SK286745B6 - Pharmaceutical compositions containing oligosaccharides, oligosaccharides and preparation thereof and the use of them - Google Patents

Abstract

The invention concerns pharmaceutical compositions containing as active principle an oligosaccharide of formula (I), in which n is an integer from 0 to 25, R1, R3, R4, R5, R6 and R8 represent an hydrogen atom or an SO3M radical, R2 and R7 represent an hydrogen atom or an SO3M or COCH3 radical and M is sodium, calcium, magnesium or potassium, a mixture of said oligosaccharides of formula (I) and methods for preparing them.

Description

A method for reducing the amount of nitric oxide present at the site of inflammation by administering a nitric oxide synthase inhibitor, a nitric oxide scavenger, or a tetrahydrobiopterin synthesis inhibitor is described in US 5,449,688. The present invention differs from this document in that it relates to oligosaccharide compounds, i. j. compounds which are different from those described in the cited document.

Mc Lean et al. (Eur. J. Biochem., 1984, 145 (3), 607-15) describe the enzymatic degradation of heparin as well as the disaccharides resulting from this degradation. The present invention differs from the cited method in the structure of the claimed oligosaccharides and in their use for treating or preventing inflammatory conditions.

Lcc et al. (J. Chromatogr., 1981, 212 (1), 65-73) describes an HPLC method for the analysis of the reduction of unsaturated disaccharides derived from enzymatic cleavage followed by reduction of chondroitin, dermatan and heparin sulfates and heparin with sodium borohydride. The present invention differs from said method in the structure of the claimed oligosaccharides and in their use for treating or preventing inflammatory diseases.

SUMMARY OF THE INVENTION

In the above formula (I), n is an integer from 0 to 25, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁸ , which are the same or different, are hydrogen or SO 3 M, R ² and R ⁷ , which are the same or different, is hydrogen or SO 3 M or COCH _3, and M is sodium, calcium, magnesium or potassium.

These oligosaccharides thus contain an even number of carbohydrates.

In the general formula (I), R ⁴ and R ⁶ preferably represent hydrogen atoms.

Oligosaccharides of formula (I) wherein n is 0 and either R ¹ , R ⁶ and R ⁸ are hydrogen, R ⁷ is SO 3 M or COCH 3 and M is sodium, or R ¹ and R ⁶ are hydrogen, R ⁷ represents a COCH 3 radical, R ⁸ is SO 3 M radical and M is sodium, or R ⁶ is H, R ^1, R ⁷ and R ⁸ represent an SO 3 M, and M is sodium, have been described by Lee GH, et al., J. chrome. 212, 65-73 (1981), but no pharmacological properties have been attributed to these compounds.

Oligosaccharides of formula (I) wherein n is 0 and either R ⁶ and R ⁷ are hydrogen, R ¹ and R ⁸ are SO 3 M and M is sodium, or R ¹ , R ⁶ and R ⁷ are hydrogen, R ⁸ is SO3M and M is sodium are described in MW McLean et al., Eur. J. Biochem. 1984, 145, 607, and no pharmacological properties have been attributed to these compounds.

Suitable pharmaceutical compositions are those comprising an oligosaccharide of formula (I) wherein:

- n represents an integer from 0 to 10, in particular an integer from 0 to 6 and in particular an integer from 1 to 6,

- R ^1, R ^2, R ^3, R ^5, R ^7, R ^s, which may be identical or different, represent H or SO 3 M, and in particular, R ^1, R ^2, R ^3, R ^5, R ^7, R ⁸ are SO3M and

- M represents sodium.

Particularly preferred pharmaceutical compositions are those comprising an oligosaccharide of formula (I) wherein:

- n is 0, R ¹ , R ⁷ and R ⁸ are SO ₃ M, R ⁶ is hydrogen and M is sodium,

- n is 1, R ¹ , R ² , R ³ , R ⁵ , R ⁷ , R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium,

- n is 2, R ¹ , R ² , R ³ , R ⁵ , R ⁷ , R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium,

- n is 3, R ¹ , R ² , R ³ , R ⁵ , R ⁷ , R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium,

n is 4, R ¹ , R ² , R ³ , R ⁵ , R ⁷ , R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium.

Oligosaccharides of formula (I) except those in which n is 0 and either R ¹ , R ⁶ and R ⁸ are hydrogen, R ⁷ is SO 3 M or COCH 3 and M is sodium, or R ¹ and R ⁶ are atom hydrogen, R ⁷ is COCH 3, R ⁸ is SO 3 M and M is sodium, or R ⁶ is hydrogen, R ¹ , R ⁷ and R ⁸ are SO 3 M and M is sodium, or R ⁶ and R ⁷ are hydrogen, R ^{@ 1} and R @ ⁸ are SO3M and M is sodium, or R ^{@ 1} , R @ ⁶ and R @ ⁷ are hydrogen, R @ ⁸ is SO3 M and M is sodium, are novel compounds and themselves form part of the invention.

Oligosaccharides of formula (I) may be prepared by treatment of oligosaccharides of formula (II) with alkali metal borohydride or quaternary ammonium.

wherein n is an integer from 0 to 25, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁸ , which are the same or different, represent a hydrogen atom or a SO 3 M, R ² and R ^{7 group} which are same or different, are hydrogen or SO 3 M or COCH ₃ and M is sodium, calcium, magnesium or potassium.

This reaction is carried out in an aqueous medium at a temperature near 25 ° C, at a pH between 7 and 10, preferably between 9 and 10, throughout the reaction. Maintaining the pH at said temperature is achieved by adding 0.5 M sodium hydroxide solution. The reaction is quenched by acidifying the reaction mixture, for example by adding acetic acid until a pH between 4 and 5 is reached.

The alkali metal borohydride includes lithium borohydride, sodium borohydride and potassium borohydride.

Quaternary ammonium borohydride is tetrabutylammonium borohydride.

The oligosaccharides of formula (II) may be obtained by gel chromatography of a mixture of oligosaccharides III, which is obtained by enzymatic depolymerization of heparin or by basic depolymerization of heparin benzyl ester or hemisynthesis benzyl ester heparin.

This chromatography is carried out on columns which are packed with a polyacrylamidagarose type gel, for example, commercially available under the designation Ultrogel ACA202 (Biosepra). Preferably, a battery of polyacrylamidagarose gel columns is used. The number of columns used is adapted to the volume, gel and oligosaccharides to be separated. The mixture is diluted with a solution containing phosphate buffer and sodium chloride. Preferably, the phosphate buffer solution is a 0.02 mol / L NaH ₂ PO ₄ / Na ₂ HPO _{4 solution} (pH 7) containing 0.1 mol / L sodium chloride. Detection of individual fractions is performed by UV spectrometry (254 nm) and ion spectrometry (IBF). The fractions thus obtained can then be purified, for example, by means of SAX type chromatography (Strong Anion Exchange) which is carried out in a manner known per se and in particular by the methods described in KG Rice and RJ Linhardt, Carbohydrate Research 190, 219-233 (1989); A. Lamkjaer, SH Hansen and PB Ostergaard, Carbohydrate Research, 166, 37-52 (1995); and WO 90/01501 (Example 2). The fractions are then lyophilized and then desalted in a gel-packed column, such as a Sephadex G10 gel column (Pharmacia Biochemicals).

If purification is not carried out by SAX chromatography, the lyophilisates can be purified by simple or fractional precipitation by known methods and in particular by the method described in patent FR 2548672. In general, the following procedure is carried out:

The lyophilized fraction to be purified is dissolved at 25 ° C in about ten volumes of distilled water. Addition of methanol or ethanol precipitates the desired oligosaccharide, controlling its concentration by high performance liquid chromatography (CLHK, Liquide Haute Performance Chromatography). The amount of methanol or ethanol added is determined depending on the desired purity and the desired yield of said oligosaccharide. Likewise, this operation can be carried out in the following steps, starting from the original lyophilisate solution. To this end, the desolubilizing agent (methanol or ethanol) is added in small amounts and the precipitate formed is isolated after each addition of said agent. The precipitates thus obtained are analyzed by high performance liquid chromatography. Depending on the desired purity and the desired yield, adequate fractions of the precipitate are combined.

In one variant of the invention, the lyophilized fraction to be purified may be dissolved in 10 to 200 volumes of water containing 0 to 30% sodium acetate. The percentage of sodium acetate is determined in advance depending on the nature of the oligosaccharide to be treated (depending on its size). Addition of methanol precipitates the desired oligosaccharide, controlling the degree of enrichment (and hence its concentration) by high performance liquid chromatography. The amount of methanol added is determined depending on the desired purity and the desired yield of said oligosaccharide. Likewise, this operation can be carried out in several successive steps starting from the initial lyophilisate solution. Therefore, the desolubilizing agent (methanol) is added in small amounts and the partial precipitate obtained is isolated after each addition of methanol. The resulting precipitates are analyzed by high performance liquid chromatography. Depending on the desired purity and the desired yield of oligosaccharide, the appropriate fractions of the precipitates obtained are combined.

In the case of intermediates of formula (II) in which n = 0, 1 or 2, it is preferable to start from the mixture III, which is obtained by enzymatic depolymerization.

This depolymerization is carried out using heparinase I (EC 4.2.2.7) in a phosphate buffer solution at pH 7, in the presence of sodium chloride and bovine serum albumin BSA (Albumin de Serum Bovin), at a temperature of 10-18 ° C, preferably at 15 ° C. ° C, within 8 to 10 days, preferably within 9 days. Depolymerization is interrupted, for example, by heating the reaction mixture to 100 ° C for 2 minutes, then lyophilizing. It is preferred to use 7 µl heparinase per 25 g heparin. The phosphate buffer solution generally contains 0.05 mol / l NaH ₂ PO ₄ ^{/ /} Na ₂ HPO ₄ (pH 7) in the presence of 0.1 mol / k sodium chloride. The concentration of bovine serum albumin BSA is usually 2%.

In the case of intermediates of formula (II) in which n = 0, 1, 2, 3 or 4, it is preferable to start from the mixture III, which is obtained by depolymerization of the benzyl ester of heparin.

The heparin benzyl ester can be prepared by the methods described in U.S. Patent Nos. 5,389,618, EP 40,144 and FR 2,548,672. The esterification rate is preferably 50 to 100%. Preferably, the esterification rate will be 70 to 90%.

This depolymerization is carried out in an aqueous medium using an alkali metal hydroxide (e.g. lithium hydroxide, potassium hydroxide, sodium hydroxide or cesium hydroxide) or a quaternary ammonium (e.g. tetrabutylammonium hydroxide), preferably with a molarity of between 0.1 and 0.2 mol / l. , at a temperature of 40 to 80 ° C for 5 to 120 minutes. Preferably, it is operated for 5-15 minutes at 60-70 ° C using 0.15 mol / L sodium hydroxide solution. The depolymerization reaction is interrupted by the neutralization addition of an acid such as acetic acid. After addition of 10% w / v. Sodium acetate, the oligosaccharide mixture is precipitated by the addition of methanol, preferably 2 volumes of methanol per 1 volume of the reaction mixture are added and the mixture is filtered.

In a preferred embodiment of the invention, the oligosaccharide mixture obtained after chemical depolymerization in the form of an aqueous solution is enriched by ultrafiltration on membranes with an appropriate nominal separation threshold (Pellicon type based on regenerated cellulose commercially marketed by Millipore); the membrane type is selected depending on the type of enriched oligosaccharides to be isolated.

For oligosaccharides II in which n = 0, a membrane with a nominal separation threshold of 1 kDa is used, for oligosaccharides II in which n = 1 a membrane with a nominal separation threshold of 1 kDa or 3 kDa is used, for oligosaccharides II in which n = 2, a membrane with a nominal separation threshold of 3 kDa is used, for oligosaccharides II in which n = 3 or 4, a membrane with a nominal separation threshold of 5 kDa is used. During this operation, the permeate is retained while the retained fraction is waste. Thus, the fraction of the enriched product may represent 50 to 10% of the initial oligosaccharide mixture, while maintaining at least 80% of the desired oligosaccharide.

In the case of intermediates II, in which n = 0 to 25, it is preferable to start from mixture III, which is obtained by depolymerization of the benzyl ester of the hemisynthesis sulphated polysaccharide. The hemisynthesis sulphated polysaccharide benzyl ester is prepared from hemisynthesis sulphated polysaccharides which are obtained from polysaccharide K5 by the method described in WO94 / 29352 and WO96 / 14425. The esterification, depolymerization and isolation conditions are the same as those already described for heparin benzyl ester.

In all of the above procedures, the starting heparin may be heparin of porcine, ovine, caprine or bovine origin and may come from mucus (lime) of the lungs or skin of animals. Preferably, pig or sheep mucus heparin and bovine lung or even more preferably, pig or mucus heparin is used.

The oligosaccharides of formula (I) have anti-inflammatory properties and can thus be used for the prevention and treatment of diseases associated with inflammatory processes involving cytotoxic agents such as nitric oxide (NO), its inducible form being mainly released by neutrophils or macrophages, when neutrophils and macrophages migrate and are activated at the tissue level. Neutrophil migration, activation and adhesion occurs at the level of ischemized tissue zones due to occlusion or spasm of vascularizing artery tissue. These ischemias can occur either at the cerebral level (cerebral vascular event), or at the myocardial level (myocardial infarction), or at the lower limb level (ischemia referred to as peripheral). The oligosaccharides of formula (I) may thus be used for the prevention and / or treatment of neurodegenerative diseases in which cerebral inflammation plays a malignant role which may result in death, including cerebral ischemia, cardiac ischemia (myocardial infarction), peripheral ischemia, central nervous system injuries, and in particular skull injuries, spinal injuries and combined skull and spinal injuries, multiple sclerosis, neuropathic pain and peripheral neuropathy, motoneuron diseases and amyotrophic lateral sclerosis, progressive spinal atrophy, progressive spinal atrophy, progressive spinal atrophy, neuro-AIDS, Alzheimer's disease, Parkinson's disease, Huntington's chorea and some forms of osteoarthritis, especially with joint localization.

The anti-inflammatory activity of these products was determined in vivo in a lipopolysaccharide-induced NOx (nitrite and nitrate) production test from E. Coli, which was carried out according to the protocol described by M-Yamashita et al. In Eur. J. Pharmacol, 338, 2, 151-158 (1997) or J. E. Shellito et al. in Am. J. Respir. Cell. Mol. Biol., 13, 1.45-53 (1995).

Male CD1 mice (Charles River, body weight 25-35 g) are injected intravenously with 0.5 mg / kg oligosaccharide intravenous bolus at T0 and 1 or 2 mg / kg oligosaccharide subcutaneously at T + 15 minutes. At T + 30, experimental animals are given 100 mg / kg lipopolysaccharide (LPS), which originates from E. Coli (Sigma L3129, serotype 0127: B8). At T + 3 hours, test animals are re-injected subcutaneously with 1 or 2 mg / kg of oligosaccharide. At T + 5 hours and 30 minutes, a blood sample is taken from the test animals by eye puncture and the NOx (nitrite and nitrate) concentration in the plasma is determined by the Griess colorimetric method after nitrate to nitrite reduction using nitrate reductase as follows:

The ml plasma sample is mixed with 88 ml deionized water and the resulting mixture is incubated in the dark for one hour at ambient temperature with 40 ml phosphate buffer (0.31 M, pH 7.5), 20 ml beta-NADPH (reduced nicotinamide adenine). -dinucleotide phosphate) (0.86 mM), 20 ml FDA (flavin-adenine dinucleotide) 0.11 mM) and 20 ml nitrate reductase (2U / ml) (Boehringer Mannhein). 10 ml of ZnSO ₄ (1 M) is added to precipitate the proteins and after mixing the samples are centrifuged at 20,000 g for 5 minutes. Finally, 50 ml of the supernatant is incubated for 10 minutes at ambient temperature with 100 ml of Griess reagent [1% sulfanilamide in a mixture of phosphoric acid and 0.1% naphthylethylenediamine in deionized water (v / v)]. Using a microplate spectrophotometer, the optical densities are read at 540 nm, each point being determined twice. KNO ₃ and NaNO ₂ are used as standards for the colorimetric method.

In this test, the oligosaccharides of the invention inhibit more than 50% NOx production.

The oligosaccharides of formula (I) improve the survival and growth of motoneurons and are therefore particularly suitable for the prevention and / or treatment of motoneuronal diseases, including in particular amyotrophic lateral sclerosis, progressive spinal atrophy, infantile muscular atrophy and primary lateral sclerosis.

Motoneuron cultures are known to undergo apoptosis when performed in the absence of trophic support (e.g., BDNF or NT5). Recently, it has been found that the oligosaccharides of the invention allow the survival and growth of motoneurons. This activity was tested on cultures of atrocytes and motoneutrons that are devoid of neurotrophic factor according to the following protocol.

Cultures enriched with motoneurons

Motoneuron-enriched cultures are prepared using the centrifuge method described in RK Schnaar and AE Schaffner, J. Neurosci., 1, 204-217 (1981) and modified as described in W. Cam and CE Henderson, J. Neurosci. Methods, 44, 59-70 (1992). The spinal cord of the E15 rat embryos is sterile removed and free of dorsal spinal chains. The spinal cords are then dissected and incubated for 15 minutes at 37 ° C in PBS (saline phosphate buffer: NaCl 137 mM, KCl 2.68, Na ₂ HPO ₄ 6.45, KH ₂ PO ₄

1.47 nM) to which 0.05% trypsin was added. The cell disruption is completed by spreading with the end of a 1 ml pipette in a culture medium enriched with 0.1% bovine serum albumin (BSA) and 0.1 mg / ml ADNase.

The cell suspension is spread on a 6.5% (w / v) metrisamide band in L15 medium (commercially provided by Gibco BRL) and centrifuged at 500 g for 15 minutes. The range band containing motoneurons is isolated, diluted in L15 and incubated for 45 minutes at ambient temperature in culture boxes previously impregnated with mouse anti-IgG and MC192 supernatant hybridoma [(Chandler CE et al., J. Biol. Chem. , 259, 6882 (1984)] The suspended cells are washed with L15 and the motoneurons are eluted with gentle agitation with MC192 supernatant hybridoma. sodium bicarbonate (22 MM), coalbumin (0.1 mg / ml), putrescine (0.1 mM), insulin (5 (gg / ml), sodium lenate (31 nM), glucose (20 mM) were added, progesterone (21 nM), penicillin (100 µl / ml) and streptomycin (100 µg / ml) The cultures are maintained at 37 ° C in a humidified atmosphere containing 5% CO ₂ .

Spinal cord astrocyte culture

Astrocytes are obtained from rat embryos by a slightly modified method described by R. P. Saneto and J. De Vellis, Neurochemistry and Practical Approach (A.J. Tumer and H.S. John) IRL Press, Oxford-Washington DC, p. 27-63 (1987).

The spinal cord is harvested sterile from the test animals and free from changes and dorsal ganglions. Five to ten dishes are transferred to PBS (saline phosphate buffer: NaCl 137 mM, KCl 2.68 mM, Na ₂ HPO ₄ 1.47 mM) and sectioned prior to incubation at 37 ° C for 25 minutes at PBS to which 0.25% trypsin was added. The enzymatic process is interrupted by the addition of 10 ml of modified DubelccoEagle medium (DMEM) to which 10% fetal calf serum (FCS) has been added and the cells are harvested by centrifugation. The next stage of mechanical disintegration is carried out using the end of a 1 ml pipette. Cells are plated at a density of 1.5 x 10 ⁶ cells per 25 cm ² culture medium in DMEM with 10% FCS. After two days in vitro, the cultures are supplied with nutrients every day throughout the study period. When a visible cell monolayer is obtained, the cultures are agitated for 48 hours at a stirrer rotation speed of 250 rpm and the following day the monolayers are treated with cytosine arabinoside (10 µM) for 48 hours. Astrocyte monolayers are then expanded until a density of 5 by 35 mm is reached on 25 cm ² culture flasks at the start of the study.

Said spinal astrocyte cultures are composed of more than 98% of cells that are immunoreactive to glial fibrillar acid (GFAP) protein.

Astrocyte monolayers are exposed either with PBS alone (reference experiment) or with the test product in PBS solution for 24 hours using concentrations of 0.1 to 10 ng / ml. The astrocyte monolayers are then washed with DMEM and maintained for 2 hours in a culture medium to which motoneurons are added. After two hours of culture and after 2 hours or 3 days, vehicle or test product is added to the culture medium.

Immunochemical identification of motoneurons

Cells are fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (pH 7.4 at 4 ° C for 15 minutes). The cultures are then washed and non-specific sites are blocked with 2% bovine serum albumin (BSA) in PBS and 0.1% Triton X100. These cultures are sequentially incubated with p75 ^LNGRF antibodies (Chandler cell cited above) overnight with biotinylated goat serum (1/125 Gibco) and streptavidene peroxidase (1/200, Gibco) for 60 minutes. The antibodies are visualized using a DAB / H ₂ O ₂ type reaction.

Cell counting by statistical analysis

Immunoreactive cells for the weakly active neutrophine receptor p75 ^Ingrf , which has neurites longer than 4 cell diameters, are considered to be motoneuron-living. The number of these neurons is evaluated by counting the labeled cells on an area of 0.825 cm ² under a microscope at a two-fold magnification. The values obtained are expressed as the number of motoneurons per cm ² or as a percentage of the motoneurons present in the cultures maintained in the absence of the trophic factor relative to the control reference experiment. The determination is carried out at least three times.

Statistical analyzes are performed using Student's t-test.

In pretreatment with the oligosaccharides of the invention, the number of motoneurons that grow on the astrocyte monolayer is increased by 20 to 50%.

The following non-limiting examples illustrate the preparation of the oligosaccharides of formula (I) and the corresponding intermediates.

The abbreviations in these examples have the following meanings:

AIs: (4-deoxy-2-O-sulfo-α-L-threo-hex-enopyranosyluronic acid) - (14) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, tetrasodium salt , or AUAp2D- (1-> 4) aD-GlcNp2S6S;

Is: (2-sulfo-α-L-idopyranosyluronic acid) - (1- * 4) -2-deoxy-2-sulfoamino-6-O-sulfo-α-D-glucopyranose, tetrasodium salt (2-sulfo-α-L- idopyranosyluronic) - (1- * 4) -2-deoxy-6-O-sulfo-2-sulfoamino-αD-glucopyranose, tetrasodium salt, or αL-IdoAp2S- (1- »4) -αD-GlcNp2S6S:

lis: (α-L-idopyranosyluronic acid) - (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-α-D-glucopyranose, trisodium salt, or α-L-IdoAp- (1- * 4) -α -GlcNp2S6.S:

IIIs: (2-sulfo-α-L-idopyranosyluronic acid) - (1? 4) -2-deoxy-2-sulfoamino-o-D-glucopyranose, trisodium salt, or α-L-AdoAp2S- (1- * 4) -α-D-GlcNp2S ;

IdoAp: idopyranosyluronic acid; GlcNp: 2-amino-2-deoxyglucopyranose; AUap: 4-deoxy-α-L-threo-hex-enopyranosyluronic acid;

S: sulfate.

DETAILED DESCRIPTION OF THE INVENTION

Examples of preparation of a mixture of intermediates of formula (II)

Example A

Preparation of oligosaccharides of formula (II) in which n = 0, 1 and 2, by enzymatic depolymerization and separation of g heparin are dissolved in 250 ml of a phosphate buffer solution containing 0.05 mol / l NaH ₂ PO ₄ (pH = 7) 0.2 M sodium chloride and 2% BSA (bovine serum albumin). 7 µl of heparinase I (EC 4.2.2.2.7) is introduced into the mixture thus obtained and the solution obtained is cooled to 15 ° C, which is maintained at this temperature throughout the depolymerization reaction. The progress of this reaction is monitored by taking aliquots and analyzing them by permeation gel chromatography. After 9 days, the enzymatic reaction is interrupted by heating the reaction mixture to 100 ° C for 10 minutes. The mixture is then cooled and then lyophilized. A mixture of oligosaccharides III is thus obtained.

The resulting oligosaccharide mixture is then chromatographed using the following procedure:

Chromatography is carried out on polyacrylamidagarose gel columns known as Ultrogel ACA 202, eluting with a solution containing phosphate buffer (0.02 mol / l NaH ₂ PO ₄ ) pH = 7 and 0.1 mol / l sodium chloride. Detection is performed by UV spectrometry (254 nm) and ion spectrometry (IBF). Optionally, the products obtained can be purified by SAX chromatography on a strong ion exchanger or by fractional precipitation according to the procedure described in FR 2,548,672.

This method yields 3 g of disaccharide Als, 1100 mg of a hexasaccharide mixture, which typically contains 55% of the Als-Is-Is derivative, 35% of the Als-Is-IIs and 10% of the Als-Is-IIIs derivative. The latter mixture is either purified by known methods to separate each of the components, or the mixture is used alone for transformation into the derivatives of formula (I).

Example B

Preparation of oligosaccharides of formula (II) in which n = 0, 1, 2, 3 or 4, by depolymerization of benzyl ester of heparide and separation

(a) Preparation of heparin benzyl ester

The heparin benzyl ester is prepared as described in Example 3 of US Patent No. 5,389,618.

b) Depolymerization

100 g of heparin benzyl ester are dissolved in 1.9 l of demineralized water. The mixture was then heated to 60 ° C with stirring. After obtaining a homogeneous solution, about 35 ml of 23% sodium hydroxide solution are introduced into the solution at once. After 10 minutes of reaction, the solution is cooled and neutralized with 80 ml of 2 N acetic acid solution. To this solution was added 10% (w / v) sodium acetate. The oligosaccharide mixture is precipitated by the addition of about 2 volumes of methanol. The precipitate was collected by filtration, washed twice with methanol and then dried under reduced pressure at 50 ° C. After drying, 73.8 g of a mixture of oligosaccharides II are obtained.

c) Oligosaccharide enrichment in which n = 1 g of the oligosaccharide mixture obtained in the preceding step is dissolved in about 35 volumes of water. The resulting solution is ultrafiltered through a membrane with a 3 kDa separation threshold (Pellicon). When 600 ml of permeate flows out, the retained portion is diluted with 500 ml of water. The ultrafiltration is continued until a further 450 ml of permeate flows out. The two permeate fractions are combined and concentrated to dryness under reduced pressure. 6.1 g of a yellowish white solid are obtained. Analysis of this solid product by permeation gel chromatography showed that the product contained about 30% of the oligosaccharide of formula (II) in which n = 1.

d) Fractionation of ultrafiltered oligosaccharide mixtures

The enriched mixture is fractionated in columns which are packed with a polyacrylamidagarose gel known as Ultrogel ACA 202 (two columns in series, 10 cm in diameter and 50 cm in height) are used. 5 g of the ultrafiltration-enriched mixture are dissolved in 25 ml of water and then eluted with a 0.2 mol / l sodium chloride solution at a rate of 5 ml / min. Fractions of 25 ml are collected at the bottom of the column. Detection of the product was performed by UV spectrometry (254 nm) or ion spectrometry (IBF). The product fractions in which n = 1 are isolated, lyophilized and then desalted in a column which is

Filled with sephadex gel G10. After lyophilization, 1 g of tetrasaccharide is obtained, which typically contains 70% of the κ-Is derivative of formula (II) (R ¹ , R ² , R ³ , R ⁵ , R ⁷ , R ⁸ = SO ₃ Na; R ⁴ , R ⁶ = H and M = Na). Alternatively, the Als-Is derivative can be purified by SAX chromatography using a strong anion exchanger or preferably by traction precipitation, as described in FR 2,548,672 and a variant of the process described within the scope of the invention.

Examples of Preparation of Oligosaccharides of Formula (I)

Example 1

A solution of 300 mg of the oligosaccharide of formula (II) at 25 ° C is metered into the reactor in which n equals 0, R ¹ , R ⁷ and R ⁸ are SO ₃ M, R ⁶ is hydrogen and M is sodium; 2 ml water. With stirring, 212 mg of sodium borohydride are added in one portion. The pH is adjusted to between 9 and 10 by the addition of 0.5 M sodium hydroxide solution. After 12 hours, acetic acid is added gradually until a pH of between 4 and 5 is reached. The mixture is stirred for one hour, then the pH is adjusted to a value of 6.7 by addition of 0.5 M sodium hydroxide solution. The mixture is then concentrated to dryness under reduced pressure at 50 ° C.

The concentrate obtained is dispersed in 10 ml of methanol under magnetic stirring. After sedimentation overnight, the suspension is filtered through a Whatman GF / B membrane. Dissolve the solid collected on the filter with two portions of 10 ml of distilled water. This solution is then concentrated to dryness under reduced pressure at 50 ° C. 580 mg of a white solid are obtained.

The product is then dispersed in 15 ml of methanol under magnetic stirring. After stirring for 30 minutes, the suspension is filtered through a Whatman GF / B membrane. The obtained filter cake is dissolved in two 10 ml portions of distilled water. The residue is concentrated to dryness under reduced pressure at 50 ° C. 250 mg of an oligosaccharide of formula (I) in which n is equal to 0, R ¹ , R ⁷ and R ⁸ are SO ₃ M, R ⁶ is hydrogen and M is sodium, is obtained in the form of a mixture of diastereoisomers.

I and II are constitutive disaccharide sugars, wherein I is a reduced residue and II is an unsaturated uronic acid residue [(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1-> 4) (2-deoxy-6-O-sulfo-2-sulfoamino-D-glucitol, tetrasodium salt); (4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-D-mannitol, tetrasodium) salt);

Proton D _Z O, 600 MHz, T = 305 K, d in ppm:

3.38 (1H, m, H- ²¹ '),

3.70 (1H each and Dd, J = 6 and 12 Hz and dd, J = 5 and 12 Hz respectively, 2H-1 ^(I '),

3.86 (1H, t, J = 5Hz, H-3 ^(I) ),

4.13 (1H, dd, J = 8 and 11Hz, H-6 ⁽¹⁾ ),

4.18 (1H, m, H-4 ^(I ')), 4.25 (2H, m, H-6 '),

4.25 (2H, m, H-6 ^(II) );

4.35 (1 H, m, H-5 ⁽¹⁾ ),

4.65 (1H, m, H-2 ^{(1 ')} ),

5.67 (1 H, s, H-1 ^(II) ),

6.00 (1H, d, J = 5Hz, H-4 ™)].

Example 2

A solution of 60 mg of the oligosaccharide of formula (II) at 25 ° C is poured into the reactor in which n equals 1, R ¹ , R ² , R ³ , R ⁷ , R ⁷ and R ⁸ represent SO 3 M, R ⁴ and R ⁶ are hydrogen and M is sodium, in 1.2 ml of water. While stirring, 18 mg of sodium borohydride are added in one portion. The pH is adjusted to between 9 and 10 by the addition of 0.5 M sodium hydroxide solution. After 12 hours, acetic acid is added gradually until a pH of between 4 and 5 is reached. The mixture is stirred for 1 hour, then 5 ml of methanol are added. The suspension is filtered through a Whatman GF / B membrane and the isolated solid is washed twice with 0.5 ml of methanol. After drying, 42 mg of an oligosaccharide of formula (I) is obtained in which n is 1, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO 3 M, R ⁴ and R ⁶ are hydrogen and M represents sodium, in the form of a mixture of diastereoisomers.

I to IV are constitutive sugars of tetrasaccharides, wherein I is a reduced residue and IV is an unsaturated uronic acid residue [(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyl-uronic acid- (1- »4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-> 4) -2-deoxy-2-sulfoamino-6-O-sulfo-D-glucidtol, octasodium salt); (4-deoxy-2-O-sulfo-alpha-L-treohex-4-enopyranosyluronic acid- (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- ( 1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-> 4) -2-deoxy-2-deoxy-6-O-sulfo-2-sulfoamino-D-mannitol, octasodium salt );

proton spectrum in D ₂ O, 400 MHz, T = 298 K, delta in ppm:

3.25 (1H, dd, J = 10Hz and 3Hz, H-2 ^(III) ),

3.37 (1 H, m, H-2 ^(I) ),

3.59 (1 H, m, H-3 ^(III) ),

3.75 (2H, m, 2H-1 (R)),

3.79 (1H, t, J = 9Hz, H-4 ⁽ 1H ⁾ ),

3.86 (1H, m, H-3 ⁽¹⁾ ), between 4.05 and 4.40 (10H, solid, Η-4 ^ω / 2Η-5 ^ω / 2Η-6®, II-2 ⁽ 7 II -3 ⁽ⁱⁱ 7H- ^{4i )} , 2H-6 ⁽ 11H-3 ^(iv) ),

4.58 (1 H, m, H-2 ^(IV) ),

4.60 (1 H, m, H-5 ^(II) ), 5.27 (1 H, d, J = 4 Hz, H-1 ^(II) ),

5.27 (1H, d, J = 4Hz, H-1 ^{(II >} )),

5.42 (1 H, d, J = 4Hz, H-1 ^(III) ),

5.47 (1 H, d, J = 2Hz, H-1 ^(IV) ),

5.95 (1H, d, J = 5Hz, H-4 ^(IV) ).

Example 3

A solution of 100 mg of the oligosaccharide of formula (II) at 25 ° C is poured into the reactor in which n is 2, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and ^R6 are hydrogen and M is sodium, in mL of water. 20 mg of sodium borohydride are added in one portion with stirring. The pH is adjusted to between 9 and 10 by the addition of 0.5 M sodium hydroxide solution. After 12 hours, acetic acid is gradually added until a pH of between 4 and 5 is reached. The mixture is stirred for one hour, then the pH is adjusted to 6.7 by addition of 0.5 M sodium hydroxide solution. Dilute the mixture with distilled water to give a volume of 20 ml. Sodium acetate (2.5 g) was added followed by methanol volumes. The suspension is filtered through a Whatman GF / B membrane and the isolated solid is washed twice with 2 ml of methanol. After drying, 61 mg of an oligosaccharide of formula (I) is obtained in which n is 2, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are atom and M represents sodium, in the form of a mixture of diastereoisomers.

I to VI are constitutive sugars of hexasaccharides, wherein I is a reduced residue and V1 is a residue of unsaturated uronic acid [(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic- (1-> 4)) 2-deoxy-2-sulfoamino-6-O-sulfo-alpha-D-glucopyranosyl- (1 → 4)-2-O-sulfo-alpha-L-idopyranosyluronic acid- (1- * 4) -2- deoxy-2-sulfoamino-6-O-sulfo-alpha-D-glucopyranosyl- (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1- »4) -2-deoxy-2 -sulfoamino-6-O-sulfo-D-glucitol, dodecasodium salt); (4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1-> 4) -2-deoxy-6-O-sulfoamino-alpha-D-glucopyranosyl- (1- »4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-» 4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1- »4) 2-O-sulfo-alpha-L-idopyranosyluronic acid (1- (4) -2-deoxy-6-O-sulfo-2-sulfoamino-D-mannitol dodecasodic salt);

3.25 (2H, m, H-2 ^{(III) and (V)} ),

3.38 (1 H, m, H-2 ⁽¹⁾ ),

3.61 (2H, t, J = 10Hz, H-3 ^{(III) and (V)} ), between 3.70 and 3.83 (4H, Massif, 2H-1 ^(I) and H-4 ^{(1II), and (V)} ), 3.86 (1H, t, J = 5Hz, H-3 (R)), 4.00 (2H, m, H-5 ^{(III) and (V)} ), 4.07 (1H, m, H-4 ^(IV) ), 4.08 (1H, m, H-4 ^(I) ), between 4.10 and 4.45 (13H, massif, H-5 ⁽¹⁾ / 2H-6 ^{( 1)} , H-2 ⁽ III ⁾ / H-3 ⁽ⁿ⁾ / H-4 ^(II) , 2H-6 ^(II) , H-2 ^(IV) ZH-3 ^(IV) , 2H-6 ^(V) H-3 ^(VI) ), 4.60 (1H, s, H-2 ^(VI) ),

4.62 (1H, s, H-5 ^(II) ), 4.78 (1H, s, H-5 ^([V) ), 5.17 (1H, s, H-1 ⁽ 1H ⁾ ),

5.28 (1 H, d, J = 4 Hz, H-1 ^(II)), 5.38 (1H, d, J = 3 Hz, ^H-V), 5.44 (1 H, d, J = 3 Hz, H ^{<UI )} ),

5.47 (1H, d, J = 2Hz, H-1 ^(V1) ), 5.96 (1H, d, J = 5Hz, H-4 ^(VI) ).

Example 4

A solution of 100 mg of the oligosaccharide of formula (II) at 25 ° C is poured into the reactor in which n equals 3, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ represent SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium, in 2 ml of water. With stirring, 30 mg of sodium borohydride are added in two portions. The pH is adjusted to between 9 and 10 by the addition of 0.5 M sodium hydroxide solution. After 12 hours, acetic acid is added gradually until a pH of between 4 and 5 is reached. The mixture is stirred for 1 hour, then the pH is adjusted to 6.7 by addition of 0.5M sodium hydroxide solution. The mixture is then diluted with distilled water in an amount to give a mixture of 20 ml. 2 g of sodium acetate are added and then 3 volumes of methanol are added. The suspension is filtered through a Whatman GF / B membrane and the isolated solid is washed twice with 1 ml of methanol. After drying, 68 mg of a white solid is obtained. After checking by high performance liquid chromatography, it was found that the product was not completely reduced and therefore all the previous operations were repeated. After drying, 45 mg of an oligosaccharide of formula (I) is obtained in which R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen and M is sodium , in the form of a mixture of diastereoisomers.

I to VIII are constitutive sugars of octasaccharides, wherein I is a reduced residue and VIII is a residue of unsaturated uronic acid [(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1-> 4)) ) -2-Deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1- »4) -2 -deoxy-2-sulfoamino-6-O-sulfo-alpha-D-glucopyranosyl- (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1- »4) -2-deoxy- 6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1- * 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1 → 4) -2-deoxy-6-O -sulfo-2-sulfoamino-D-glucinol, hexadecasodium salt); (4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1- »4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl) - (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-> 4) -2-deoxy-6-O-sulfoamino-alpha-D-glucopyranosyl- (1- »4) -2-O-sulfo-alpha-L-idopyranosyluronic acid- (14) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1- »4) -acid 2- O-sulfo-alpha-L-idopyranosyluronic- (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-D-mannitol, hexadecasodium salt); proton spectrum in D ₂ O, 600 MHz, T = 305 K, delta in ppm:

3.25 (3H, m, H-2 ^(III) , H-2 ^(V) , H-2 ^(VII) ),

3.38 (1 H, m, H-2 ^(I) ),

3.61 (3H, m, H-3 ^(III) , H-3 ^(V) , H-3 ^(VII) ), between 3.70 and 3.83 (5H, massif, 2H-1 ^(I) and H-4 ^(III) , H-4 ^(V) , H-4 ^(VII) ), 3.86 (1 H, t, J = 5 Hz, H-3 ^(I) ), 4.00 (3H, m , Η-5 ^(ΙΠ), 5-H ^00, H-5 ^(VII)), 4.08 (3H, m, H-4 ^(l), II-4 ^(IV), II-4 ^{(V 1))} , between 4.10 and 4.45 (17H, massif, H-5 ^w / 2H-6®, H-2 ^{( )} / H-3 ^{< )} / H-4 ^{( )} , 2H-6 ^(n,) , H-2 ^(V) / H-3 ^(1V) , 2H-6 ^W , H2 ^(VI) / H-3 ^(VI) , 2H-6 ^(VII) , H-3 ^(VIII) ), 4.59 (1H, s, H-2 ^(VIII) ), 4.62 (1H, s, HR ⁽ 1H ⁾ ), 4.78 (2H, s, H-5 ^([V) , H-5 ^{(VI)) I)} , 5.17 (2H, s, H ^(V) , H-1 ^(VI1) ), 5.44 (111, d, J = 3 Hz, H-1 ^(II1) ),

5.47 (1 H, s, H-1 ^(VIII) ),

5.96 (1H, d, J = 5Hz, H-4 ^(V1II) ).

Example 5

The reactor was poured onto a solution of 65 mg of an oligosaccharide of formula (II) at 25 ° C, wherein n is 4, R ^1, R ^2, R ^3, R ^5, R ⁷ and ^R represent an SO 3 M, R ^4, and R 0 represents a hydrogen atom and M represents sodium, in 1.2 ml of water. While stirring, 18 mg of sodium borohydride are added in one portion. The pH is adjusted to between 9 and 10 by the addition of 0.5 M sodium hydroxide solution. After 12 hours, acetic acid is added gradually until a pH of between 4 and 5 is reached. The mixture is then stirred for one hour, then the pH of the reaction mixture is adjusted to 6.7 by addition of 0.5M sodium hydroxide solution. The mixture was then diluted with 3 mL of 10% aqueous sodium acetate and 3 volumes of methanol (12 mL) were added. The suspension is filtered and the isolated solid product is washed with 3 ml of methanol. After drying, 54 mg of an oligosaccharide of formula (I) is obtained in which n is 4, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO 3 M, R ⁴ and R ⁶ are hydrogen and M represents sodium, in the form of a mixture of diastereoisomers.

I to X are constitutive sugars of decasaccharides, wherein I is a reduced residue and X is an unsaturated uronic acid residue [(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1-> 4)) -2-deoxy-2-sulfoamino-6-O-sulfo-alpha-D-glucopyranosyl- (1 · 4 · 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (D> 4) -2 -deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1- »4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-» 4) -2-deoxy- 6-O-sulpho-2-sulfoamino-alpha-D-glucopyranosyl- (1-> 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1-> 4) -2-deoxy-6 -O-sulfo-2-sulfamino-alpha-D-glucopyranosyl- (1- * 4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1 → 4) -2-deoxy-6-O- sulfo-2-sulfoamino-D-glucitol, eicosodium salt); (4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid- (1? 4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- ( 1- (4) 2-O-sulfo-alpha-L-idopyranosyluronic acid- (1- * 4) -2-deoxy-6-O-sulfoaminoalpha-D-glucopyranosyl- (1-> 4 (-acid 2- O-sulfo-alpha-L-idopyranosyluronic- (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1- * 4) -acetic acid 2-O-sulfo -alpha-L-idopyranosyluronic- (1- »4) -2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl- (1-» 4) -acetic acid 2-O-sulfo-alpha- L-idopyranosyluronic- (1-> 4) -2-deoxy-6-O-sulfo-2-sulfoamino-D-mannitol, eicosodium salt; proton spectrum in D ₂ O, 600 MHz, T = 303 K, delta v ppm:

3.23 (4H, m, H-2 ^(III) , H-2 ⁽ H ⁾ , H-2 ^(VII) , H-2 ^(IX) ),

3.35 (1 H, m, H-2 ⁽ )))

3.59 (4H, m, H-3 ⁽ H ⁾ , H-3 ^(v) , H-3 ^(VII) , H-3 ^(IX) ), between 3.65 and 3.80 (6H, m),

3.85 (2H, m, H-3 ^(I) ), between 3.90 and 4.40 (29H, m),

4.57 (1 H, m, H-2 ^(X) ),

4.59 (1 H, m, H-5 ^(II) ),

4.57 (3H, m, H-5 ^(IV) , H-5 ^(VI) , H-5 ^(VI) ),

5.15 (3H, m, H-1 ^(IV) , H-1 ^(VI) , H-1 ^(VIII) ),

5.25 (1H, m, H ^{< n)} ),

5.37 (3H, m, H-1 ^(V) , H 1 ^{(V n} ', H-1 ^(IX) ),

5.42 (1H, m, HI ^(III) );

5.45 (1 H, m, H-1 ^(X) ),

5.93 (1H, d, J = 5Hz, H-4 ^(X) ).

The medicaments according to the invention comprise as active ingredient at least one oligosaccharide of the formula I or a mixture of oligosaccharides of the formula I in the form of a composition in which the at least one oligosaccharide is in combination with any other pharmaceutically compatible product which may be inert or physiologically active . The medicaments of the invention may be administered intravenously, subcutaneously, orally, rectally, topically or pulmonally (by inhalation).

Sterile compositions for intravenous or subcutaneous administration are generally aqueous solutions. These compositions may also contain additives, in particular wetting agents, isotonizing agents, emulsifying agents, dispersing agents and stabilizing agents. Sterilization can be accomplished in various ways, for example, by aseptic filtration, incorporation of sterilizing agents into the composition, or by irradiation. These compositions may also be prepared in the form of sterile solid compositions, which may be dissolved in sterile water or any other sterile injectable medium immediately prior to use.

As solid compositions for oral administration, tablets, pills, powders (in gelatin capsules or sachets) or granules may be used. In these compositions, the active ingredient is mixed under a stream of argon with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica. The compositions may also contain substances other than diluents, such as one or more lubricants, for example magnesium stearate or talc, an agent that promotes oral absorption, a colorant, a coating (dragees) or a lacquer.

As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil may be used. These compositions may also contain substances other than diluents, such as wetting, sweetening, thickening, flavoring or stabilizing agents.

Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active ingredient, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols.

Compositions for topical administration may be, for example, creams, lotions, oral disinfectants, patches, nasal drops or aerosols.

The doses of active ingredient administered will depend on the desired effect, the duration of treatment and the route of administration. These doses are generally between 0.5 and 10 mg / kg / day for subcutaneous administration, which is a daily dose of 3 to 60 mg for an adult patient weighing 60 kg.

In general, it will be the attending physician who determines the dosage of the active ingredient depending on the age, body weight and any other personal factors of the patient being treated.

The invention also relates to the use of the oligosaccharides of the invention for the preparation of a medicament for the prevention or treatment of diseases associated with the inflammatory process, which includes the formation of nitric oxide (NO), or for the survival and growth of motoneurons.

The invention is particularly advantageous for the use of the oligosaccharides of formula (I) for the preparation of medicaments for the prevention and treatment of cerebral, cardiac or peripheral vascular ischemia, osteoarthritis, central nervous system, skull, spine and combined injuries of skull and spine, sclerosis pain and peripheral neuropathies, motoneuron diseases, amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's chorea.

The invention also relates to a method for the prevention and / or treatment of diseases associated with the inflammatory process, which involves the production of cytotoxic agents such as nitric oxide (NO), and diseases associated with the survival and growth of motoneurons. The oligosaccharides of formula (I) may thus be used for the prevention and / or treatment of neurodegenerative diseases in which cerebral inflammation plays a malignant and possibly death event, including cerebral ischemia, cardiac ischemia (myocardial infarction), peripheral ischemia, nervous system injuries, and in particular spinal and combined skull and spinal injuries, multiple sclerosis, neuropathic pain and peripheral neuropathy, motoneuron diseases such as amyotrophic lateral sclerosis, progressive spinal atrophy, neuro-lateral atrial atrophy, neuroarthritis Alzheimer's disease, Parkinson's disease and Huntington's chorea and some forms of osteoarthritis, particularly with joint localization.

The invention also relates to a method of preventing and / or treating motoneuronal diseases such as amyotrophic lateral sclerosis, progressive spinal atrophy, infantile muscle atrophy and primary lateral sclerosis.

Claims (25)

hydrogen or SO ₃ M, R ² and R ⁷ , which are the same or different, are hydrogen or SO ₃ M or COCH ₃ and M is sodium, calcium, magnesium or potassium, or a mixture of these oligosaccharides, except oligosaccharides of formula (I) in which n is equal to 0 and - either R ¹ , R ⁶ and R ⁸ are hydrogen, R ⁷ is SO ₃ M or COCH ₃ and M is sodium, or R ^{@ 1} and R @ ⁶ are hydrogen, R @ 1 is COCH3, R @ ⁸ is SO3 M and M is sodium, - or R ⁶ represents a hydrogen atom, R ¹ , R ⁷ and R ⁸ represent a SO ₃ M group and M represents sodium, - or R ⁶ and R ⁷ represent hydrogen atoms, R ¹ and R ⁸ represent SO ₃ M and M represents sodium, - or R ^1, R ^s and R ⁷ are hydrogen, R ⁸ is SO ₃ M and M is sodium. Pharmaceutical composition according to claim 1, characterized in that it comprises an oligosaccharide of the formula (I) in which R ⁴ and R ^c represent a hydrogen atom or a mixture of these oligosaccharides. Pharmaceutical composition according to either of Claims 1 or 2, characterized in that it comprises an oligosaccharide of the formula (I) in which n represents an integer from 0 to 10, or a mixture of these oligosaccharides. Pharmaceutical composition according to one of claims 1 or 2, characterized in that it comprises an oligosaccharide of the general formula (I) in which n represents an integer from 0 to 6, or a mixture of these oligosaccharides. Pharmaceutical composition according to one of claims 1 or 2, characterized in that it comprises an oligosaccharide of the formula (I) in which n represents an integer from 1 to 6, or a mixture of these oligosaccharides. Pharmaceutical composition according to claim 1, characterized in that it comprises an oligosaccharide of the general formula (I) in which n equals 1, R ¹ , R ² , R ³ , R ³ , R ⁷ and R ⁸ represent SO ₃ M group. R ⁴ and R ⁶ are hydrogen and M is sodium. Pharmaceutical composition according to claim 1, characterized in that it comprises an oligosaccharide of the general formula (I) in which n is 2, R ¹ , R ² , R ³ , R ³ , R ⁷ and R ⁸ represent SO ₃ M group. R ⁴ and R ⁶ are hydrogen and M is sodium. Pharmaceutical composition according to claim 1, characterized in that it comprises an oligosaccharide of the formula (I) in which n is 3, R ¹ , R ² , R ³ , R ³ , R ⁷ and R ⁸ represent SO ₃ M R ⁴ and R ⁶ are hydrogen and M is sodium. Pharmaceutical composition according to claim 1, characterized in that it comprises an oligosaccharide of the formula (I) in which n is 4, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ represent SO ₃ M R ⁴ and R ⁶ are hydrogen and M is sodium. SK 286745 Β6 An oligosaccharide of formula (I) wherein n is an integer from 0 to 25, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁸ , which are the same or different, represent a hydrogen atom or a SO 3 M group, R ² and R ⁷ , which are the same or different, represent a hydrogen atom, or a SO 3 M group or a COCH ₃ group, and M represents sodium, calcium, magnesium or potassium, with the exception of oligosaccharides of formula I wherein n is 0 and either R ¹ , R ⁶ and R ⁸ are hydrogen, R ⁷ is SO 3 M or COCH 3 and M is sodium, or R ¹ and R ⁶ are hydrogen, R ⁷ is COCH 3, R ⁸ is SO 3 M and M R ⁶ is hydrogen, R ¹ , R ⁷ and R ⁸ are SO 3 M and M is sodium, or R ⁶ and R ⁷ are hydrogen, R ¹ and R ⁸ are SO 3 M and M is sodium, or R ¹ , R ⁶ and R ⁷ are hydrogen, R ⁸ is SO 3 M and M is sodium. The oligosaccharide of formula (I) according to claim 10, wherein R ⁴ and R ⁶ are hydrogen. The oligosaccharide of formula (I) according to any one of claims 10 or 11, wherein n is an integer from 0 to 10. An oligosaccharide of formula (I) according to any one of claims 10 or 11, wherein n is an integer from 0 to 6. The oligosaccharide of formula (I) according to any one of claims 10 or 11, wherein n is an integer from 1 to 6. An oligosaccharide of formula (I) according to claim 10, wherein n is 1, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen. and M represents sodium. An oligosaccharide of formula (I) according to claim 10, wherein n is 2, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen. and M represents sodium. The oligosaccharide of formula (I) according to claim 10, wherein n is 3, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen. and M represents sodium. An oligosaccharide of formula (I) according to claim 10, wherein n is 4, R ¹ , R ² , R ³ , R ⁵ , R ⁷ and R ⁸ are SO ₃ M, R ⁴ and R ⁶ are hydrogen. and M represents sodium. Process for preparing oligosaccharides of formula (I) according to claim 10, characterized in that an alkali metal or quaternary ammonium borohydride is reacted with oligosaccharides of formula (II) of hydrogen or a SO ₃ M, R ² and R ^{6 group} which: are the same or different, are hydrogen or SO ₃ M or COCH ₃ and M is sodium, calcium, magnesium or potassium, and the oligosaccharides obtained are then isolated. The process of claim 19, wherein the reaction is carried out in an aqueous medium, at a temperature near 25 ° C and at a pH of 7 to 10. Process according to either of Claims 19 and 20, characterized in that the reaction is carried out at a pH of 9 to 10. Process according to claims 19 to 21, characterized in that the alkali metal or quaternary ammonium borohydride is lithium, sodium or potassium borohydride or tetrabutylammonium borohydride. Use of oligosaccharides of formula (I) is hydrogen or SO ₃ M, R ² and R ⁷ , which are the same or different, are hydrogen or SO ₃ M or COCH ₃ and M is sodium, calcium, magnesium or potassium, or mixtures of these oligosaccharides for the preparation of a medicament for the prevention or treatment of diseases associated with an inflammatory process that includes the formation of nitric oxide (NO). Use of oligosaccharides of the general formula (I) according to claim 23 for the preparation of medicaments for the prevention and treatment of cerebral, cardiac or peripheral vascular ischemia, osteoarthritis, central nervous system injury, skull, spine injury and combined skull and spine injury, multiple sclerosis, and peripheral neuropathies, motoneuron diseases, amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer's disease, Parkinson's disease and Huntington's chorea. Use of oligosaccharides of general formula (I) in which n represents an integer from 0 to 25, R ¹ , R ³ , R ⁴ , R ³ , R ⁶ and R ⁸ , which are the same or different, represent a hydrogen atom or a SO 3 M group , R ² and R ⁷ , which are the same or different, are hydrogen or SO 3 M or COCH ₃ and M is sodium, calcium, magnesium or potassium, or mixtures of these oligosaccharides for the preparation of a medicament for the prevention and / or treatment of diseases which are associated with the survival and growth of motoneurons.