MXPA97008289A - Preparation and use of oligosacaridos sulfata - Google Patents

Abstract

The present invention relates to sulphated oligosaccharides, wherein the oligosaccharide has the general formula (I), wherein R1 and R2 and each Rx represent a unit of monosaccharide, which may all be the same or different, the units of monosaccharide by the glycosidic linkages 1 - > 2, 1 - > 3, 1 - > 4, and / or 1 - > 6 and n is an integer from 1 to 6, and the use thereof as anti-angiogenic, anti-metastatic and anti-inflammatory agents

Description

PREPARATION AND USE OF SULFATED OLIGOSACA FIELD OF THE INVENTION This invention relates to sulfated oligosaccharides, their preparation and use as anti-angiogenic, anti-metastatic and / or anti-inflammatory agents.

BACKGROUND OF THE INVENTION Heparan sulfates belong to the glycosaminoglycan family of polysaccharides. They are present in most multicellular animals and have a ubiquitous distribution, expressed on the cell surface and extracellular matrices (ECM) of most tissues (1, 2). Heparan sulfates usually exist as proteoglycans and have made considerable progress in sequencing and cloning the polypeptides from the center of the molecule. In addition, for example, at least eight core polypeptides of heparan sulfate proteoglycans (HSPG) have been identified on the cell surface (3). Initially HSPGs were considered to play a structural role on the cell surface and extracellular matrices. However, heparan sulfate chains exhibit a remarkable structural diversity (2,4) which suggests that they can provide important signaling information for many biological processes. Thus, although heparan sulfate chains are initially synthesized as a simple alternating repeat of glucuronosyl and N-acetylglucosaminyl residues linked by β-4 and Oíl-4 bonds there are many subsequent modifications. The polysaccharide is N-deacetylated and N-sulphated and subsequently undergoes C5 epimerization of glucuronosyl units in iduronosyl units, and several O-sulfations of the uronosyl and glucosaminyl residues. The variability of these modifications allow about thirty different disaccharide sequences which, when accommodated in different orders along with the heparan sulfate chain, can theoretically result in a large number of different heparan sulfate structures. With respect to this, the anticoagulant polysaccharide heparin present only in mast cell granules represents an extreme form where epimerization and sulfation have been maximized. Most heparan sulfates contain short stretches of highly sulfated residues joined by relatively long stretches of unsulfated units. Now there is clear evidence that heparan sulfates play a critical role in a wide range of biological processes (2-4). In particular, they can act as ligands for adhesion molecules involved in cell-cell interactions (5,6), participate in cell-extracellular matrix interactions (5,6) and act as essential cell surface receptors for growth factors as a factor of basic fibroblast growth (bFGF) (7,8) and vascular endothelial growth factor (VEGF) (9). HSPGs are also key components of basement membranes, which represent an important barrier to cell migration (10). Basement membrane barriers can only be broken when the cells display a range of degrading enzymes (11) that include an endoglycosidase, heparanase termed, which cleaves the chains of heparan sulfate (12,13). It has been shown that many of the biological processes in which heparan sulfates participate involve the recognition of unique heparan sulfate structures, with the position of the sulphates in the polysaccharide chain of critical importance (3). For example, it has been shown that the defined heparan sulfate sequences are recognized by acidic and basic fibroblast growth factor (14-16) and dissociated by heparanases. Based on these observations, it has been an objective of the present inventors to synthesize sulfated oligosaccharides which block the recognition of heparan sulfate by growth factors, and inhibit the dissociation of heparan sulfates by heparanases. In the case of blockage of growth factors, it was considered that low molecular weight imitations of heparan sulfate should be particularly effective, since it is now believed that cell surface heparan sulfates mediate the cross-linking of growth factors bound to their receptors (17). In addition, the sulphated oligosaccharides should be effective heparanase inhibitors acting as non-dissociable substrates of this enzyme. The sulphated oligosaccharides with inhibitory activity of the growth factor have several clinical uses. Heparin / heparan sulfate that binds growth factors, such as bFGF and VEGF, are potent inducers of angiogenesis (18). In adults, angiogenesis is a relatively rare occurrence except during wound healing. However, there are several "angiogenesis-dependent diseases" in adults where angiogenesis is critically important (18-20). The most important of these is angiogenesis associated with the growth of solid tumors, proliferative retinopathies and rheumatoid arthritis. Sulphated oligosaccharides that block the action of key angiogenic growth factors, such as bFGF and VEGF, would be particularly useful for the treatment of these angiogenesis-dependent diseases. Similarly, oligosaccharides that inhibit the action of heparanase have several clinical applications. The subendothelial basement membrane represents an important physical barrier for the passage of endothelial cells, tumor cells and leukocytes through the wall of blood vessels. The enzyme heparanase, combined with a range of proteolytic enzymes (eg plasmin, metalloproteinases on matrix), plays an essential part in the degradation of the basement membrane by invading cells (11-13, 21). Thus, by preventing degradation of the basement membrane, sulphated oligosaccharides with heparanase inhibitory activity should exhibit anti-metastatic and anti-inflammatory activity, and also inhibit early stages of angiogenesis. The use of sulfated oligosaccharides which simultaneously inhibit the action of angiogenic growth factor and heparanase enzyme would be preferred in many clinical situations, for example, treatment of highly metastatic solid tumors and rheumatoid arthritis. The above International Patent Application No.

PCT / AU88 / 00017 (Publication No. WO 88/05301) describes the use of sulfated polysaccharides such as heparin and modified heparin, fucoidin, pentosan sulfate, dextran sulfate and lambda carrageenan, which block or inhibit heparanase activity , in the anti-metastatic and / or anti-inflammatory treatment of an animal or human patient. In the work that led to the present invention, the inventors have prepared sulphated oligosaccharides using either natural oligosaccharides or fully synthetic oligosaccharides comprising hexose-containing homopolymers.

Some of these compounds have been shown to be potent inhibitors of human angiogenesis, tumor metastasis and inflammation. The information obtained is consistent with the sulphated oligosaccharides that exhibit their biological effects by inhibiting angiogenic growth factor and / or heparanase function and certain sulphated oligosaccharides have been obtained which are potent inhibitors of both angiogenesis and activity of heparanase.

SUMMARY OF THE INVENTION According to one aspect, the present invention provides sulphated oligosaccharides, wherein the oligosaccharide has the general formula I: R (R?) N-R2 (I) where Rj and R2 and each R? represents a monosaccharide unit, which may be the same or different, the adjacent monosaccharide units being linked by 1? 2, 1? 3, 1? 4 and / or 1? 6 glycosidic bonds; and n is an integer from 1 to 6. The sulphated oligosaccharides according to this invention are based on polymers of monosaccharide units, which can be linked by glycosidic bonds of 1? 2, 1? 3, 1? 4 and / or 1? 6 and which may consist of 3 to 8 monosaccharide units. Preferably, the oligosaccharides consist of 3 to 6 monosaccharide units (ie n is 1 to 4), more preferably 5 to 6 monosaccharide units (n is 3 to 4). The polymers may comprise homopolymers containing only one type of monosaccharide linkage or heteropolymers containing two or more different types of monosaccharide units. The monosaccharide units that are bonded together to form the oligosaccharides are preferably hexoses, and may be either furanoses such as fructose or pyranose such as glucose, mannose, altrose, allose, talose, galactose, idosa or gulose. The hexoses may be in the D- configuration or the L- configuration. In a particular aspect of the present invention, novel synthetic oligosaccharides having the general formula II are provided: R "(Ry) n-Ry (II) wherein each group Ry is the same and each represents a monosaccharide unit, the adjacent monosaccharide units being joined by 1? 3, 1? 4 and / or? 6 glycosidic bonds; and n is an integer from 1 to 6. In this particular aspect, the invention also provides sulphated oligosaccharides wherein the oligosaccharide has the general formula II above. Preferably, in the homopolymeric oligosaccharides of formula II, the monosaccharide unit is a hexose such as glucose, mannose, altrose, allose, talose, galactose, idosa or gulose. Preferably also, in these oligosaccharides n is from 1 to 4, more preferably from 3 to 4. The oligosaccharides of general formulas I and II also include compounds wherein the monosaccharide units are derivatives, in particular where the units are phosphate, acetyl derivatives or another ester of the monosaccharides. In general, the sulphated oligosaccharides of this invention can be prepared by sulfation of the oligosaccharides by methods known per se in the art to give their corresponding 0-sulphated derivatives. Suitable sulfation methods are exemplified below. The oligosaccharides to be sulfated may be natural products including natural oligosaccharides such as (for example raffinose and stachyose), as well as oligosaccharides prepared by enzymatic or chemical degradation of natural polysaccharides (for example maltotetrose, maltopentose and maltohexose).; glucotriosa, glucotetrosa, and glucopentosa; tetra, hexa- and octasaccharides of chondroitin; and mannitolose phosphate from the yeast Pichia holstii). As previously described, sulfated oligosaccharides falling within the scope of this invention have been shown to exhibit heparanase inhibitory activity and / or growth factor inhibitory activity, and accordingly in yet another aspect of the present invention extends to the use of sulphated oligosaccharide as described above with an anti-angiogenic, anti-metastatic and / or anti-inflammatory agent in the treatment of a warm-blooded animal patient (including a human). T the present invention extends to a method for the anti-angiogenic, anti-metastatic, and / or anti-inflammatory treatment of a human or other warm-blooded animal patient in need of such treatment, which comprises administering to the patient an effective amount of at least one sulfated oligosaccharide as described above. The active component is administered in therapeutically effective amounts. A therapeutically effective amount means the amount necessary to at least in part achieve the desired effect, or delay the outbreak of, inhibit the progress of, or stop, the outbreak or progress of the particular condition being treated. These amounts will, of course, depend on the particular condition being treated, the severity of the condition and the patient's individual parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed only with routine experimentation. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to deep medical judgment. Technicians with ordinary experience in the field will understand, however, that a lower dose or tolerable dose may be administered for medical reasons, physiological reasons or virtually for any other reason. The invention also extends to the use in the manufacture of a medicament for the anti-angiogenic, anti-metastatic and / or anti-inflammatory treatment of a human or other warm-blooded animal patient of at least one sulfated oligosaccharide as described above. In addition, this invention also provides a pharmaceutical or veterinary composition for anti-angiogenic, anti-metastatic and / or anti-inflammatory treatment, which comprises at least one sulfated oligosaccharide as described above, together with a pharmaceutically and veterinarily acceptable carrier or diluent for it. The formulation of these therapeutic compositions is well known to persons skilled in the art. Suitable pharmaceutically or veterinarily acceptable vehicles and / or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption retardant agents, and the like. The use of these media and agents for active pharmaceutical and veterinary substances is well known in the art, and is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any medium or agent is incompatible with the active ingredient, use thereof is contemplated in pharmaceutical and veterinary compositions of the present invention. Supplementary active ingredients may also be incorporated into the compositions. It is especially convenient to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. The unit dosage form as used herein refers to physically discrete units suitable as unit doses for the human or animal being treated: each unit contains a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the vehicle and / or pharmaceutical or veterinary diluent required. The specifications for the unit dosage forms of the invention are dictated by and directly depend on (a) the unique characteristic of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of composing this active ingredient for the particular treatment. The sulphated oligosaccharides of this invention can be used in the treatment of angiogenesis-dependent diseases including angiogenesis associated with the growth of solid tumors, proliferative retinopathies and rheumatoid arthritis, as well as the treatment of inflammatory diseases and conditions in which the inhibitory activity of the heparanase of sulfated oligosaccharides would be particularly useful for inhibiting leukocyte infiltration, including chronic inflammatory diseases where leukocyte infiltration is a key element such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, inflammatory bowel diseases such as ulcerative colitis and Chron's disease, graft rejection and chronic asthma. In all this specification, unless the context requires otherwise, the word "understand", or variations such as "comprises" or "comprising", shall be understood as implying the inclusion of an integer or group of declared integers but not exclusion. of any other integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION As amply described above, the present invention relates to sulfated oligosaccharides with their use as anti-angiogenic, anti-metastatic, and / or anti-inflammatory agents. Some oligosaccharides can be obtained from natural sources for subsequent sulfation, however, a simple procedure for synthesizing oligosaccharides of defined chain length and stereochemistry is highly desirable. The present invention provides an improved method for synthesizing and isolating oligomers of hexose sugars from simple chromatography techniques. Many examples can be found describing methods for preparing sugar polymers and oligomers in the scientific and patent literature. For example, in a commonly used process an unmodified sugar monomer, either alone or in the presence of a solvent, can be heated in the presence of a catalyst to give branched and linear polymeric products with several and sometimes undefined chemical bonds (22). , 2. 3) . Another method where the sugar is melted in the presence of cation exchange resins (24) also gives highly branched polymers of high molecular weight. In these two examples, the polymers are formed with the concomitant loss of one molecule of water for each polymer bond formed. Another example is a known method of staggered polymerization "which involves using the Koenings-Knorr reaction where the sugars that have non-hydroxyl groups (such as a bromine or chlorine atom) in the position and protective groups (such as acetyl) in other hydroxyl groups of sugar the hydroxyl groups are reacted in position 1 with a hydroxyl group in another sugar (24). In these methods a molecule other than water, for example HBr, is lost during polymer bond formation. This method of preparing oligosaccharides is tedious, requires the preparation of complex initial materials and gives poor total yields (25). Similarly it is known that a hexose sugar containing a primary alcohol group on carbon 6 and O-protecting groups (such as acetyl) at positions 2, 3 and 4 and a leaving group such as bromine at position 1 will self-condense, especially in the presence of a catalyst such as silver oxide, to give bound polymers 1.6 ,; a series of gentiodextrins have been prepared in this way to give 1-bromo-2,3,4-tri-O-acetyl-oí-D-glucose: however the oligosaccharide productions were low due to the formation of 1, 6-anhydro-β-D-glucose derived from intramolecular condensation; yields of the dimer (14 percent) and trimer (22 percent) were not good and yields of the tetramer and pentamer were worse (<5 percent) and the hexamer was isolated with only a 1 percent yield (26). More recent publications describe chemical synthesis of polymers of 1, 6-bond-jS-pyranosyl units (27) and D-dextran (28), made by the ring-opening polymerization reaction of anhydrous sugar derivatives. This method is hampered by considerable effort to prepare the initial anhydrosugar material and there is no evidence that the oligosaccharides can be easily prepared by this method even when the reactions are carried out, for example -60 ° C. Another method has employed acid catalyzed melt polymerization of 1,2,3,4-tetra-O-acetyl-0-D-glucose to prepare a mixture of 1, 6-linked oligosaccharides acetates which, after deacetylation and undergoing chromatographic examination, has been shown to contain at most mono and disaccharides, namely glucose (15 percent), levoglucosan (4 percent) and gentiobiose (16 percent) while the yield of oligosaccharide was unacceptably low, specifically, gentiotriosa ( 4 percent) and gentiotetrosa (0.6 percent (29).) This method was described later in greater detail (30) and although the yield of polymerized products improved slightly it resulted in only very low yields of the expected oligomers. literature that, although there are already several methods for preparing several oligosaccharides, no method for synthesizing homo-oligosaccharides in good yield from readily available and inexpensive starting material has been described to date. In work that led to this invention, the inventors discovered a process by which hexopyran oligosaccharides can be synthesized in good yield from readily available and inexpensive starting materials. In accordance with this aspect of the invention, there is provided a process for the preparation of the hexopyran oligosaccharides which comprises heating an acetyl or other ester derivative of a hexose in an inert solvent under reduced pressure and in the presence of a Lewis acid or other catalyst. According to this process, the oligomerization of derived hexose sugars includes, but is not restricted to, 1, 2, 3, 4-tetra-0-acetyl derivatives of glucose, mannose, galactose, altrose, talose, gulose, iodine and allose it can be made to take place in a controlled manner to give O-acetylated hexose oligosaccharides. In this process, the degree of oligomerization (chain length) can be easily controlled by manipulating the temperature at which the oligomerization reaction is carried out and varying the time over which the reaction is allowed to proceed. Following the oligomerization reaction, the crude product mixture can be subjected to another acetylation, in order to acetylate the remaining free hydroxyl groups of the oligosaccharides. The acetylated oligosaccharides can then be easily separated by chromatography by adsorption. The acetyl groups have ultraviolet light absorbance, facilitating the use of spectrophotometry to identify the acetylated oligosaccharides as they are sequentially leached from the column. The acetyl protection groups can also be removed from the oligosaccharide mixture, and the resulting oligosaccharides separated according to size by gel filtration chromatography (size exclusion). In the examples described herein, the sulphated oligosaccharides are isolated and used as their respective sodium salts. It will be understood that other pharmaceutically acceptable salts, such as calcium or pharmaceutically acceptable amine salts, can be isolated and used in the corresponding manner. Accordingly, references herein to "sulphated oligosaccharide" will be understood to include sodium or other pharmaceutically acceptable salts of the sulfated oligosaccharide. Other features of the present invention are described more fully in the following Examples. It will be understood, however, that this detailed description is included solely for the purpose of exemplifying the present invention and should not be construed in any way as a restriction on the broad description of the invention as stipulated above. Examples 1, 2 and 3 exemplify the preparation of synthetic oligosaccharides by the novel process described herein, Examples 4, 5 and 6 exemplify a process for the sulphation of oligosaccharides, and Example 7 exemplifies the use of sulphated oligosaccharides as agents anti-antiogenic, anti-metastatic and / or anti-inflammatory. (In Examples 1 and 2, "ND" represents "not determined.") In the accompanying drawings: Figure 1 shows the effect of maltohexose sulfate on human angiogenesis in vi tro. The upper figure is a digital image of angiogenesis control 14 days after the start of the culture. The lower figure represents angiogenesis in the presence of 20 μg / milliliter of maltohexose sulfate. Figure 2 shows the effect of different concentrations of maltose sulfate (D), maltotetrose sulfate (O) and maltohexose sulfate (M) on human angiogenesis in vi tro. the information obtained from digital images of the angiogenic response 14 days after the start of the culture. Each average value + standard error (n = 4). Figure 3 shows the effect of different concentrations (μg / milliliter) of mannitol phosphate from Pichia holstii in human angiogenesis in vitro. The information obtained from the digital images of the angiogenic response 19 days after the start of the culture. Each average value + standard error (n = 4). Figure 4 shows the effect of sulphated maltose oligosaccharides of different chain length on the metastasis of rat mammary adenocarcinoma 13762 MAT. The control animals received 13762 MAT cells in the absence of oligosaccharide. In panel A the treated animals received 2 milligrams intravenously of each compound at the time of injection of the tumor cell. In panel B treated animals received 4 milligrams, subcutaneously, of each compound at the time of tumor cell injection. The vertical bars represent standard errors of the means. Figure 5 is an assessment of the ability of sulphated maltose oligosaccharides of different chain lengths to inhibit heparan sulfate binding at the cell surface in BALB / c 3T3 cells for immobilized aFGF. The bound 3T3 cells were quantified by Rose Bengal staining and by measuring dye absorbency at 540 nm. The degree of sulphation of the different maltose oligosaccharides is listed in Table 2. Figure 6 shows the effect of sulphation degree of maltohexose sulfate on its ability to inhibit metastasis of rat mammary adenocarcinoma 13762 MAT. The numbers along the X axis refer to the number of sulfate / maltohexose molecule groups. The control animals received tumor cells in the absence of the compounds. The oligosaccharides were administered at a dose of 2 milligrams / rat, intravenously, at the time of tumor cell injection. The vertical bars represent standard errors of the means. Figure 7 shows the effect of maltohexose with different numbers of sulfate / molecule groups on human angiogenesis in vi tro. The oligosaccharides were added at 200 μg / milliliter and the test was performed in serum-free medium. A similar angiogenic response was observed in this experiment whether the test was performed on medium containing serum (20 percent FCS) or without serum (without FCS). Maltohexose with 20 sulphates / molecule represents the molecule sulfated to the maximum. The information is average + standard error of 4 determinations. Figure 8 shows the effect of different sulphated oligosaccharides containing mannose on human angiogenesis in vi tro. The values in square brackets represent percentage of sulfation of the oligosaccharides. Oligosaccharides were added at 200 μg / milliliter and the test was performed in medium containing serum. The information is average + standard error of four determinations. Figure 9 shows the effect of oligosaccharides of sulfated mannose of different chain length on the metastasis of rat mammary adenocarcinoma 13762 MAT. The values in brackets represent the percentage of sulfation of the oligosaccharides. The control animals received 13762 MAT cells in the absence of oligosaccharides. The treated animals received either 2 milligrams (A) or 4 milligrams (B) subcutaneously of each compound immediately after the intravenous injection of tumor cell. The vertical bars represent the standard errors of the means. Figure 10 shows the effect of sulphated galactose and glucose oligosaccharides of different chain length on the metastasis of rat mammary adenocarcinoma 13762 MAT. The values in square brackets represent percentage of sulfation of the oligosaccharides. The control animals received 13762 MAT cells in the absence of oligosaccharide. The treated animals received 2 milligrams subcutaneously of each compound immediately after the intravenous injection of tumor cell. The vertical bars represent standard errors of the means.

EXAMPLE 1 The mannose oligosaccharides were obtained in the following manner: 1, 2, 3, 4-tetra-O-acetylmanose (31) (15.0 grams, 43 mmol) and zinc chloride (1.5 grams) were completely mixed in tetramethylene sulfone ( 7 milliliters), this mixture was heated under reduced pressure with stirring at about 110 ° C for 6 hours; at this point the mass of the reaction hardened and the generation of steam (acetic acid) ceased. A soda and lime tube was placed between the reaction vessel and the vacuum source throughout the reaction time. The reaction mixture was allowed to cool and a portion (11.0 grams) of the product mixture was dissolved in dry pyridine (20 milliliters) and to this solution was added acetic anhydride (2 milliliters), this mixture was protected from atmospheric humidity and it was heated to about 50 ° C with stirring for 2 hours. After cooling, ethanol (10 milliliters) was added and the mixture was allowed to stand for 2 hours. The pyridine, ethanol and any ethyl acetate formed were evaporated under reduced pressure and the residue was washed thoroughly with water to remove zinc chloride, tetramethylene sulfone and pyridine. The residue was dissolved in dichloromethane, washed with water and the organic layer was dried over anhydrous sodium sulfate. The derived oligosaccharides were initially separated into two fractions by applying the dichloromethane solution to a short column (4 x 40 centimeters) of silica gel 60 (130 grams) which was first eluted with chloroform and then with acetone. Elution with chloroform gave a mixture of the completely acetylated monosaccharide and oligomers containing from 3 to 5 units of sodium (Mixture A). Subsequent elution with acetone gave a mixture of completely O-acetylated oligomers containing mainly from 6 to 12 mannose units per molecule (Mixture B). Mixture A (4 grams) is applied to a column (3.3 x 135 centimeters) packed with grade tic silica gel (H). The column was eluted with acetone / light petroleum (boiling point 60-80 °) with a gradient starting at 1: 5 and the percentage of acetone increased until a final ratio of 1: 1 was reached. The flow rate was ^ 0.5 milliliters / minute. By collecting and combining the appropriate fractions (determined by silica gel tic analysis) and removing the solvent under reduced pressure oligosaccharides were obtained from completely O-acetylated mannitol Id as follows (n = number of residues of mannose, yield, molecular rotation (c = 2, CHCI3)): la, (3, 0.7 grams, 4.2 percent, [a] 27D = + ND); Ib, (4; 4.1 grams, 8.4 percent, [] 27D = + 50); le, (5; 1.2 grams, 7.9 percent, [OÍ] 27D = + 47.3); Id, (6, 0.8 grams, 5.2 percent, [a] D = + 36.0). Mix B (7.0 grams) was chromatographed in a manner similar to Mixture A but the elution gradient started with acetone / light oil (boiling point 60-80 °) 4: 5 and the percentage of acetone increased until it was 100 percent. In this way, the oligosaccharides were obtained from completely 0-acetylated ld-lj as follows (n = number of mannose residues, -performance, molecular rotation (c = l, CHCI3)): Id, (6; 1.6 grams, 10.4 percent, [a] 27D = + ND); le, (7; 3.2 grams, 21.2 percent, [a] 27D = + 50.0); lf, (8; 0.5 grams, 3.4 percent, [a] 27D = + 47.0); lg, (9; 0.7 grams, 4.7 percent, ta] 27D = + 59); lh, (10; 0.9 grams, 5.7 percent, [a] 27D = + 54); li, (11; 0.02 grams, 0.1 percent, [a] 27D = + ND); lj, (12; 0. 03 grams, 0.2 percent, [a]. The compound (1.55 grams) was dissolved in dry methanol (40 milliliters) and 1M of methanolic sodium methoxide (8.6 milliliters) was added with stirring at room temperature. The resulting precipitate was filtered, washed thoroughly with methanol and dried. The product (2a; 0.61 grams, 70 percent [o]] 27D = + 72 °) was identified as manotriose by elemental analysis (CHN values were ± 0.4 percent of what was expected), mass spectrometry by electroaspersion (M + 504) and electron magnetic resonance spectroscopy. The lb-lj oligomers were similarly treated to give the following mannose oligosaccharides (n = number of mannose residues, yield percentage of acetylated derivatives, molecular rotation (c = 1, H20)): 2b, (4, 75 percent) , [a] 27D = + 78 °); 2c, (5, 85 percent, [aj 27D = + 80 °); 2d, (6, 98 percent, [a] 27D = 84 °); 2e, (7, 98 percent, [a] 27D = 86.3 °); 2f, (8; 99 percent, [a] 27D = + 98.0); 2g, (9; 99 percent, [a] 27D = + 106 °); 2h, (10; 99 percent, [a] 27D = + 100 °); 2i, (11; 98 percent, [a] 27D = ND); 2j, (12, 98 percent, [a].) Alternatively, these mannose oligosaccharides can be isolated by gel filtration chromatography (size exclusion), so that a mixture was obtained by heating a completely stirred mixture of 1, 2, 3 , 4-tetra-0-acetylmanose (15.0 grams, 43 mmol) and zinc chloride (1.5 grams) in tetramethylene sulfone (7 milliliters), under reduced pressure at approximately 110 ° C for 6 hours as described above. the reaction was allowed to cool, and water (50 milliliters) was added and the reaction mixture was stirred at room temperature for 5 minutes and the water layer was discarded.This washing procedure was repeated and the mass was subsequently dissolved in chloroform , washed with water and dried over anhydrous sodium sulfate.After filtering, the chloroform was removed under reduced pressure to give the mixture of crude acetylated oligosaccharide. (11.3 grams). This mixture was dissolved in isopropanol (20 milliliters) and methanol (60 milliliters) and then 1 M sodium methoxide in methanol (8 milliliters) was added and the mixture was allowed to stand at room temperature for 1 hour. The resulting precipitate was filtered and washed twice with methanol (30 milliliters). After drying this product mixture (6.5 grams) was applied to the top of a gel filtration column (BioRad) of fine grade P2 gel (coated, 5x90 centimeters) which had been stabilized by operating for two days with water ( flow rate 0.5 milliliters per minute at 60 ° C. The column was eluted with water, at a flow rate of 0.5 milliliters per minute.The products eluting from the column were identified as peaks by differential refractometry and the fractions were collected at Thus, the fractions corresponding to the areas under 11 separate peaks were collected, each of these fractions was rechromatographed on an identical P2 gel column at 60 ° C and eluted with water at a flow rate of 0.5. milliliters / minute Thus, by way of example, the fraction identified as corresponding to the mannitol (0.9 grams) of the first gel filtration operation was rechromatographed for Give a main central peak with one shoulder on each side. The product eluting in the central peak was isolated by removing the water under reduced pressure to give (0.5 grams) of material that was rechromatographed on an identical P2 gel column at 60 ° C at a flow rate of 0.3 milliliters / minute. In this way, manopentose (0.3 grams) identical to the previous 2c was obtained. Found C 38.4; H, 6.7, C30H52O26 • 6 H20 requires C 38.5; H 6.8 percent. The value of nitrogen was 0 percent found and 0 percent expected. The compound was found to be substantially pure by high performance liquid chromatography (HPLC).

This was determined in a Dionex high performance liquid chromatography system configured as follows: Column: Code-CPMA1 # 1291 (+ guard # H72). A column of Ion Exchange of Quaternary Ammonium. Detector: Electrochemical detector (ED40: IAMP). Flow rate: 1 milliliter / minute. Solvents: Solution A: 0.1M NaOH Solution B: 1M acetate in 0.1M NaOH Gradient: Time (min)% A% B Action 0 95 5 Elution 20 90 10 Elution 25 0 100 Elution / Wash 30 0 100 Elution / Washing Spectrometry of mass by electroaspersion showed that this compound had a mass M + of 828, the right molecular weight of the mannitol. In a similar manner manotriosa, manotetrosa, manohexosa and manoheptosa identical to 2a, 2b, 2d and 2e were isolated.

EXAMPLE 2 This example shows the effect of carrying out the polymerization reaction at a lower temperature than in Example 1. The glucose oligosaccharides were obtained by polymerization of 1, 2, 3, 4-tetra-0-acetylglucose from the same as for the mannose oligosaccharides produced by the method set forth in Example 1 above, except that in this case, the polymerization reaction was carried out at 90 ° C for 8 hours. The reaction mixture was treated as in Example 1 and the products were isolated by column chromatography, where the column (7 centimeters x 155 centimeters) was packed with tic grade silica gel (H). Using similar methods of elution to those described in Example 1, the oligosaccharides of fully O-acetylated glucose 3a-3e were obtained as follows: (n = number of glucose residues, yield, molecular rotation (c = 2, CHC13)): 3a, (3, 4.14 grams, 24.9 percent, [a] 27D = + 37.5 °); 3b, (4; 2.92 grams, 18.4 percent, [a] 27D = + 44 °); 3c, (5; 2.99 grams, 19.1 percent, [a] 27D = + 37.5 °); 3d, (6; 1.37 grams, 8.9 percent, [a] 27D = + 36 °); 3e, (7, 0.18 grams, 1.2 percent, [a] 27D = + 39 °). Compound 3a (1.0 grams) was dissolved in dry methanol (30 milliliters) and 1M methanolic sodium methoxide (5.5 milliliters) was added with stirring at room temperature. The resulting precipitate was filtered, washed thoroughly with methanol and dried. The product (4a; ([of] 27D = 68.5 °) was identified as glucotriosa by elemental analysis, electrospray mass spectrometry (M + = 504) and neutron magnetic resonance spectroscopy.The 4b-4e oligomers were treated similarly to give the following glucose oligosaccharides (n = number of glucose residues, yield percentage, molecular rotation (c = 2, H20)): 4b, (4; 85 percent, [Qf] 27D = + 83 °); 4c, (5, 90 percent, [a] 27D = + 84 °), 4d, (6, 90 percent, [CÜ] 27D = 86 °), 4e, (7, 89 percent, [a] 27D = + 92.5 °) Alternatively, these glucose oligosaccharides can be isolated by gel filtration chromatography (size exclusion) Thus, a mixture of acetylated glucose oligosaccharides was obtained after heating a completely stirred mixture of 1, 2, 3, 4-tetra-O-acetylglucose (15.0 grams, 43 mmol) and zinc chloride (1.5 grams) in tetramethylene sulfone (7 milliliters), under reduced pressure to approximate at 110 ° C for 6 hours as described above. The reaction mass was dissolved in dichloromethane, washed with water and dried over anhydrous sodium sulfate. The dichloromethane was removed under reduced pressure and the product was weighed and dissolved in isopropanol (20 milliliters) and methanol (60 milliliters) and then 1 M sodium methoxide in methanol (9 milliliters) was added and the mixture was allowed to stand at room temperature. environment for 1 hour. The resulting precipitate was filtered and washed twice with methanol (30 milliliters). A portion (7.6 grams) of this mixture was dissolved in water (10 milliliters) and applied to a 5 x 90 cm water-coated chromatography column with thin grade P2 exclusion gel (BioRad). The column was packed, preheated and operated at 60 ° C for two days before use. After the addition of the glucose oligosaccharide mixture the column was maintained at 60 ° C and eluted with water (1 milliliter / minute). The products eluting from the column were identified as peaks by differential refractometry and the fractions were collected accordingly. In this way, the fractions corresponding to the areas under 10 separate peaks were collected. Each of these fractions was rechromatographed on an identical P2 gel column at 60 ° C and eluted with water at a flow rate of 0.5 milliliters / minute. Thus, by way of example, the fraction identified as corresponding to glucopentose (0.59 grams) was rechromatographed to give a main central peak with one shoulder on each side. The product eluting at the central peak was isolated by removing the water under reduced pressure to give (0.3 grams) of material which was again rechromatographed on a P2 gel column identical to 60 ° C at a flow rate of 0.3 milliliters /minute. In this way glucopentose (0.2 grams) was obtained, identical with previous 4c. Similarly, glucotryose, glucotetrose, glucohexose and glycoheptose identical to 4a, 4b, 4d and 4e were isolated.

EXAMPLE 3 Oligosaccharides of other hexose sugars including galactose, altrose, talose, gulose, iodine and allose can be obtained following the methods described in Examples 1 and 2. For example, the galactose oligosaccharides were obtained as follows: 1, 2 , 3, 4-tetra-0-acetylgalactose (21.0 grams) and zinc chloride (2.1 grams) were completely mixed in tetramethylene sulfone (10 milliliters), this mixture was heated under reduced pressure with stirring at about 90 ° C for 17 hours; At this time the mass of the reaction hardened and the generation of steam (acetic acid) stopped. A soda and lime tube was placed between the reaction vessel and the vacuum source throughout the reaction time. The reaction mixture was allowed to cool, the reaction mass was subsequently dissolved in dichloromethane, washed with water and dried over anhydrous sodium sulfate. The dichloromethane was removed under reduced pressure and the product was weighed and dissolved in isopropanol (30 milliliters) and methanol (70 milliliters) and then 1M sodium methoxide in methanol (10 milliliters) was added and the mixture was allowed to stand at room temperature during the hour. The resulting precipitate was filtered and washed twice with methanol (30 milliliters). This mixture was separated by gel filtration chromatography as described for the polymers of mannose and glucose in Examples 1 and 2 above. The products eluting from the column were identified as peaks by differential refractometry and the fractions were collected accordingly. In this way, 8 fractions were collected. Seven of these fractions were rechromatographed twice on an identical P2 gel column at 60 ° C and eluted with water at a flow rate of 0.5 milliliters / minute during the first operation and 0.3 milliliters / minute in the second. In this way the following galactose oligosaccharides were obtained: galactotriose, 1.03 grams, 5.3 percent; galactotetrose, 1.15 grams, 6.0 percent; galactopentose, 1.21 grams, 6.3 percent; galactohexose, 4.26 grams 22.1 percent, galactoheptose, 2.11 grams, 11 percent; galacto-octose, 1.91 grams, 9.9 percent and galactonose, 0.08 grams, 0.4 percent.

EXAMPLE 4 To a solution of sulfur trioxide-pyridine complex (0.8 grams) (Aldrich) in fresh distilled DMF (1 milliliter) at 80 ° C was added dropwise a solution of manopentose (2c) (0.1 grams) in dry pyridine (3 milliliters), and everything was heated at 80 ° C for another 90 minutes. The supernatant was decanted while still hot and the sticky residue was washed thoroughly with methanol (2 milliliters) three times. After decanting the residual methanol, the product was dissolved in water (5 milliliters) and neutralized (to pH of about 6) with barium acetate (approximately 0.4 grams in 2 milliliters of water) with vigorous stirring. After centrifugation (3,000 x g), the superimposed liquid was decanted and retained and the precipitated barium sulfate agglomerate was thoroughly washed with water (3 x 10 milliliters). The retained superimposed liquid and the washings were combined and placed on a column (1.0 x 14 centimeters) of DOWEX 50W-X8-400 cation exchange resin (H + form). The column was eluted with water until the eluate remained neutral. The eluate (approximately 50 milliliters) was stirred and neutralized (to a pH of about 7) with sodium acetate (0.7 grams). The solution was diluted with acetone (200 milliliters) and centrifuged (1,750 x g) to separate the product. The agglomerate was finely pulverized by grinding under methanol, and then stirred while still under methanol and then filtered. The solid was washed several times with methanol to give the pure compound (without inorganic salt) (0.2 grams, 66 percent). The product was not contaminated with barium ion (by microanalysis and flame ionization) or nitrogen (microanalysis). It was found that the product, sulfated mannitol, had 11 out of a total of 17 possible sulphated positions. Found C 14.2; H, 3.0; S 14.1; Na 7.3. C3oH60059S l ^ ag. 36 H20 requires C 14.1; H 5.2; S 13.8; Na 7.2 percent. The values of nitrogen and barium were 0 percent found and 0 percent expected.

EXAMPLE 5 To a mixture of sulfur trioxide complex, pyridine (Aldrich Chemical Company) (4 grams) in dry DMF (5 milliliters) was added dry pyridine (10 milliliters) under an atmosphere of dry nitrogen. This mixture was heated to 50 ° C and stirred rapidly while the glycohexose (4d) (0.5 grams), isolated by the method described in Example 2 above, was added in a single addition. Additional pyridine (5 milliliters) was added and the mixture was then heated, with continuous stirring at 80 ° C for 90 minutes. The reaction mixture was then kept at 4 ° C overnight. The liquid was decanted from the reaction vessel, methanol (3 milliliters) was added and the semi-solid mass was broken and thoroughly mixed with the methanol. After the methanol was settled, it was decanted and this procedure was repeated. Water (5 milliliters) was added to the remaining solid and the resulting solution was placed in a 50 milliliter test tube. The reaction vessel was flushed with additional water (5 milliliters) which was combined with the first solution. The resulting solution was adjusted to a pH of about 7-8 with 40 percent NaOH, after which methanol (40 milliliters) was added. The resulting cloudy solution was centrifuged (3,000 x g) for 25 minutes and the clear solution of the precipitate was decanted. The remaining solid was dissolved again in water (10 milliliters), methanol (40 milliliters) was added, and the tube was centrifuged as before. After decanting the clear superimposed solvent, the solid was dissolved in water (10 milliliters) and passed through a P2 gel desalting column (2.5 centimeters x 250 centimeters; fine grade P2 gel - BioRad) to give the salt sodium concentration of the sulfated derivative of 1,6-glucohexose.

EXAMPLE 6 Although the synthesis of hexose homopolymers is described in Examples 1, 2 and 3, it is usually extremely difficult to synthesize most of the oligosaccharide structures. Thus, a simpler approach is to sulfate oligosaccharides of defined structure from natural sources. The natural product oligosaccharides used in this example were of two kinds. The first class contained oligosaccharides that did not require further degradation and fractionation. Examples of this class are maltose, raffinose and stachyose. The second class consisted of oligosaccharides obtained from natural polysaccharides that were partially degraded enzymatically or chemically, and fractionated by size. Examples of this class are oligosaccharides derived from amylose, chondroitin and dextran and manopentose phosphate from the yeast Pichia holstii. Maltose, raffinose and stachyose were purchased from Sigma Chemical Co., St. Louis, MO. Maltotriose, maltotetrose, maltopentose, maltohexose and maltoheptose were obtained from Seikagaku, Tokyo, Japan and represent purified oligosaccharides from limited amylase digesters of the al-4 glucose homopolymer bound amylose. The tetra-, hexa- and octasaccharides of chondroitin were purified by fractionation by gel filtration of chondroitin-6-sulfate bovine testicular hyaluronidase digester as previously described (32). Cyclohexa-, hepta- and octa-amyloses were obtained from Sigma. These oligosaccharides can be sulfated as described in Example 5. By way of example, maltohexose sulfate was prepared in the following manner. To a solution of sulfur trioxide-pyridine complex (4.0 grams) (Aldrich) in fresh distilled DMF (5 milliliters) at 80 ° C was added dropwise a solution of maltohexose (0.5 grams) in dry pyridine (15 milliliters), and everything was heated at 80 ° C for another 90 minutes. The supernatant was decanted while still hot and the sticky residue was washed thoroughly with methanol (10 milliliters) three times. After decanting the residual methanol, the product was dissolved in water (15 milliliters) and neutralized (to pH of about 6) with barium acetate (approximately 2.0 grams in 10 milliliters of water) with vigorous stirring. After centrifugation (3, 000 x g), the superimposed liquid was decanted and retained and the precipitated barium sulfate agglomerate was washed completely with water (3 x 10 milliliters). The retained superimposed liquid and the washings were combined and placed on a column (2.5 x 14 centimeters) of DOWEX cation exchange resin 50W-X8-400 (form H +). The column was eluted with water until the eluate remained neutral. The eluate (approximately 250 milliliters) was stirred and neutralized (to a pH of about 7) with sodium acetate (3.5 grams). The solution was diluted with acetone (1 liter) and centrifuged (1,750 x g) to separate the product. The agglomerate was finely pulverized by grinding under methanol, and stirred while still under methanol and then filtered. The filtrate was washed several times with methanol to give the pure compound (without inorganic salt) (0.88 grams, 55 percent). The product was not contaminated with barium ion (determined by microanalysis and flame ionization) or nitrogen (microanalysis). It was found that the product had 14 of a total of possible sulphated positions. Found C 13.9; H, 2.2; S 14.3; Na 6.7. C36H71073S14Na9. 45 H20 requires C 13.8; H 5.1; S 14.3; Na 6.6 percent. The nitrogen and barium values were 0.32 and 0 percent found and 0 percent expected for each one.

Information of H NMR (magnetic resonance of electrons) (300 MHZ - Gemini 300, referenced of acetone 2. 25 ppm low TMS field); for the previous maltohexose sulfate indicated that 14 out of 20 possible positions were sulfated. This was determined from chemical changes of the hydrogens centered around 4.15 ppm (integrating for 20 H) against the centered ones around 4.4 ppm (integrating for 16 H). It can be assumed that all groups Primary OH, that is, those in position 6, will be sulfated since it is the least sterically hindered position. It is further assumed that in the internal sugar residues only another position will be sulphated. The terminal sugar residues, in addition to that of position 6, will each have two other sulphated positions. Manopentose phosphate was prepared from the exopolysaccharide produced by the diploid yeast Pichia holstii (strain NRRL Y-2448 formerly Hansenula holstii). The method for the cultivation of P. holstii and the isolation of the mannitol phosphate was based on what was previously described (33, 34). Briefly, the crude exopolysaccharide was isolated from culture supernatants of yeast grown aerobically as a potassium salt by ethanol precipitation. Then acid hydrolysis was used to liberate the mannitol phosphate from the phospho-manne monoester center (PPME) of the exopolysaccharide. The phosphomannan monoester and mannitolose phosphate were then separated from each other as barium salts by differential precipitation by ethanol and subsequently by gel filtration. The oligosaccharide has the structure P-6-Man-a- (1? 3) -Man-a- (1? 3) -Man-ar- (1? 3) -Man-a- (1? 2) Man ( 3. 4). The yeast mannitol phosphate sulfate (33, 34) isolated from the yeast exopolysaccharide was prepared in the following manner. A suspension of yeast manopentose phosphate (0.09 grams) in DMF (2 milliliters) and pyridine (3 milliliters) was added to a solution of sulfur trioxide-pyridine complex (0.8 grams) (Aldrich) in DMF (1 milliliter). . The mixture was heated at 80 ° C for 2 hours. The supernatant was decanted while still hot and the sticky residue was washed thoroughly with methanol (2 milliliters) three times. After decanting the residual methanol, the product was dissolved in water (5 milliliters) and neutralized (to pH 6) with barium acetate (approximately 0.7 grams in 5 milliliters of water) with vigorous stirring. After centrifugation (3,000 x g), the superimposed liquid was decanted and the precipitated barium sulfate agglomerate was washed thoroughly with water (3 x 10 milliliters). The superimposed liquid and the washings were combined and placed on a column (2.5 x 14 centimeters) of cation exchange resin DOWEX 50W-X8-400 (form H +). The column was eluted with water until the eluate remained neutral. The eluate (approximately 50 milliliters) was stirred and neutralized (to pH 7) with sodium acetate (approximately 0.4 grams). The solution was diluted with acetone (150 milliliters) and centrifuged (1,750 x g) to separate the product. The agglomerate was finely pulverized by grinding under methanol, and stirred while still under methanol and then filtered. The solid was washed several times with methanol to give sulphated yeast mannopentophate phosphate (0.18 grams). The product was not contaminated with barium ion (determined by microanalysis and flame ionization) or nitrogen (microanalysis). It was found that the product had 10 out of a total of 16 possible sulphated positions. Found C 15.35; H, 2.7; P 1.2; S 13.7; Na 8.5. C ^ Q ^^ gPS ^ Nag. 25 H20 requires C 15.3; H 3.5; P 1.3; S 13.6; Na 8.8 percent. The values of nitrogen and barium were 0.16 and 0 percent found and 0 percent expected for each. 1-6C-glucose oligosaccharides were prepared by acid hydrolysis of dextran (average molecular weight 71,000; Sigma Chemical Co.). Thus, dextran (5 grams) was dissolved in distilled water (100 milliliters) and this solution was adjusted to pH 1.8 with 1M hydrochloric acid. The mixture was refluxed (100 ° C) for 48 hours. The mixture was dried under reduced pressure and was added to 100 milliliters with distilled water and dried a second time under reduced pressure. Absolute ethanol (100 milliliters) was added to the residue and evaporated under reduced pressure. The residue was made 4 milliliters with distilled water and applied to a 5 x 90 cm water-coated chromatography column packed with thin grade P2 exclusion gel (BioRad). The column was packed, heated and operated at 60 ° C for two days before use. After the addition of 1,6-a-glucose oligosaccharides to the mixture, the column was maintained at 60 ° C and eluted with water (1 milliliter / minute). The products eluting from the column were identified as peaks by differential refractometry and the fractions were collected accordingly. In this way, the fractions corresponding to areas under separate peaks were collected. Each of these fractions was rechromatographed on an identical P2 gel column at 60 ° C and eluted with water at a flow rate of 0.5 milliliters / minute. Thus, by way of example, the fraction identified as corresponding to 1,6-a-glucohexose (0.19 grams) was rechromatographed to give a main central peak with one shoulder on each side. The product eluting at the central peak was isolated by removing the water under reduced pressure to give (0.16 grams) of material that was rechromatographed on an identical P2 gel column at 60 ° C at a flow rate of 0.3 milliliters / minute . In this way glucohexose (0.14 grams) was obtained (electroaspersion M + = 990). In a similar manner, 1, 6-Qf-glucotriosa, 1, 6-o; -glucotetrosa (0.21 grams) and 1,6-a-glucopentose (0.17 grams) were isolated.

EXAMPLE 7 A. MATERIALS AND METHODS Activity Anticoagulant of Sulphated Oligosaccharides The anticoagulant activity of each oligosaccharide was assessed as previously described (35), using both the thrombin time procedure and the activated partial thromboplastin time. The activity of each preparation was compared to a heparin control and the anticoagulant activity was expressed as a percentage of heparin activity.

Human Antiogenesis Test The test method used is described in International Patent Application No. PCT / AU95 / 00105, the disclosure of which is incorporated herein by reference. The blood vessels, approximately 1-2 millimeters in diameter and 2-5 centimeters in length, were separated from the surface of human placentas within the first 6 hours of birth. The vessels were placed in Hank's BSS solution containing 2.5 milligrams / milliliter of fungizone and cut into fragments 1-2 millimeters in length using dissection forceps and iridectomy scissors. The fragments were freed of residual clots and soaked in Hank's BSS solution before use. The dissection and cutting of the vessels was carried out with the help of a magnifying lens lamp (Maggylamp, Newbound, Balmain, NSW, Australia). Similar angiogenic responses were obtained from blood vessels of venous or arterial origin but, for each test, single-vessel vessel fragments were used. The angiogenesis tests were performed in 24 or 48 well culture trays (Costar, Cambridge, MA). In the 24-well format, 30 μl of bovine thrombin (50 NIH units / milliliter in 0.15 M NaCl, Sigma Chemical Co., St Louis, MO) was added to each well followed by 1.0 milliliter / well of 3 milligrams / milliliter of bovine fibrinogen (Sigma) in Medium 199. Thrombin and fibrinogen were rapidly mixed and a vessel fragment was rapidly placed in the center of the well before clot formation. Usually the fibrin gel formation was presented in 30 seconds and the vessel fragment was left suspended in the gel. After gel formation, 1.0 mil / well of Medium 199 supplemented with 20 percent fetal calf serum (FCS) was added, 0.2 milligrams of e-aminocaproic acid, L-glutamine and antibiotics (gentamicin and fungazone) were added. In the 48 well format all the reagent volumes were put in half. The vessels were cultured at 37 ° C in a humid environment for 14-21 days by changing the medium twice a week. Angiogenesis was quantified by computer-based image analysis, using NIH Image software, of digital images of cultures obtained with a Dycam digital camera mounted on an inverted microscope (Olympus, Tokyo, Japan). Heparanase test The heparanase test is based on the observation that serum protein, histidine-rich glycoprotein (HRG), binds to heparan sulfate chains and masks the dissociation site of heparanase. Based on the finding that heparan dissociated heparan sulfate does not bind to a histidine-rich glycoprotein, a heparanase test has been developed that involves digesting heparan sulfate chains labeled with JH with heparanase, binding digested heparan sulfate with granules histadin-rich glycoprotein traps and measuring the unbound 3H tag. With increasing digestion of the substrate an increasing amount of 3H tag ceases to bind to histidine-rich glycoprotein granules. Thus, this method represents a simple and rapid procedure to measure heparanase activity in tissue extracts and assess the inhibition of heparanase by several compounds. Initially, bovine intestinal heparan sulfate (Mr av 32 kDa) was partially de-N-acetylated by heating it in hydrazine hydrate (36) and reacetylated with 3H acetic anhydride. The glycoprotein rich in chicken histadin, purified by the method of Rylatt et al. (1981) (37), was coupled with Sepharose 4B (Pharmacia) activated CNBr according to the instructions of the manufacturers. Heparanase activity of human platelets was determined by incubation (37 ° C, 30 minutes) of human platelet heparanase (which has been shown to have the same activity towards heparan sulfate as heparanase activity present in cell lines of highly cultured human metastatic carcinoma HCT 116, adenocarcinoma 13762 rat MAT and mouse melanoma B16) with 60 pmoles of bovine intestinal heparan sulfate radiolabelled with JR. The activity was determined by the speed of production of smaller heparan fragments (approximately 5 kDa) that were not bound after passing through the incubation mixture (100 μl) through minicolumns of histidine-rich glyphosate Sepharose (200 μl packed granules) retaining the largest substrate not dissociated and partially dissociated. In the heparanase inhibition tests, different concentrations of inhibitor were added to the enzyme before the addition of radiolabelled substrate, the inhibitor being retained in the reaction mixture throughout the incubation period.

Metastasis test The antimetastatic activity of different sulphated oligosaccharides was assessed using the highly metastatic rat mammary adenocarcinoma 13762 MAT (35). The tumor cells were maintained in vi tro as previously described (35). Three hundred forty-four female Fisher rats, (10-13 weeks of age) were injected intravenously with 2 x 10 5 cells 13762 MAT in 0.6 milliliters of RPMI 1640 medium containing 10 percent FCS. At the time of tumor injection the animals were also injected with 2 milligrams of sulphated oligosaccharide, obtaining similar results if the oligosaccharide was injected intravenously, i.p. or subcutaneously. The lungs of the rats were removed 13 days after the injection of the tumor cells, placed in Bouin's solution for at least 24 hours and the lung metastases were evaluated under a dissecting microscope. The number of metastases in rats treated with sulfated oligosaccharide was compared with that observed in the control animals, with a minimum of four animals in each group.

Effect of Sulphated Oligosaccharides on FGF-Heparin / Heparan Sulfate Interaction A binding assay, which is described in detail elsewhere (38), was used to measure the binding of an aFGF and bFGF to 'heparin and assesses the capacity of different sulphated oligosaccharides to inhibit this interaction. Briefly, the EGF were immobilized in the wells of 96-well PVC trays and the binding of radiolabeled heparin to the immobilized FGF was assessed. In inhibition assays, a series of oligosaccharide dilutions were examined for their ability to inhibit the interaction of FGF-heparin. The inhibition results were expressed as the concentration of sulphated oligosaccharides required to inhibit the binding of heparin to immobilized FGFs by 20 percent or 50 percent. Unlabelled heparin was included as a control in all the binding-inhibition experiments. The interaction of FGF-heparan sulfate was assessed, as previously reported (39), by measuring the binding of BALB / c 3T3 fibroblasts to FGF immobilized in PVC, with cell binding detected by Rose Bengal staining of the adherent cells. The sulphated oligosaccharides were examined for their ability to inhibit this process of cell adhesion, which is totally dependent on the structures of heparan sulfate on the surface of BALB / c 3T3 cells (39). The data were expressed as the concentration of sulphated oligosaccharide that inhibits cell adhesion by 50 percent (IC50).

Inflammation model with air bags The trial is based on a previously reported procedure (40). Subcutaneous air pockets formed in the backs of mice by injecting 5 milliliters of sterile air under the skin of a shaved area between the shoulder blades of an anesthetized mouse on day 1. On day 3, the bag was reinflated by injection of 2.5 milliliters of air. Inflammation was induced on day 6 by injecting 1.0 milliliters of 56 milligrams / milliliter of thioglycolate or 1.0 milliliter of saline directly into the bag as control. Approximately 17-20 hours after injection of thioglycollate the animals were sacrificed by cervical dislocation and the cell contents of the bag was recovered by injecting 2.5 milliliters of PBS / 5 percent FCS on ice. The ability of the sulphated oligosaccharides to inhibit the inflammatory reaction was tested by being injected subcutaneously (50 μl in PBS) in a separate site immediately following the administration of the thioglycolate. Prednisolone was used as an anti-inflammatory drug to control, being injected subcutaneously in oil at 25 milligrams / kilogram. The total cellular content of each bag was determined using a Coulter Counter and different subpopulations of leukocytes were assessed by immunofluorescent flow cytometry.

Mouse Asthma Model A previously reported mouse asthma model (41) was used to test the ability of sulphated oligosaccharides to inhibit aeroallergic (ovalbumin, OVA) induced eosinophil infiltration in the lungs. Mice (C57BL / 6, 6-10 weeks old) were sensitized by i.p. with 50 milligrams of OVA / 1 milligram of Alhydrogel (CSL Ltd, Parkville, Australia) in 0.9 percent sterile saline on days 0 and 12. On day 24, the mice were exposed to an OVA spray (10 milligrams / milliliter) in 0.9 percent saline for 30 minutes three times (at one hour intervals), and then exposed to a similar challenge on days 26 and 28. The aerosol was generated at 6 liters / minute by a nebulizer that produced an average particle of 39μm in diameter in a closed chamber of 800 cubic centimeters. On day 29 the mice were sacrificed by cervical dislocation. The trachea was cannulated and the airway lumens were washed with 4 x 1 milliliter of 0.9 percent saline solution containing BSA (0.1 percent weight / volume) at 37 ° C. Approximately 0.8 milliliters of the instilled fluid was recovered by washing. The bronchoalveolar lavage fluid (BALF) obtained from an animal was combined and the cell numbers were determined using a standard hemocytometer. The BALF cells were also cytocentrifuged and differentially stained with May-Grunwald-Giemsa solution, the eosinophils were identified using morphological criteria. The data were calculated as number of eosinophils / milliliter of BALF. Sulphated oligosaccharides were administered to the animals either systemically by i.p., inserted Alzet miniosmotic pumps, or via the lungs as an aerosol. The miniosmotic pumps were inserted on day 23, 24 hours before the OVA stimulus, and the drug continuously supplied until the animals were sacrificed on day 29. In the case of administration by aerosol, the mice were exposed to an aerosol of sulphated oligosaccharides in 0.9 percent saline for 30 minutes three times (at 1 hour intervals) on days 23, 25, and 27.

Model of Experimental Autoimmune Encephalomyelitis (EAE) Spleen cells were prepared for the adopted transfer of experimental autoimmune encephalomyelitis as previously described (43). Briefly, Lewis rats were sensitized to the myelin basic protein, the immune spleen cells activated by ConA in vi tro and 30 x 10 activated EAE autoimmune effector encephalomyelitis cells transferred Cona i.v. to each container. Miniosmotic pumps (Alzet) containing sulphated oligosaccharides were implanted subcutaneously at the time of cell transfer and a dose of 70 milligrams / kilogram / day was administered for 14 days. Clinical experimental autoimmune encephalomyelitis was classified according to the following scheme: 0 asymptomatic, 1, half flaccid tail, - 2 total tail flaccidity; 3 ataxia, difficulty straightening; 4, weakness in hind limbs; and 5, hind limb paralysis.

Inflammatory Bowel Disease Inflammatory bowel disease was induced in mice with the addition of drinking water with 5 percent (w / v) dextran sodium sulfate (DSS) provided by TdB Consultora, Uppsala, Sweden. The solution was adjusted to a pH of 8.0 and filtered through a membrane medium of 0.45μ. The sodium dextran sulphate DSS solutions were collected daily, refiltered and the volume was adjusted with fresh dextran sodium sulfate material. Male BALB / c mice from 6 to 7 weeks of age were selected for body weight and those between 20-23 grams were grouped in cages of 5 mice / cage. Mice were injected with sulfated mannitol phosphate (20 milligrams / kilogram / day) or vehicle (sterile water) at 8-hour intervals from day 0 to day 10. The volume of the injection was standardized to 100 μl and injected subcutaneously into the nape of the neck. The consumption regime of dextran sodium sulphate, body weight and symptoms were recorded daily for all mice. The symptoms of diarrhea and rectal bleeding were recorded as light or strong and were given numerical values of 1 and 4, respectively. The presence of mucus was also noted and included as a grade of mild diarrhea. The sum of the diarrhea and rectal bleeding scores was divided by the number of surviving animals in that group on that day. The total score is the sum of the grades of diarrhea and rectal bleeding.

B. RESULTS Anti-angiogenic and anti-metastatic activity of the Natural Sulphated Oligosaccharides. Once a range of natural sulphated oligosaccharides was synthesized, they were examined in the range of biological assays. Table 1 summarizes the results obtained with the sulfated forms of 12 natural oligosaccharides. The biological activities of suramin, (a compound having moderate anti-angiogenic and inhibitory activity of heparanase) (42), and heparin are also included in Table 1 for comparison. Initially, it was shown that all tested oligosaccharides have negligible anticoagulant activity, ie, heparin activity < 2 percent (Table 1). This was an important property since heparin, a potent anti-metastatic compound, has limited clinical utility for this indication due to its potent anticoagulant activity. Three of the sulfated natural oligosaccharides were quite potent inhibitors of human angiogenesis, namely, sulfated mannitol phosphate (derived from P. holstii), maltotetrose sulfate and maltohexose sulfate. Manopentose and maltohexose phosphate were the most potent of these compounds with a 50 percent inhibitory concentration of 2 μg / milliliters while maltotetrose gave 50 percent inhibition at 20 μg / milliliter. An example of the pronounced inhibition of angiogenesis induced by 20 μg / milliliter of maltohexose sulfate is depicted in Figure 1. It is interesting to note that heparin had little anti-angiogenic activity. Thus, it seems likely that sulphated oligosaccharides of relatively short chain lengths are required for this type of activity. A more complete titration of the inhibition of angiogenesis by the maltose series is represented in Figure 2 and by the sulphate-bearing mannose phosphate in Figure 3. It can be seen that, with the maltose series, the maltose sulfate has little activity inhibitory, while the sulfates of maltotetrose and maltohexose were quite powerful inhibitors (Figure 2).

All the angiogenesis experiments presented in Table 1 involve the addition of oligosaccharide to the culture medium at the beginning of the angiogenesis assay. However, preliminary studies (data not shown) indicate that the addition of maltohexose sulfate, after the start of the angiogenesis response, may also inhibit the extra outgrowth of the vessels although the most effective inhibition occurs when the compound is added to the start. of the crop. The sulphated oligosaccharides also differ markedly in their inhibitory activity of heparanase, with the most potent inhibitors being sulfate-sulphate phosphate and maltohexose sulfate, the activity of these two compounds resembling that of heparin (Table 1). In an interesting way, these two compounds are also effective anti-angiogenic compounds. However, the inhibition of angiogenesis does not correlate with the heparanase inhibitory activity of many compounds. For example, the sulphated cycloamyl groups were very potent heparanase inhibitors, but poor inhibitors of angiogenesis. The maltose series was very informative regarding the length of the chain and the inhibition of heparase. Table 3 shows the inhibitory activity of heparanase for the complete maltose series, varying from disaccharide (maltose) to heptasaccharide (maltoheptose).

Maltose was non-inhibitory, maltotriose was weakly inhibitory, maltotetrosa exhibited a modest inhibitory activity, while penta-, hexa- and hepta-saccharides exhibited high inhibitory activity. Thus, a sulphated or higher pentasaccharide is required for optimal inhibition of heparanase. Many of the sulfated sugars have been tested in vivo for their anti-metastatic activity (Table 1). In general, there is a reasonably good correlation between the inhibition of heparanase and the antimetastatic activity. A) Yes, sulphate-treated mannitol phosphate and maltohexose sulfate, the two compounds with the highest inhibitory activity of heparanase, have the greatest antimetastatic activity, in fact, they do not differ significantly from heparin in the ability to avoid metastasis (Table 1 ). Two other compounds, cyclo-octa-amylose sulfate and stachyose sulfate were reasonably effective antimetastatics, a property consistent with their modest heparanase inhibitory activity. Collectively, these data suggest that sulphated mannitol phosphate and maltohexose sulfate simultaneously possess considerable anti-angiogenic, antimetastatic and heparanase inhibitory activities. The antimetastatic activity of the maltose series of the sulphated oligosaccharides is presented in greater detail in Figure 4. With the length of the growing chain there was a regular increase in the antimetastatic activity of the oligosaccharides, being the penta-, hexa-, and hepta-saccharides the most active. When administered intravenously at a dose of 2 mg / rat, maltose sulfate had no effect on metastasis (Figure 4A) but when administered subcutaneously at 4 mg / rat significant inhibition of metastasis was observed (Figure 4B). Subsequent experiments revealed that, irrespective of the injection route, ie, i.v., subcutaneous or i.p.) the sulfated oligosaccharides showed comparable antimetastatic activity (data not shown). In fact, the antimetastatic activity of maltose sulfate was observed only when high doses were administered to animals. Since maltose sulfate is a very bad heparanase inhibitor, this result suggests that the inhibition of heparanase may not be the only way for oligosaccharides to inhibit tumor metastasis, particularly when using high doses of oligosaccharides. The cycloamyl groups were sulfated and included in the study as they represent non-linear oligosaccharides. It is interesting to note that these compounds were only modestly active (Table 1), implying that linear oligosaccharides may be required for optimal activity. In addition, the most active sulphated oligosaccharides were much more effective inhibitors of angiogenesis, metastasis and heparanase activity than suramin (Table 1), a drug that is used in clinical cases as an anti-angiogenic compound (42). Since the anti-angiogenic activity of the compounds does not always correlate directly with their heparanase inhibitory activity, it seems likely that the sulfated oligosaccharides can inhibit angiogenesis by some other mechanism. As mentioned above, it is very likely that some sulfated oligosaccharides may disrupt the action of angiogenic growth factors by disrupting heparan sulfate growth factor interactions. Previous analyzes (see International Patent Application No. PCT / AU95 / 00105) have shown that the human angiogenesis assay used in this Example is highly dependent on the endogenous bFGF basic fibroblast growth factor, and to a lesser extent, on the action of aFGF and VEGF. Thus, the various oligosaccharides were examined to see their ability to act as competitors for the interaction of bFGF, aFGF and VEGF with heparin or heparan sulfate. It was found that, with the growth of chain length, the maltose series of the sulphated oligosaccharides become more effective inhibitors of the interaction of bFGF and aFGF with cell surface heparan sulfates (Table 2), that is, maltose was weakly inhibitory, while penta-, hexa- and hepta-saccharides were the most active. Sulphated mannopentose sulfate also exhibited considerable inhibitory activity in this system (Table 2). Complete inhibition curves for the inhibition of heparan aFGF-sulfate interaction by the maltose series of the sulfated oligosaccharides are presented in Figure 5. Further studies showed that maltohexose sulfate was also a potent inhibitor of the binding of the radiolabelled heparin with bFGF and aFGF (data not shown). Since maltohexose sulfate was one of the most active anti-angiogenic and anti-metastatic compounds, the influence of the degree of sulphation on its biological activity was examined in some detail. Initially it was noted that even when some anticoagulant activity was detected with the more highly sulfated maltohexose, this activity was still extremely low compared to heparin (Table 2). However, with the increase in sulphation, there was a regular increase in maltohexose's ability to inhibit the activity of heparanase and the binding of FGF with heparan sulfate (Table 2). However, the inhibitory activity was established as a plateau in both systems when sulfation was 85 percent or higher. Metastasis inhibition studies (Figure 6) also showed that with increasing degrees of sulfation, maltohexose sulfate becomes a more effective antimetastatic drug. On the contrary, there is a suggestion that the highly highly sulfated maltohexose (90-100 percent sulfated) was a less effective inhibitor of angiogenesis (Figure 7). These data suggest that there are subtle differences in the optimal structure of the sulfated oligosaccharide required to inhibit angiogenesis and metastasis. However, several sulphated oligosaccharides, derived from natural oligosaccharides, have been identified that simultaneously have potent antimetastatic and anti-angiogenic activity. These compounds are sulphate pentapentose phosphate from P. holstii and maltopentose sulphate, maltohexose and maltoheptose.

Anti-Angiogenic and Anti-Metastatic Activity of Sulphated Synthetic Oligosaccharides The sulfated synthetic oligosaccharides described in Examples 1-5 were tested for their biological activity. Table 3 summarizes the ability of synthetic sulphated oligosaccharides containing mannose, galactose or glucose to inhibit coagulation, the action of heparanase and the growth factor-link of heparan sulfate. All the synthetic sulphated oligosaccharides examined had negligible anticoagulant activity. However, with the exception of the mannose and glucose trisaccharides, all the other sulphated oligosaccharides were reasonably effective inhibitors of the heparanase activity and the heparan growth factor-heparan sulfate linkage. In fact, the overall conclusion is that sulphated synthetic oligosaccharides containing 4-6 hexose units (ie, D-mannose, D-galactose or D-glucose) are highly active in these assays. One exception is galactotriose sulfate, which was essentially as active as other members of the galactose series. When tested in the human angiogenesis assay, the sulphated mannose oligosaccharides were inhibitory, although the penta- and hexa-saccharides were more active than the tetrasaccharide (Figure 8), resembling the sulfated mannitolose phosphate in its efficacy. Similarly, sulfated mannose from tetra-, penta- and hexa-saccharide were as effective as mannitolose phosphate as an antimetastatic drug (Figure 9). Sulphated oligosaccharides containing galactose and glucohexose sulfate also inhibited metastasis (Figure 10), although these tended to be slightly less active than compounds containing mannose.

Anti-Inflammatory Activity of Sulphated Oligosaccharides As mentioned above, a key barrier to the entry of leukocytes into inflammatory sites is the subendothelial basement membrane. In order to cross this membrane, leukocytes must use a battery of degrading enzymes (11). Of particular relevance is the endoglycosidase, heparanase, which dissociates the basement membrane associated with heparan sulfate chains and is essential for the extravasation of leukocytes (12,13). In fact, as with metastasis inhibition studies (35), sulfated polysaccharides that inhibit the activity of heparanase are potent inhibitors of inflammation (43,44). Based on these observations, those skilled in the art would anticipate that sulfated oligosaccharides that were potent anti-angiogenic and anti-metastatic agents would be highly effective anti-inflammatory compounds. Of particular importance in this regard are maltohexose sulfate and mannitolose sulfate. In addition, since angiogenesis is associated with chronic inflammatory diseases such as rheumatoid arthritis (18), the anti-angiogenic activity of these compounds would be of additional value in the treatment of inflammation. The evidence in favor of this prediction has been obtained in many animal models of inflammation. First, maltohexose sulfate, manopentose sulfate, and mannitolose phosphate were able to significantly inhibit air bag inflammation induced by thioglycollate (Table 4). In fact, in one experiment a single injection of manopentose sulfate was effective as prednisolone in inhibiting leukocyte infiltration, which was predominantly neutrophilic in nature, whereas maltohexose sulfate was somewhat less effective. A greater inhibition of the inflammatory response was still observed when the sulphated oligosaccharides were injected in two equal doses separated by 6 hours. Second, the oligosaccharides were examined for their ability to inhibit a mouse model of chronic asthma. This model is characterized by a massive influx of eosinophils into the lungs of mice that was induced by an aeroallergenic stimulus (41). Such an inflammatory response is characteristic of chronic asthma in humans. When administered via miniosmotic pumps, maltohexose sulfate and manopentose sulfate significantly inhibited the accumulation of eosinophils in mouse lungs (Table 5). Maltohexose sulfate also exhibited some anti-inflammatory activity when administered as an aerosol (40 milligram / milliliter solution). Third, both manopentose sulfate and sulfated mannitol phosphate significantly inhibited experimental autoimmune encephalomyelitis in a rat model of the disease (Table 6). In fact, some animals treated with the sulfated oligosaccharides stopped developing the symptoms of the disease. These data are consistent with previous studies that show that polysaccharides that inhibit the activity of heparanase can reduce the severity of experimental autoimmune encephalomyelitis (43). Finally, manopentose phosphate was examined for its ability to inhibit a model of inflammatory bowel disease in mice. This model, which is induced by dextran sulfate in drinking water, induces a colitis that resembles ulcerative colitis and, to a lesser degree, Chron's disease. It was found that 20 milligrams / kilogram / day of sulfated mannitol phosphate produced a marked attenuation of acute colitis and also prevented the loss of body weight caused by the disease (Table 7). The controls in this experiment received injections of the sulfated oligosaccharide, but not the dextran sulfate in the drinking water.

TABLE 1 INHIBITION OF HUMAN ANGIOGENESIS, HEPARANASE ACTIVITY AND METASTASIS THROUGH SULFATED FORMS OF DIFFERENT NATURAL OLIGOSACARIDES Percentage of control of metastasis + standard error of the mean (n = 4) in lungs of rats receiving 13762 MAT cells i.v. and 2 mg / rat of each oligosaccharide at the same time as tumor cell injection. Underlined values represent compounds with the greatest anti-metastatic effect. c Mannopenose phosphate isolated from the yeast Pichia holstii. ND Not determined.

TABLE 2 INHIBITION OF THE HEPARANASE ACTIVITY AND THE LINKED GROWTH FACTOR WITH HEPARAN SULFATES BY SULFATED MANOPESE PHOSPHATE AND THE SULFATED OLIGOSACARIDOS OF THE MALTOSA SERIES Actual number of sulfate groups bound / theoretical maximum number of sulfate groups that can be coupled to each molecule Anticoagulant activity as a percentage of heparin activity (100%) Compound concentration required to 50% inhibit the heparanase activity of the human platelets, or binding of mouse 3T3 cells to immobilized aFGF / bFGF In the case of the heparanase assay, the IC50 for heparin was 2 μg / ml d Mannopentose phosphate isolated from the yeast Pichia holstii ND = Not determined TABLE 3 INHIBITION OF THE ACTIVITY OF HEPARANASE, LINK OF THE GROWTH FACTOR WITH HEPARAN SULFATES THROUGH SULFATED FORMS OF DIFFERENT SYNTHETIC OLIGOSACARIDOS Actual number of sulfate groups bound / theoretical maximum number of sulfate groups that can be coupled with each molecule. Anticoagulant activity as a percentage of heparin activity (100%). Compound concentration required to inhibit heparanase activity of human platelets at 50%, or binding of mouse 3T3 cells to immobilized FGF / bFGF. In the case of the heparanase assay, the IC 50 for heparin was 2 μg / ml. ND = Not determined.

TABLE 4 EFFECT OF SULFATED OLIGOSACRIDES ON INFLAMMATION OF AIR BAG3 Air bag inflammation induced by thioglycolate injection and leukocyte influx assessed 17 hours later. The drug treatments were injected subcutaneously at the same time as the thioglycolate for Exp. 1. In Exp. 2, the sulfated oligosaccharides were injected subcutaneously 0 hours and 7 hours after the injection of thioglycolate. Data presented as percentage number of leukocytes control an air bag infiltrate ± standard error of the mean. The controls were injected with thioglycollate but did not receive drug treatment, only one injection of saline. The antecedent leukocyte infiltrate in air pockets that were injected with saline alone was 9 + 2% of that observed after the injection of thioglycolate. = not determined.

TABLE 5 EFFECT OF SULFATED OLIGOSACRIDES ON OVALBUMIN (OVA) ACCUMULATION OF INDUCED EOSINOPHILES IN RATON LUNGS Mice sensitized to OVA and then an influx of eosinophils in lungs induced by administration of OVA by aerosol. Sulphated oligosaccharides administered either i.p. with miniosmotic pumps or via the lungs as an aerosol. Data expressed as number of control eosinophils in percentage in bronchoalveolar lavage fluid (BALF) ± standard error, being the animal controls stimulated with OVA and receiving saline either by miniosmotic pumps or via the lungs as an aerosol. Concentration of sulphated oligosaccharide in the aerosol solution.

TABLE 6 EFFECT OF OLIGOSACARIDOS SULFATADOS EN EXPERIMENTAL EXPERIMENTAL TRANSFERRED ENERPHONOMYELITIS (EAE) EAE induced in Lewis rats with 30 xlO6 activated EAE effector cells ConA. Sulphated oligosaccharides, administered subcutaneously, miniosmotic pumps inserted at the time of cell transfer, administered dose of 70 mg / kg / day. Average day of EAE outbreak in animals that developed the disease. The severity of the disease represents the cumulative score of the animals.

TABLE 7 EFFECT OF MANOPESE PHOSPHATE ON INFLAMMATORY BOWEL DISEASE IN MICE3" Inflammatory bowel disease induced by the administration of dextran sulfate in drinking water. Days after the start of the administration of dextran sulfate. Untreated animals received vehicle injections three times daily while treated animals received sulfated mannitol phosphate injections at a dose of 20 mg / kg / day three times a day. The mean score of the disease represents the sum of the scores of both diarrhea and rectal bleeding for the animals at each time point.

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Claims (24)

1. A sulphated oligosaccharide, wherein the oligosaccharide has the general formula I: R (R?) N -R2 (I) wherein R ^ and R2 and each R? they represent a unit of monosaccharide, which can be all the same or different, the adjacent monosaccharide units being linked by the glycosidic bonds 1? twenty-one ? 3, 1 - * 4, and / or 1? 6 and n is an integer from 1 to 6.

2. A sulphated oligosaccharide according to claim 1 wherein n is from 1 to 4, preferably 3 or 4.

3. A sulfated oligosaccharide according to claim 1 or claim 2 , wherein the monosaccharide units are hexoses selected from the group consisting of fructose, glucose, mannose, altrose, allose, talose, galactose, idosa and gulose.

4. A sulphated oligosaccharide according to claim 1 wherein the oligosaccharide has the general formula II: R y-R y ^ -R y (ü) wherein each R y group is the same and each represents a unit of monosaccharide, the adjacent monosaccharide units by the glycosidic linkages 1? 3, 1 - * 4, l? 6; and n is an integer from 1 to 6. A sulfated oligosaccharide according to claim 4, wherein n is from 1 to 4, preferably 3 or 4. 6. A sulfated oligosaccharide according to claim 4, or claim

5. , wherein Ry represents a monosaccharide unit which is a hexose selected from the group consisting of glucose, mannose, altrose, allose, talose, galactose, idosa and gulose. 7. A sulphated oligosaccharide according to claim 6, wherein Ry represents glucose, mannose or galactose. 8. A sulphated oligosaccharide according to claim 1, wherein the oligosaccharide is a natural oligosaccharide. 9. A sulphated oligosaccharide according to claim 8, wherein the oligosaccharide is selected from raffinose and stachyose. 10. A sulphated oligosaccharide according to claim 1, wherein the oligosaccharide is prepared by the enzymatic or chemical degradation of a natural polysaccharide. 11. A sulphated oligosaccharide according to claim 10, wherein the oligosaccharide is an oligosaccharide derived from amylose, chondroitin or dextran. 12. A sulphated oligosaccharide according to claim 11, wherein the oligosaccharide is selected from maltotetrose, maltopentose, maltohexose, glucotriosa, glucotetrosa, glucopentose, and tetra-, hexa- and octasaccharides of chondroitin. 13. A sulphated oligosaccharide according to claim 10, wherein the oligosaccharide is mannopentose phosphate from the yeast Pichia holstii. 14. A method for the anti-angiogenic, anti-metastatic and / or anti-inflammatory treatment of a human or other warm-blooded animal patient in need of such treatment, comprising administering to the patient an effective amount of at least one oligosaccharide sulfated according to any of claims 1 to 13. 15. The use of a sulfated oligosaccharide according to any of claims 1 to 13, as an anti-angiogenic, anti-metastatic and / or anti-inflammatory agent in the treatment of a warm-blooded animal patient (including a human). 1

6. A method or use according to claim 14 or claim 15, wherein the treatment comprises the treatment of an angiogenesis-dependent disease, including angiogenesis associated with the growth of solid tumors, proliferative retinopathies and rheumatoid arthritis. A method or use according to claim 14 or claim 15, wherein the treatment comprises the treatment of inflammatory diseases and conditions in which the inhibitory activity of heparanase inhibits the infiltration of leukocytes, including chronic inflammatory diseases such as arthritis rheumatoid, multiple sclerosis, insulin-dependent diabetes mellitus, diseases such as ulcerative colitis and inflammatory bowel disease of Crohn's, allograft rejection and chronic asthma. 18. A pharmaceutical or veterinary composition for anti-angiogenic, anti-metastatic and / or anti-inflammatory treatment, comprising at least one sulfated oligosaccharide according to any of claims 1 to 13, together with a pharmaceutically or veterinarily acceptable carrier or diluent. for the same. The use in the manufacture of a medicament for the anti-angiogenic, anti-metastatic and / or anti-inflammatory treatment of a human patient or other warm-blooded animal of at least one sulfated oligosaccharide according to any of claims 1 to 13 20. An oligosaccharide of the general formula II: Ry- (Ry) n-Ry (II) wherein each group Ry is the same and each represents a unit of monosaccharide, the adjacent monosaccharide units being linked by the glycosidic linkages 1 ? 3, 1? 4, and / or 1? 6; and n is an integer from 1 to 6. 21. An oligosaccharide according to claim 20, wherein n is from 1 to 4, preferably 3 or 4. 22. An oligosaccharide according to claim 20 or claim 21, wherein Ry represents a monosaccharide unit which is a hexose selected from the group consisting of glucose, mannose, altrose, allose, talose, galactose, idosa and gulose. 23. An oligosaccharide according to claim 22, wherein Ry represents glucose, mannose or galactose. 24. An oligosaccharide according to claim 20 wherein Ry is a fully O-acetylated monosaccharide, or wherein Ry is a fully 0-esterified monosaccharide with an acyl moiety other than acetyl. RRgjrMtw Sulphated oligosaccharides, where the oligosaccharide has the general formula Rl * (Rx) n "R2 (I) wherein Rj and R2 and each R? Represent a unit of monosaccharide, which may be all the same or different, the adjacent monosaccharide units being linked by the glycosidic linkages 1? 2. , 1? 3, 1? 4, and / ol? 6 and n is an integer from 1 to 6, and the use of them as an i-angiogenic, anti-metastatic and anti-inflammatory agents. * * * * *