide units of the mentioned N,O-sulfated heparosan. 15. Preparation of N,O-sulfated heparosan, characterized in that it contains at least 70 mass percent of N,O-sulfated heparosan according to one of claims 1-4. 16. Process for obtaining a preparation containing 70% to 100% of N,O-sulfated heparosan according to claim 1, characterized in that it contains the sequence of the following phases: phase a) cultivation of Escherichia coli colony (K5), phase b) isolation and purification, with or without preliminary purification of N-acetylheparosan to obtain a preparation containing 70% to 100% of N-acetylheparosan comprising chains or mixtures of chains with a molecular weight between 1500 and 15000 Da, with a repeating disaccharide structure of the formula II: [image] phase c) partial deacetylation of this composition of N-acetylheparosan, to obtain a preparation containing 70% to 100% of heparosan comprising chains or mixtures of chains with a molecular weight between 1500 and 15000 Da, with a repeating disaccharide structure of formula III: [image] in which R' represents acetyl in 0 to 80% of the disaccharide units and a sight atom in the remaining disaccharide units. phase d) - or partial N-O-sulfation of this heparosan composition - or partial N,O-sulfation of this heparosan composition, followed by a phase of complete N-sulfation, - either complete or partial N-sulfation followed by a phase of complete or partial O-sulfation, and optionally includes one or more stages of fractionation of molecular masses, carried out at the end of stage a, b, c or d. 17. The method according to claim 16, characterized in that the Escherichia coli (K5) SEBR 3282 colony (deposited at the CNCM Pasteur institute, Paris, France, under number I-1013) or a mutant of that colony, arose spontaneously or was induced. 18. The method according to claim 16, characterized in that the cultivation of the species Escherichia coli (K5) continues for at least two hours after stopping the growth of the biomass. 19. The method according to claim 16, characterized in that the isolation and purification of N-acetylheparosan comprises at least one phase of precipitation with alcohol and at least one phase of ion exchange chromatography. 20. The process according to claim 16, characterized in that the isolation and purification of N-acetylheparosan is carried out using a process that includes the following stages: phase a1: precipitation with ethanol, phase b1: dialysis, phase c1: precipitation with ethanol, then dehydration and drying, phase d1: purification by anion exchange chromatography, phase e1: ethanol precipitation of the eluate obtained in phase d1, dehydration, drying and grinding in which phase a1 or c1 can be omitted. 21. The process according to claim 16, characterized in that the isolation and purification of N-acetylheparosan is carried out using a process that includes the following stages: phase a1': dialysis phase b1': purification in an acidic environment, elimination of impurities insoluble in aqueous solutions pH=3.5 and pH=1.8 phase c1': precipitation with ethanol, then dehydration and drying, phase d1': alkaline hydrolysis and dialysis phase e1': purification by exclusion chromatography. 22. The method according to claim 16, characterized in that the N-acetylheparosan obtained by cultivating a colony of Escherichia coli (K5) is subjected to preliminary purification by a process that includes the following stages before isolation and purification. phase a"1: centrifugation of the suspension obtained at the end of cultivation phase b"1: bringing the supernatant into contact with an alkaline solution phase c"1: preliminary filtering, phase d"1: concentration on the membrane of a predetermined cut-off threshold phase e"1: dialysis, of which phase e"1 can be omitted. 23. The method according to claim 16, characterized in that after the phase of partial N,O-sulfation comes complete N-sulfation, which is carried out with the use of a complex of sulfuric anhydride with an organic base in an aqueous solution of basic pH. 24. The method according to claim 16, characterized in that molecular mass fractionation is carried out after N-sulfation. 25. Pharmaceutical preparation, characterized in that it contains, as an active basic component, N,O-sulfated heparosan according to one of claims 1-14 bound with or in a mixture with a pharmaceutically acceptable internal excipient. 26. Pharmaceutical preparation, characterized by the fact that as an active basic component it contains the composition of N,O-sulfated heparosan according to claim 15 bound with or in a mixture with a pharmaceutically acceptable internal excipient. 27. Pharmaceutical preparation according to one of claims 25 and 26, characterized in that it can be used to regulate coagulation. 28. Heparosans comprising a mixture of chains with a molecular weight between 1500 and 15000 Da, characterized in that they have a repeating disaccharide structure of formula II: [image] 29. Heparosans according to claim 28, characterized in that R' represents an acetyl group in 0 to 60% of the disaccharide units of said heparosans. 30. Heparosan preparation, characterized in that it contains at least 70 mass percent of heparosan according to claim 28 or 29.