NO323409B1 - New oligosaccharides, their preparation, pharmaceutical preparations containing the oligosaccharides and their use. - Google Patents

Abstract

The invention concerns oligosaccharides of formula (I), their mixtures, their diastereomers, methods for preparing them and pharmaceutical compositions containing them.

Description

The present invention relates to oligosaccharides with the formula:

their mixtures, their diastereoisomers, their preparation pharmaceutical preparations containing the oligosaccharides, as well as their use.

Sulfated disaccharides with a reducing end having a 1,6-anhydro structure are described by H.P. Wessel in "J. Carbohydrate Chemistry", 11(8), 1039-1052 (1992); however, no pharmacological activity is mentioned.

Sulfated trisaccharides with a 1,6-anhydro moiety are likewise described in EP 84999 by Y. Ichikawa et al. in "Carbohydr. Res.", 141, 273-282 (1985) as an intermediate for the production of higher oligosaccharides. These trisaccharides have a low anti-Xa activity.

In formula (I), n is an integer from 0 to 25, Ri, R3, R4 and R5 are the same or different and mean a hydrogen atom or a SOsM residue, R2 and R$ are the same or different mean a hydrogen atom or a SO3M - or COCH3 residue and M is sodium, calcium, magnesium or potassium.

These oligosaccharides thus have an even number of saccharides.

In formula (I), R 4 is preferably a hydrogen atom.

Preferably, n is an integer from 0 to 10 and in particular 0 to 6, and most preferably 1 to 6.

The invention relates to a method for producing oligosaccharides of formula (I) characterized by reacting an alkali metal or quaternary ammonium hydroxide with oligosaccharides of the formula:

there

ner an integer from 0 to 25,

Ri, R3, R4 and Rs are the same or different and mean a hydrogen atom or a SC^M residue', R2 and Re are the same or different and mean a hydrogen atom, or a SO3M or COCH3 residue and

M is sodium, calcium, magnesium or potassium, or a mixture of these,

and isolates the oligosaccharides or their mixtures.

This reaction takes place in an aqueous medium at a temperature from 40 to 80 °C and at a pH value from 0 to 13.

Sodium, potassium, lithium or cesium hydroxide can be mentioned as alkali metal hydroxide that can be used.

Tetrabutylammonium hydroxide can be mentioned as quaternary ammonium hydroxide that can be used.

The amount of alkali metal or quaternary ammonium hydroxide must be sufficient so that the pH value in the reaction mixture remains stable throughout the duration of the reaction. It is thus necessary during the entire reaction to continuously add alkali metal or quaternary ammonium hydroxide.

Alkali metal or quaternary ammonium hydroxide is preferably present in the form of an aqueous 1 to 5% solution.

Preferably, the reaction takes place at a temperature of 60 to 70 °C.

Preferably, the pH value of the reaction is 11 to 12.5.

The reaction is stopped by acidifying the reaction medium, for example by adding an acidic resin such as the resin Amberlite IR120® (Fluka).

The oligosaccharides of formula (I) can optionally be purified by permeation on a gel of the polyacrylamide-agarose type such as the one marketed under the name Ultrogel ACA202®

(Biosepra) according to the protocol described below for the separation of intermediate oligosaccharides of formula (II). The oligosaccharides of formula (I) where n is 0 or 1 can also optionally be purified on an alumina column where a mixture of water and ethanol is used as eluent.

The intermediate oligosaccharides of formula (II) and their mixtures can be obtained by separation by chromatography on gel of a mixture of the oligosaccharides (III), obtained by enzymatic depolymerization of heparin or basic depolymerization of the benzyl ester of heparin or a hemisynthesis benzyl ester of heparin.

This chromatography takes place on columns filled with gel of the polyacrylamide-agarose type such as that marketed under the brand name Ultrogel ACA202® (Biosepra). A column battery of polyacrylamide agarose gel is preferably used. The number of columns used is adapted as a function of the volume, the gel and the oligosaccharides to be separated. The mixture is eluted with a solution containing a phosphate buffer and sodium chloride. Preferably, the phosphate buffer solution is a 0.02 mol/l solution of NaH2PO4:Na2HPO4 (pH 7) containing 0.1 mol/l sodium chloride. The detection of the different fractions is realized by UV spectrometry (254 nm) and ion spectrometry (IBF). The fractions thus obtained can then optionally be purified, for example by SAX chromatography (strong anion exchange) according to methods well known to those skilled in the art and in particular according to the methods described by K.G. Rice and R.J. Linhardt in "Carbohydrate Research", 190, 219-233 (1989); by A. Lamkjær, S.H. Hansen and P.B. Østergaard in "Carbohydrate Research", 266,37-52 (1995) as well as in WO 90/01501 (example 2). The fractions are then lyophilized and then desalted on a column filled with a gel, for example a Sephadex G10® column (Pharmacia Biochemicals).

When the purification is not carried out by SAX chromatography, the lyophilisates can possibly be purified by simple precipitation or fractionated according to well-known methods for the person skilled in the art and in particular according to the method described in FR 2548672. In general, one works according to the following protocol: This lyophilized fraction to be purified is dissolved at 25 °C in around 10 volumes of distilled water. By adding methanol or ethanol, the desired oligosaccharide is precipitated by controlling its enrichment by HPLC. The addition of methanol or ethanol is determined as a function of the purity and the desired yield of the relevant oligosaccharide. Furthermore, this operation can be carried out in several successive steps from the lyophilisate starting solution. For this purpose, an insolubilizing agent (methanol or ethanol) is added in small portions and the resulting precipitate is isolated after each addition. The thus obtained precipitates are analyzed by HPLC. According to the desired purity and yield, the adequate precipitate fractions are collected.

For the intermediate products of formula (II) where n = 0.1 or 2, these are preferably obtained from a mixture (III), obtained by enzymatic depolymerization.

This depolymerization takes place with the help of heparin I (EC 4.2.2.7) in a phosphate buffer solution with pH 7 in the presence of sodium chloride and BSA (bovine serum albumin), at a temperature between 10 and 18 °C, preferably 15 °C, for a period of time between 8 and 10 days and preferably 9 days. The depolymerization is stopped by heating the reaction medium to 100 °C for 2 minutes and the mixture is recovered by lyophilization. It is preferred to use 7 IU (international units) of heparinase I for 25 g of heparin. The phosphate buffer solution generally comprises 0.05 mol/l NaH2PO4:Na2HP04 (pH 7) in the presence of 0.1 mol/l sodium chloride. The BSA concentration is generally 2%.

For the intermediate products of formula (II) where n = 0, 1, 2, 3 or 4, it is preferred to proceed from a mixture (III) obtained by depolymerization of a benzyl ester of heparin.

The benzyl ester of heparin can be prepared according to the methods described in US 5 389 618, EP 40 144 and FR 2 548 672. The degree of esterification is preferably between 50 and 100%. Preferably, it lies between 70 and 90%.

The depolymerisation takes place in an aqueous medium using an alkali metal hydroxide (lithium, sodium, potassium or cesium hydroxide) or a quaternary ammonium hydroxide (for example tetrabutylammonium hydroxide), preferably at a molarity between 0.1 and 0.2 mol/l, at a temperature between 40 and 80 °C and preferably for 5 to 120 minutes. In a preferred working mode, one works for 5 to 15 minutes at a temperature between 60 and 70 °C with a sodium hydroxide solution of 0.15 mol/l. The depolymerisation reaction is stopped by neutralization by adding an acid such as acetic acid. After adding 10% by weight of sodium acetate, the oligosaccharide mixture is precipitated by adding methanol, preferably 2 volumes per 1 volume of reaction medium, and the whole is filtered.

According to a preferred aspect of the invention, the oligosaccharide mixture obtained after chemical depolymerization, in the form of an aqueous solution, is enriched by ultrafiltration on membranes with a nominally suitable cut-off threshold (type Pellicon of regenerated cellulose, marketed by Millipore); the type of membrane is adapted as a function of the type of enriched oligosaccharides to be recovered. For the oligosaccharides (II) where n = 0, a membrane with a nominal cut-off threshold of 1 kDa is used, for the oligosaccharides (II) where n = 1, a 1 kDa membrane or 3 kDa membrane is used, for the oligosaccharides (II ) where n = 2, a 1 kDa membrane is used, for the oligosaccharides (ii) where n = 3 or 4, an S kDa membrane is used. During this operation, the permeate is recovered and the retentate is discarded. Thus, the enriched product fraction may represent 50 to 10% of the starting mixture of oligosaccharides, while preserving at least 80% of the desired oligosaccharide.

For the intermediate products of formula (II) where n = 0 to 25, it is preferred to proceed from a mixture (III) which is obtained by depolymerization of a benzyl ester of sulfated hemi-synthesis polysaccharide. The benzyl ester of sulfated hemisynthetic polysaccharide is produced from sulfated hemisynthetic polysaccharides obtained from polysaccharide K5 and according to the methods described in WO 94/29352 and WO 96/14425. The esterification, depolymerization and recovery conditions are the same as described above for the benzyl ester of heparin.

In all the previous processes, the starting heparin can be of porcine, ovine, caprine or bovine origin and can originate from the mucus, lungs or skin of the animals. Preferably, a porcine or ovine mucus heparin or a bovine lung heparin is used, most preferably heparin from porcine mucus.

The oligosaccharides of formula (I) exhibit anti-inflammatory properties and can thus be used for the prevention or therapy of diseases associated with an inflammatory process which implies the production of cytotoxic substances such as nitrogen monoxide (NO), the inducible form of which is released in particular by neutrophils or macrophages when these migrate and are activated in a tissue. The activation, migration, adhesion and infiltration of neutrophils occurs in ischemic tissue zones as a result of an occlusion or a spasm in an artery that vascularizes this tissue. These ischaemias can occur either at the cerebral level (cerebral vascular accident) or in the myocardium (myocardial infarction) or in the lower limbs (so-called peripheral ischaemias). The oligosaccharides of formula (I) can also be used for prevention and/or therapy of neurodegenerative diseases in which cerebral inflammation plays a destructive role that can lead to death, among which can be mentioned cerebral ischaemia, cardiac ischaemia (myocardial infarction), peripheral ischaemia, traumatism in the central nervous system and in particular cranial, spinal or cranio-spinal trauma, plaque sclerosis, neuropathic pain and peripheral neuropathies, motor neurone diseases such as amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's chorea, and certain forms of osteoarthritis, in particular with articular location.

The invention provides a pharmaceutical preparation characterized in that it contains as active ingredient at least one oligosaccharide according to the invention.

The invention also relates to the use of oligosaccharides of formula (I) according to the invention for the production of a drug for use in the prevention or therapy of diseases associated with an inflammatory process which includes the production of nitric oxide (NO).

The anti-inflammatory activity of these products has been demonstrated in vivo in a test on the production of NOx (nitrite and nitrate) induced by lipopolysaccharide (LPS) originating from E. coli according to the protocol described by M. Yamashita et eel. in "Eur. J. Pharmacol.", 338.2, 151-158 (1997) or by J.E. Shellito et al. in "Am. J. Respir. Cell Mol. Biol.", 13,1,45-53 (1995).

CD1 male mice (Charles River, 25-35 g) are injected at TO, by intravenous bolus, 0.5 mg/kg oligosaccharide, at T+15 minutes, subcutaneously, 1 or 2 mg/kg oligosaccharide. At T+30 minutes, 100 mg/kg lipopolysaccharide (LPS) from E. coli (Sigma L3129, serotype 0127:B8) is administered. At T+3 hours, inject again, subcutaneously, 1 or 2 mg/kg oligosaccharide. At T+5 hours and 30 minutes, a blood sample is taken by puncturing the eye and the NOx (nitrite and nitrate) concentrations in plasma are determined by colorometric methods according to Griess after reduction of nitrate to nitrite with nitrate reductase in the following way: 12 µl plasma sample is mixed with 88 µl deionized water and incubated in the dark for 1 hour at ambient temperature with 40 µl phosphate buffer (0.3IM, pH 7.5), 20 µl p-NADPH (reduced nicotinamide adenine dinucleotide phosphate) (0.86 mM), 20 µl FDA (flavin adenine dinucleotide) (0.11 mM) and 20 µl nitrate reductase (2 U/ml) (Boehringer Mannheim). 10 ul of ZnSO 4 (IM) is added to precipitate the protein and after mixing the samples are centrifuged at 20,000 g for 5 minutes. Finally, 50 µl of supernatant is incubated for 10 minutes at ambient temperature with 100 µl of Griess reagent (1% sulfanilamide in a mixture of hypophosphoric acid and 0.1% naphthyethylenediamine in deionized water (v/v)). The optical density is read at 540 nm with a microplate spectrophotometer and each point is determined twice. KNO3 and NaNC>2 are used as standards for the colorimetric method.

In this test, the oligosaccharides according to the invention inhibit more than 50% of the formation of NOx.

Among the oligosaccharides of formula (I) which are preferred, particular mention should be made of those oligosaccharides where: n is equal to 0, Ri and Rg are a SOjNa residue and M is sodium and the mixture of these

diastereoisomers.

n is equal to 1, Ri, R2, R3, R$ and R<$ means a SOsNa residue, R» means a hydrogen atom and M

is sodium, as well as the mixture of these diastereomers.

n equals 2, Ri, R2, R3, R5 and Re means a SOsNa residue, R4 means a hydrogen atom and M

is sodium and the mixture of these diastereoisomers.

n is equal to 2, Ri, R2, R3 and Re means a SOsNa residue, R5 means a hydrogen or a SO3Na residue, R4 means a hydrogen atom and M is sodium and the mixture of these diastereoisomers (the derivative 1,6-anhydro AIs- Is-IIs).

The following examples are representative of the preparation of the oligosaccharides of formula (I) and their intermediates.

In the examples, the abbreviations have the following meanings:

Als: (4-deoxy-2-0-sulfo-α-L-threo-hex-enopyranosyluronic acid)-(l-»-4)-2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, tetrasodium salt, or also AUAp2S-(1 ->4)-α-D-GlcNp2S6S

Is: (2-sulfo-α-L-isopyranosyluronic acid)-(l->4)-2-deoxy-2-sulfoamino-6-0-sulfo-α-D-glucopyranose, tetrasodium salt, or also α-L-IdoAp2S-(l-»4 )-ct-D-GlcNp2S6S

Ils: (α-L-idopyranosyluronic acid)-(1-»4)-2-deoxy-2-sulphamo-6-0-sulfo-α-D-gluco-pyranose, trisodium salt, or also cc-L-Ido-Ap-1-» 4)-α-D-GlcNp2S6S

His: (2-sulfo-α-I^idopyranosyluric acid)-(1^4)-2-deoxy-2-sulfoammo-α-D-gluco^pyranose, trisodium salt, or also α-L-IdoAp2S-(l->4)-α-D-GlcNp2S

IdoAp: idopyranosyluronic acid

GlcNp: 2-deoxy-2-aminoglucopyranose

AUap: 4-deoxy-α-L-threo-hex-enopyranosyluronic acid

S: sulfate

EXAMPLES OF PREPARATION OF MIXTURES OF FORMULA (II)

Example A - Preparation of oligosaccharides of formula (II) where n = 0.1 and 2

with enzymatic depolymerization and separation

25 g of heparin are dissolved in 250 ml of a phosphate buffer solution containing 0.05 mol/l NaH2PO4:Na2HP04 (pH 7), 0.2 mol/l sodium chloride and 2% BSA. 7IU heparinase I (EC 4.2.2.2.7) is added to the mixture and the resulting solution is cooled to 15 °C and kept at this temperature for the entire duration of the depolymerisation reaction. The course of the reaction is followed by samples of aliquots and analyzes by gel permeation chromatography. After around 9 hours, the enzymatic reaction is purified by heating the reaction medium to 100 °C for 10 minutes. The cooled mixture is then lyophilized. In this way, a mixture of oligosaccharides (III) is obtained.

The resulting mixture of oligosaccharides (III) is then chromatographed according to the following method: The chromatography takes place on columns filled with polyacrylamide-agarose gel known under the name Ultrogel AC A202 and the mixture is eluted with a solution containing a phosphate buffer (0.02 mol/ l NaH2P04:Na2HP04) pH 7 and 0.1 mol/l sodium chloride. The detection takes place by UV spectrometry (254 nm) and ion spectrometry (IBF). The products can optionally be purified by SAX chromatography or by fractional precipitation according to the method described in FR 2548672. The fractions of the recovered products are lyophilized and desalted on this column filled with Sephadex G10® (Pharmacia Biochemicals).

This method yields 3 g of disaccharide Is, 1100 mg of a mixture of hexasaccharide characteristically containing 55% of the derivative AIs-Is-Is, 35% AIs-Is-IIs and 10% AIs-Is-IIIs. This last mixture can be purified according to methods known per se to separate each of the components or can be used as such for conversion to the 1,6-anhydro derivative of formula (I). It should be noted that during this transformation the hexasaccharide AIs-Is-IIIs cannot lead to the formation of compounds of formula (I).

Example A - Preparation of oligosaccharides of formula (II) where n = 0,1,2,3

or 4 with depolymerization of the benzyl ester of heparin and

separation

a - Preparation of the benzyl ester of heparin

The benzyl ester of heparin is prepared according to example 3 and US 5,389,618.

b - Depolymerization

100 g of benzyl ester of heparin are dissolved in 1.91 demineralized water. The mixture is brought to 60 °C with stirring. After achieving a homogeneous solution, introduce approx. 35 ml of 23% sodium hydroxide solution. After 10 minutes of reaction, the solution is cooled and neutralized with 80 ml of an approx. 2N acetic acid solution. 10% weight/volume sodium acetate is added to this solution. The mixture of oligosaccharides is precipitated by adding approx. 2 volumes of methanol. The precipitate is isolated by filtration, washed with methanol 2 times and dried under reduced pressure at 50 °C. After drying, 73.8 g of a mixture of oligosaccharides (II) is obtained.

c - Enrichment of the oligosaccharide where n = 1

30 g of the oligosaccharide mixture obtained above is dissolved in approx. 35 volumes of water. This solution is ultrafiltered on 3 kDa membranes (Pellicon). When 600 ml of permeate has been drawn through, the retentate is diluted with 500 ml. The operation is followed until withdrawal of 450 ml of additional permeate. The two permeate fractions are combined and concentrated to dryness under reduced pressure. 6.1 g of a yellowish, white solid is obtained. The analysis of the solid by gel permeation chromatography suggests that it contains approx. 30% oligosaccharide of formula (II), where n = 1.

d - Fractionation of the ultrafiltered oligosaccharide mixtures

This enriched mixture is fractionated on columns filled with polyacrylamide-agarose gel of the type Ultrogel ACA202 (4 columns in series with a diameter of 10 cm and a height of 50 cm are used). 5 g of mixture enriched by ultrafiltration is dissolved in 25 ml of water and eluted with a solution of 0.2 mol/l sodium chloride at a rate of 5 ml/min. Fractions of 25 ml are recovered at the bottom of the column. Detection of the products is carried out by UV spectrometry (254 nm) and ion spectrometry (IBF). The product fractions are n = 1 recovered, lyophilized and desalted on a column filled with Sephadex G10 gel. After lyophilization, 1 g of tetrasaccharide is obtained which characteristically contains 70% of the derivative AIs-Is with formula (II) (Ri, R2, R3, R$, Re = SO3Na; R1 = H and M = Na). The derivative AIs-Is can optionally be purified by SAX chromatography or according to a preferred aspect, by fractional precipitation according to a method described in FR 2 548 672.

Example 1

5 ml of a sodium hydroxide solution of 0.0063 mol/l is introduced into a reactor kept at 66 °C. The pH value in the solution is measured and used as the target value (pH = 11.35). While stirring, 30 mg of oligosaccharide of formula (II) where n is equal to 0, Ri and Re mean a SO 3 Na residue and M is sodium is added all at once. The pH value is regulated and maintained at pH 11.35 by continuous addition of a 0.5 moi/l sodium hydroxide solution. After 10 hours, the addition of sodium hydroxide is stopped and the reaction mixture is cooled to 25 °C. The pH value in the solution is then brought to between 6 and 7 by adding the resin Amberlite IR120. The mixture is filtered on a Whatman GF/B membrane and concentrated to dryness at 2.7 kPa and a temperature near 25°C. The product is taken up in 0.5 ml of distilled water and lyophilized. 29 mg of a mixture of diastereoisomers of the oligosaccharide of formula (I) is obtained, where n is equal to 0, R] and Re is a SOjNa residue and M is sodium.

Proton spectrum in D20.400 MHz, T = 298K, S in ppm: (4-deoxy-2-0-suIfo-α-L-threo-hex-4-enopyranosyluronic acid-(1-^4)-1,6-anhydro-2 -deoxy-2-sulfoamino-p-D-mannopyranose.trisodium salt): 3.15 (1H, s, H2), 3.75 (2H, m, H6 and H3), 3.88 (1H, m, H4), 4 .20 (1H, d, J = 8 Hz, H6), 4.22 (1H, t, J = 5 Hz, H3'), 4.58 (1H, m H2<*>), 4.75 (1H , m, H5), 5.53 (1H, s, Hl), 5.60 (1H, dd, J = 6 and 1 Hz, Hl'), 6.03 (1H, d, J= 5 Hz, H4 *); (4-deoxy-2-0-sulfo-α-L-threo-hex-4-enopyranosyluronic acid-(l ->4)-1,6-anhydro-2-deoxy-2-sulfoamino-β-D-glucopyranose.trisodium salt): 3 .34 (1H, dd, J = 7 and 2 Hz, H2), 3.72 (1H, t, J = 8 Hz, H6), 3.90 (1H, m, H3), 4.03 (1H, s, H4), 4.20 (1H, d, J = 8 Hz, H6), 4.23 (1H, t, J = 5 Hz,

H3'), 4.58 (1H, m, H2'), 4.78 (1H, m, H5), 5.50 (1H, s, Hl), 5.60 (1H, dd, J = 6 and 1 Hz, H1'), 6.03 (1H, d, J = 5 Hz, H4').

Example 2

33.3 ml of a 0.0063 mol/l sodium hydroxide solution are introduced into a reactor kept at 62 °C. The pH value in the solution is measured and used as the target value (pH = 11.15). While stirring, 200 mg of oligosaccharide of formula (II) is added once where n is equal to 1, Ri, R2, R3, R5 and Ré means an SC^Na residue, R4 means a hydrogen atom and M is sodium. The pH value is regulated and maintained at pH 11.15 by continuous addition of 0.5 mol/l sodium hydroxide solution. After 12 hours, the addition of sodium hydroxide is stopped and the reaction mixture is cooled to 25 °C. The pH value in the column is then brought to between 6 and 7 by adding Amberlite IR120 resin. The mixture is filtered on the Whatman GF/B filter and concentrated to dryness at 2.7 kPa and a temperature near 25 °C. The product is taken up with 3 ml of distilled water and lyophilized. One thus obtains 230 mg of oligosaccharide of formula (I) where n is equal to 1, R1, R2, R3, R5 and R3 means a SO3Na residue, R4 means a hydrogen atom and M is sodium, in the form of a mixture of diastereoisomers.

Proton spectrum in D20,400 MHz, T = 298K, 8 in ppm: (4-deoxy-2-0-sulfo-α-L-threo-hex-4-enopyranosyIuronic acid-(1^4)-2-deoxy-2-sulfo- amino-6-0-sulfo-α-D-glucopyranosyl-(l ->4)-2-0-sulfo-α-L-idopyranyluronic acid-(1^4)-1,6-anhydro-2-deoxy-2-sulfoamino-fi -D-mannopyranose.heptanasodium salt): 3.15 (1H, s H2), 3.25 (1H, m, H2"), 3.60 (1H, m, H3"), between 3.70 and 4.70 (14H, solid, H3/H4/H6, H27H37H47H5\ H4"/H5'7H6", H2"7H3'"), 4.75 (1H, m, H5), between 5.20 and 5.40 (2H, m, Hl * and Hl"), 5.45 (1H, m, Hl"'). 5.56 (1H, m, H1), 5.94 (1H, d, J = 5 Hz, H4); (4-deoxy-2-0-sulfo-α-L-threo-hex-4-enopyranosyluronic acid-(l->4)-2-deoxy-2-sulfo-amino-6-0-sulfo-α-D-glucopyranosyluronic acid-(l -(4)-2-O-sulfo-α-L-idopyranosyluronic acid-(1-»4)-1,6-anhydro-2-deoxy-2-sulfoamino-β-D-glucopyranose.hepta-sodium salt): 3.25 (1H , m, H2"), 3.42 (1H, dd, J = 4 and 1H, H2), 3.60 (1H, m, H3"), between 3.70 and 4.70 (14H, massive, H3 /H4/H6, H2'/H37H4<*>/H5\ H4'7H5'7H6", H2"'/H3"'), 4.75 (1H, m, H5), between 5.20 and 5.40 (2H, m, Hl' and Hl"), 5.45 (1H, m, Hl"<*>), 5.52 (1H, m, Hl), 5.94 (1H, d, J = 5 Hz , H4).

Example 3

16.7 ml of a 0.0063 mol/l sodium hydroxide solution are introduced into a reactor kept at 62<*>C. The pH value in the solution is measured and used as a measurement value (pH = 11.7). While stirring, 100 mg of oligosaccharide of formula (II) is added at once where n is Uk 2, Ri, R2, R3, R5 and Re means a SC^Na residue and R* means a hydrogen atom and M is sodium. The pH value is regulated and maintained at 11.7 by continuous addition of a 0.5 mol/l sodium hydroxide solution. After 10 hours, the addition of sodium hydroxide is stopped and the reaction mixture is cooled to 25 °C. The pH value in the solution is brought to between 6 and 7 by adding Amberlite IR120 resin. The reaction mixture is filtered on a Whatman GF/B filter and then concentrated to dryness at 2.7 kPa and a temperature close to 25 °C. The product is taken up in 3 ml of distilled water and lyophilized. 108 mg of oligosaccharide of formula (I) is obtained, where n is equal to 2, R1, R2, R3, R5 and R5 means a SO 3 Na residue, R 4 means a hydrogen atom and M is sodium, in the form of a mixture of diastereoisomers. Note that from A to F the sugars are hexasaccharides, A is the 1,6-anhydro residue and F is the unsaturated uronic acid residue.

Proton spectrum in D20.600 MHz, T - 298K, 8 in ppm: (4-deoxy-2-0-sulfo-α-L-threo-hex-4-enopyranosyIuronic acid-(1->4)-2-deoxy-2-sulfo -amino-6-0-sulfo-α-D-glucopyranosyl-(1->4)-2-0-sulfo-α-L-idopyranyluronic acid-(1->4)-2-deoxy-2-sulfoamino-6-0-sulfo -α-D-glucopyrasyl-(1->4)-2-0-sulfo-α-L-idopyranosyluronic acid-(1->4)-1,6-anhydro-2-deoxy-2-sulfoamino-p-D-manno-pyranose undecasodium salt ): 3.15 (1H, s, H2(A)), 3.25 (2H, m, H2(C+E)), 3.60 (2H, m, H3(C+E)), between 3 .65 and 4.50 (19H, solid, H2(B+D)/H3(A+B+D+F)/H4(A+B+C+D+E)/ H5(C+E)/H6 (A+C+E)), 4.60 (1H, s, H2(F)), 4.80 (3H, m, H5(A+B+D)), 5.18 (1H, s, H1 (D)), 5.30 (1H, s, H1(B)), 5.34 (1H, d, H1(C)), 5.36 (IH, d, H1(E)), 5.46 (1H, s, H1(F)), 5.57 (1H, s, H1(A)), 5.95 (1H, d, J=5 Hz, H4(F)); (4-deoxy-2-0-sulfo-α-L-threo-hex-4-enopyranosyluronic acid-(l->4)-2-deoxy-2-sulfo-amino-6-0-sulfo-α-D-glucopyranosyl-(l ->4)-2-0-sulfo-a-L-idopyranosyluronic acid-(l->4)-2-deoxy-2-sulfoamino-6-0-sulfo-a-D-glucopyranosyl-(l^4)-2-0- sulfo-α-L-iodopyranosyluronic acid-(1-»4)-1,6-anhydro-2-deoxy-2-sulfoamino-β-D-gluco-pyranose.uhdecasodium salt): 3.25 (2H, s, H2(C+E) ), 3.42 (IH, m, H2(A)), 3.60 (2H, m, H3(C+E)), between 3.65 and 4.50 (19H, massive, H2(B+D )/H3(A+B+D+F)/H4(A+B+ C+D+E)/H5(C+E)/H6(A+C+E)), 4.60 (IH, s, H2(F)), 4.80 (3H, m,

H5(A+B+D)), 5.18 (IH, s, H1(D)), 5.31 (IH, s, H1(B)), 5.34 (IH, d, H1(C) ), 5.36 (IH, d, H1(E)), 5.46 (IH, s, H1(F)), 5.52 (IH, s, H1(A)),

5.95 (IH, d, J=5 Hz, H4(F)).

Example 4

4 ml of a 0.0316 mol/l sodium hydroxide solution is added to a reactor kept at 62 °C. The pH value in the solution is measured and used as the measurement value (pH = 11.8). While stirring, 100.8 mg of a mixture of oligosaccharides of formula (II) where n is equal to 2, comprising 55% of the derivative AIs-Is-Is (R), R2, R3, Rj and Re means a SO3Na are added at once -residue, R4 means a hydrogen atom and M is sodium), 35% AIs-Is-IIs (Ri, R2,R3 and Re means a SO3Na residue, Rj means either a SO3Na residue or a hydrogen atom, R» means a hydrogen atom and M is sodium) and 10% AIs-Is-IIIs (Ri, R2, R3, R5 and Re means a SO2Na residue, R4 means a hydrogen atom and M is sodium, where the SC^M function on the C6 carbon atom is replaced with a hydrogen). The pH value is then adjusted to and maintained at 11.8 by continuous addition of a 0.5 mol/l sodium hydroxide solution. After 11 hours, the addition of sodium hydroxide is stopped and the reaction mixture is cooled to 25 °C. The pH value of the solution is then brought to between 6 and 7 by adding the resin Amberlite IRI 20. The mixture is filtered over a Whatman GF/B filter and then concentrated to dryness at 2.7 kPa and a temperature close to 25 °C. The product is taken up with 1.5 ml of distilled water and lyophilized. One obtains in this way 110 mg of a mixture of oligosaccharides of formula (I), where n is equal to 2, in particular containing the 1,6-anhydro derivative AIs-Is-Is (Ri, R2, R3, R5 and Re means a SC^Na residue, R4 means a hydrogen atom and M is sodium), and the 1,6-anhydro-derivative Als-Is-IIs (Ri, R2,R3 and Re means a SOsNa residue, R5 means either a SC^Na -residue or a hydrogen atom, R4 means a hydrogen atom and M is sodium). HPLC analysis in ion-pair mode allows following the transformation to the derivative of formula (I). In this case, the HPLC analysis shows that the transformation is carried out for the derivatives AIs-Is-Is, AIs-Is-IIs.

Example 5

To a reactor maintained at 66 °C, 8.6 ml of a 0.025 mol/l lithium hydroxide solution is introduced. The pH value of the solution is measured and used as the measurement value (pH = II, 68). While stirring, 50 mg of oligosaccharide of formula (II) where n is equal to 0, R) and Re means a SOsNa residue and M is sodium is added at once. The pH value is then adjusted to and maintained at pH 11.68 by continuous addition of a 0.466 mol/l lithium hydroxide solution. After 8 hours, the lithium hydroxide addition is stopped and the reaction mixture is cooled to 25<*>C. HPLC analysis in ion-pair mode allows to follow the transformation to the derivative of formula (I), where n is equal to 0, Ri and Re mean a SOsNa residue and M is sodium or lithium. In this case, the HPLC analysis shows that the transformation proceeds 100%. The yield for external calibration is 81.2%.

Example 6

8.3 ml of a 0.0063 mol/l potassium hydroxide solution are placed in a reactor which is kept at 66 °C. The pH value in the solution is measured and used as the measurement value (pH = 11.1). While stirring, 50 mg of oligosaccharide of formula (II) where n is equal to 0, Ri and R* mean a SC^Na residue and M is sodium are added at once. The pH value is then adjusted to and maintained at 11.1 by continuous addition of a 0.515 mol/l potassium hydroxide solution. After 24 hours, the addition of potassium hydroxide is stopped and the reaction mixture is cooled to 25 °C. In the case of HPLC in ion-pair mode, it is possible to follow the transformation to the derivative of formula (I) where n is equal to 0, Ri and Re mean an SC^Na residue and M is sodium or potassium. In this case, HPLC shows that the transformation proceeds 100%. The yield for external calibration is 75.6%.

Example 7

8.3 ml of a 0.0063 mol/l cesium hydroxide solution is added to a reactor maintained at 66 °C. The pH value in the solution is measured and used as the measurement value (pH = 10.75). While stirring, 50 mg of oligosaccharide of formula (II) where n is equal to 0, Ri and Re mean an SC^Na residue and M is sodium is added at once. The pH value is then adjusted to and maintained at 10.75 by continuous addition of a 0.476 mol/l cesium hydroxide solution. After 20 hours, the addition of cesium hydroxide is stopped and the reaction mixture is cooled to 25 °C. HPLC analysis in ion-pair mode allows following the transformation to the derivative of formula (I) where n is equal to 0, Ri and Re mean an S(>jNa residue and M is sodium or cesium. In this case, HPLC shows that the transformation proceeds to 90 .3% The yield with external calibration is 73%.

Example 8

8.3 ml of a 0.0063 mol/l tetrabutyl ammonium hydroxide solution is added to a reactor maintained at 66 °C. The pH value in the solution is measured and used as the measurement value (pH = 10.95). While stirring, 50 mg of oligosaccharide of formula (II) where n is equal to 0, Ri and Re mean an SChNa residue and M is sodium are added at once. The pH value is then adjusted to and maintained at 10.95 by continuous addition of a 0.521 mol/l tetra-butylammonium hydroxide solution. After 16 hours, the addition of cesium hydroxide is stopped and the reaction mixture is cooled to 25 °C. HPLC analysis in ion-pair mode allows following the transformation to the derivative of formula (I) where n is equal to 0, Ri and Re mean a SOaNa residue and M is sodium or tetrabutylammonium. In this case, HPLC analysis shows that the transformation proceeds 96.7%. The yield for external calibration is 65%.

Medicines may contain as active ingredient at least one oligosaccharide of formula (I) or a mixture of oligosaccharides of formula (I) in the form of a preparation in which it is associated with any other pharmaceutical, compatible product which may possibly be inert or physiologically actively. The drugs can be used intravenously, subcutaneously, orally, rectally, topically or pulmonary (inhalation).

Sterile preparations for intravenous or subcutaneous administration are generally aqueous solutions. These preparations may also contain adjuvants and in particular wetting agents, isotonizing agents, emulsifiers, dispersants and stabilisers. Sterilization can take place in several ways, for example by aseptic filtration, by incorporating sterilizing agents into the mixture or by irradiation. They can likewise be produced in the form of solid, sterile preparations which can be dissolved at the time of use in sterile water or any other sterile injectable medium.

Tablets, pills, powders (gelatin capsules, bags) or granules can be used as solid preparations for oral administration. In these preparations, the active ingredient is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or lithium dioxide, under an argon stream. These preparations may likewise include substances other than the diluents, for example one or more lubricants such as magnesium stearate or talc, an agent favoring oral absorption, dyes, coatings (dragés) or a varnish.

As liquid preparations for oral administration, one can use pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil. These preparations may include substances in addition to the diluents, for example wetting agents, odorants, diluents, aroma substances and stabilizers.

Preparations for rectal administration are suppositories or rectal capsules which, in addition to the active ingredient, contain laxatives such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

Preparations for topical administration can be, for example, creams, lotions, eye drops, mouthwashes, nasal drops or aerosols.

The doses depend on the intended effect, the duration of the treatment and the route of administration used; they are generally between 0.5 and 10 mg/kg/day subcutaneously or 3 to 60 mg/day for a 60 kg adult.

Generally speaking, the doctor determines the appropriate dosage as a function of age, weight and all other relevant factors in the individual to be treated.

The oligosaccharides of formula (I) can thus be used for prevention or therapy of neurodegenerative diseases for which the cerebral inflammation plays a harmful role that can lead to death and among which can be mentioned ischaemia in the central nervous system, cerebral ischaemia, ischaemia in the retina and cochlea, cardiac ischaemia (myocardial infarction), peripheral ischaemia, traumatism in the central nervous system and particularly cranial, spinal or cranio-spinal traumatism, traumatism in the retina and cochlea, plaque sclerosis, neuropathic pain and peripheral neuropathies, motoneuron diseases such as amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease, Huntington's chorea and certain forms of osteoarthritis, especially in articular locations.

Claims (15)

1. Oligosaccharides, characterized by the formula: there n an integer from 0 to 25, Ri, R3, R4 and R5 are the same or different and mean a hydrogen atom or a SOsM residue, R2 and Re are the same or different mean a hydrogen atom or a SO3M or COCH3 residue M is sodium, calcium, magnesium or potassium, mixtures of such oligosaccharides and their diastereoisomers. 2. Oligosaccharides of formula (I) according to claim 1, characterized in that R4 is a hydrogen atom, mixtures of such oligosaccharides and their diastereoisomers. 3. Oligosaccharides according to claim I or 2, characterized in that n is an integer from 0 to 10, mixtures of such oligosaccharides and their diastereoisomers. 4. Oligosaccharides according to claim 1 or 2, characterized in that n is an integer from 0 to 6, mixtures of such oligosaccharides and their diastereoisomers. 5. Oligosaccharides according to claim 1 or 2, characterized in that n is an integer from 1 to 6, mixtures of such oligosaccharides and their diastereoisomers. 6. Process for the production of oligosaccharides of formula (I) according to claim 1, characterized in that an alkali metal or quaternary ammonium hydroxide is reacted with oligosaccharides of the formula: there ner an integer from 0 to 25, Ri, R3, R4 and R5 are the same or different and mean a hydrogen atom or a SOjM residue, R2 and Re are the same or different and mean a hydrogen atom, or a SO3M or COCH3 residue M is sodium, calcium, magnesium or potassium, or a mixture of these, and isolates the oligosaccharides or their mixtures. 7. Method according to claim 6, characterized in that the reaction is carried out in an aqueous medium at a temperature of 40 to 80 °C and a pH value of 10 to 13. 8. Method according to claim 6 or 7, characterized in that an aqueous 1 to 5% hydroxide solution of an alkali metal or a quaternary ammonium is used. 9. Method according to any one of claims 6 to 8, characterized in that the reaction takes place at a temperature of 60 to 70 °C. 10. Method according to any one of claims 6 to 9, characterized in that the pH value in the reaction is 11 to 12.5. 11. Method according to any one of claims 6 to 10, characterized in that the hydroxide of potassium metal or quaternary ammonium is a sodium, potassium, lithium or cesium hydroxide or tetrabutyl ammonium hydroxide. 12. Pharmaceutical preparation, characterized in that it contains as active ingredient at least one oligosaccharide according to claim 1. 13. Pharmaceutical preparation, characterized in that it contains as active ingredient at least one oligosaccharide according to any one of claims 1 to 5. 14. Use of oligosaccharides of formula (I) according to one of claims 1 to 5 for the preparation of a medicament for use in the prevention or therapy of diseases associated with an inflammatory process which includes the production of nitric oxide (NO). 15. Use of oligosaccharides of formula (I) according to claim 14 for the production of drugs for use in the prevention or therapy of cerebral, cardiac or peripheral vascular ischaemia, osteoarthritis, traumatism in the central nervous system, cranial, spinal or cranio-spinal traumatism, plaque -sclerosis, neuropathic pain and peripheral neuropathies, motor neuron diseases, amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's chorea.