Derivatives of N-desulfated glycosaminoglycans and use as drugs

Abstract

A glycosaminoglycan derivative which is obtainable by a process that includes the steps of N-desulfation of from 25% to 100% of the N-sulfated residues of a glycosaminoglycan, and oxidation, by periodate at a pH of from 5.5 to 10.0, of from 25% to 100% of the 2-N-, 3-O-non-sulfated glucosamine residues, and of the 2-O-non-sulfated uronic acid residues of said glycosaminoglycan, under conditions effective to convert adjacent diols and adjacent OH/NH.sub.2 to aldehydes. The process further includes reduction, by sodium borohydride, of said oxidized glycosaminoglycan, under conditions effective to convert said aldehydes to alcohols, where the glycosaminoglycan is heparin, low molecular weight heparin, heparan sulfate or fractions thereof.

Claims

1. A glycosaminoglycan derivative which is obtainable by the following process: a) N-desulfation of from 25% to 100% of the N-sulfated residues of a glycosaminoglycan; b) oxidation, by periodate at a pH of from 5.5 to 10.0, of from 25% to 100% of the 2-N-, 3-O-non-sulfated glucosamine residues, and of the 2-O-non-sulfated uronic acid residues of said glycosaminoglycan, under conditions effective to convert adjacent diols and adjacent OH/NH.sub.2 to aldehydes; c) reduction, by sodium borohydride, of said oxidized glycosaminoglycan, under conditions effective to convert said aldehydes to alcohols; wherein the glycosaminoglycan is heparin, low molecular weight heparin, heparan sulfate or fractions thereof; and wherein the glycosaminoglycan derivative has a molecular weight of from 3,500 to 12,000 Da.

2. The glycosaminoglycan derivative of claim 1, wherein the process further comprising: d) 2-O-desulfation of up to 50% of the 2-O-sulfated residues of the glycosaminoglycan, before or after N-desulfation.

3. The glycosaminoglycan derivative of claim 2, wherein said d) 2-O-desulfation of up to 25% of the 2-O-sulfated residues of the glycosaminoglycan.

4. Oligosaccharides compound which is obtainable by enzymatic or chemical partial depolymerization of the glycosaminoglycan derivative of claim 1.

5. A derivative of N-desulfated glycosaminoglycan characterized in that said derivative comprises the following three structures: ##STR00001## wherein each R.sub.2 is SO.sub.3.sup.− or H; R.sub.3 is SO.sub.3.sup.− or H; R.sub.4 is SO.sub.3.sup.− or H; wherein the starting glycosaminoglycan that the derivative of N-desulfated glycosaminoglycan is derived from is selected from a natural or synthetic glycosaminoglycan; wherein the natural or synthetic glycosaminoglycan is heparin, low molecular weight heparin, heparan sulfate or fractions thereof; and wherein said derivative has a molecular weight of from 3,500 to 12,000 Da.

6. The derivative of N-desulfated glycosaminoglycan of claim 5 further comprises the following two structures: ##STR00002## wherein each R.sub.2 is SO.sub.3.sup.− or H; wherein R.sub.3 is SO.sub.3.sup.− or H.

7. The derivative of N-desulfated glycosaminoglycan of claim 5, wherein the natural or synthetic glycosaminoglycan is unfractionated heparin, or heparin having a molecular weight of from 3,500 to 8,000 Da.

8. The derivative of N-desulfated glycosaminoglycan of claim 5, wherein the percentage, over the total of glycosaminoglycan residues, of glycol split residues (RO), in which adjacent diols and OH/NH.sub.2 have been converted into the corresponding aldehyde and then into the corresponding alcohols, is between 31% and 54%.

9. The derivative of N-desulfated glycosaminoglycan of claim 5, wherein the percentage, over the total of glycosaminoglycan residues, of 2-O-sulfated L-iduronic acid (IdoA2S) is between 29% and 40%.

10. The derivative of N-desulfated glycosaminoglycan of claim 5, wherein the percentage, over the total of glycosaminoglycan residues, of N-acetyl D-glucosamine (GlcNAc) is between 6% and 30%.

11. The derivative of N-desulfated glycosaminoglycan of claim 5, wherein the percentage, over the total of glycosaminoglycan residues, of 2-NH.sub.2 glucosamine (GlcNH.sub.2) is between 4% and 13%.

12. Pharmaceutical composition comprising the derivative of N-desulfated glycosaminoglycan of claim 5.

13. A method of treating tumor metastasis or tumor in a patient, comprising administering to the patient in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the glycosaminoglycan derivative of claim 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient, or diluent.

14. The method of claim 13, wherein the tumor is myeloma.

15. A method of treating tumor metastasis or tumor in a patient, comprising administering to the patient in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the glycosaminoglycan derivative of claim 5, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient, or diluent.

16. The method of claim 15, wherein the tumor is myeloma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings incorporated in and forming a part of the specification illustrate several aspects of the present invention and, together with the description, serve to explain the principles of the invention. In the drawings:

(2) FIG. 1: prevalent structures generated by periodate oxidation and borohydride reduction of a glycosaminoglycan: (1) disaccharidic unit of a glycosaminoglycan polymer comprising one uronic acid (iduronic and/or glucuronic) and one glucosamine (2-N-acetylated, 2-N-unsubstituted and/or 2-N-sulfated), in which the hydroxyl group (R.sub.4) can be substituted by a sulphate group or non-substituted. After N-desulfation, glycosaminoglycan polymers can comprise disaccharidic units comprising 2-N-acetylated glucosamines (2) and 2-NH.sub.2 glucosamines (natural and/or N-desulfated) (3). Periodate oxidation and borohydride reduction lead to the conversion of adjacent diols of 2-O-non-sulfated uronic acid residues (5, 6, 8) and adjacent OH/NH.sub.2 groups of 2-N- and 3-O-non-sulfated glucosamine (7, 8) to the corresponding aldehydes (by oxidation) and then to the corresponding alcohols (by reduction). Note that in disaccharide units containing residues of an N-desulfated glucosamine and a 2-O-non-sulphate uronic acid, both residues are converted to dialdehydes and then to glycol split residues (8).

(3) While the invention will be described in connection with certain preferred embodiments, there is no intent to limit it to those embodiments. On the contrary, the intent is to cover all alternatives, modifications and equivalents as included within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

(4) The present invention relates to novel chemically modified glycosaminoglycans, in particular heparin and LMWHs, which strongly inhibit heparanase and its heparan sulfate degrading activity.

(5) The novel compounds of the present invention, endowed with heparanase inhibitory activity, are derivatives of N-desulfated and optionally 2-O-desulfated glycosaminoglycans, in which at least part of the adjacent diols and OH/NH.sub.2 have been converted into the corresponding aldehyde, which aldehydes have been then reduced to the corresponding alcohol. The conversion to aldehydes is preferably carried out employing periodate, in conditions suitable for breaking both the linkage of adjacent diols of uronic acid residues and the C.sub.2-C.sub.3 linkage of glucosamine, bearing respectively the amine and hydroxyl substituents. The starting compounds can also contain naturally 2-O-non-sulfated uronic acid residues. In particular, N-desulfation occurs on the N-sulfated glucosamine residues, while O-desulfation is on 2-O-sulfated uronic acid residues.

(6) Preferably, the glycosaminoglycan derivatives of the present invention originate from natural or synthetic glycosaminoglycans, the latter being chemically or enzymatically prepared (Naggi, A. et al., 2001, Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli KS polysaccharide, Seminars in Thrombosis and Hemostasis, 27: 5437), such as unfractionated heparins, low molecular weight heparins (LMWHs), heparan sulfates or fractions thereof, more preferably the glycosaminoglycan derivatives derive from natural or synthetic heparins or LMWHs.

(7) Specific N-desulfation of N-sulfated glucosamine residues substantially makes said residues susceptible to conversion to corresponding aldehydes (and then to corresponding alcohols), provided that these residues are also 3-O-non-sulfated. In FIG. 1 all of the disaccharidic units that can be present in a glycosaminoglycan chain and their change after N-desulfation, oxidation, and reduction reaction, are shown in a schematic view.

(8) As an example, heparin chains can naturally comprise from about 5% to about 35% of 2-O-non-sulfated uronic acid residues, from 0% to 50% of N-acetylated glucosamine residues and from about 0% to 6% of N-unsubstituted (neither N-sulfated, nor N-acetylated) glucosamine residues. Different compositions depend on the heparin source (animal species, organ sources) and on the extraction procedures.

(9) Every non-sulfated residue, both on carbon 2 and carbon 3, of a glycosaminoglycan is susceptible of conversion to aldehyde. Consequently, the extensive N-desulfation comprised in the process of the present invention provides further residues susceptible of said conversion over the percentage of susceptible units in natural glycosaminoglycans, with an increase in the percentage of units in which adjacent OH/NH.sub.2 are converted to the corresponding aldehydes (and then to the corresponding alcohols), however preserving the natural content of 2-O-sulfated iduronic acid residues. Optionally, chemically induced 2-O-desulfation of glycosaminoglycans allows to modulate the ratio of glucosamine and uronic acid susceptible to the oxidation and reduction reactions according to the present invention.

(10) The invention further relates to said glycosaminoglycan derivatives N- and optionally 2-O-desulfated, in which adjacent diols and OH/NH.sub.2 have been converted into the corresponding aldehyde, with opening of the ring, which aldehydes have been then reduced to the corresponding alcohol. The invention further relates to a process for preparing said glycosaminoglycan derivatives and also to their use as active ingredients of medicaments for treating pathological conditions such as multiple myeloma and other cancers, including their metastatic forms. Furthermore, the invention relates to the use of said glycosaminoglycan derivatives in any therapeutic indication gaining benefit from the inhibition of heparanase. The invention also relates to pharmaceutical compositions containing said N-desulfated and optionally 2-O-desulfated glycosaminoglycan derivatives, in which adjacent diols and OH/NH.sub.2 have been converted into the corresponding aldehyde, with opening of the ring, which aldehydes have been then reduced to the corresponding alcohol.

(11) The N- and, optionally, 2-O-desulfated glycosaminoglycan derivatives of the present invention are obtainable by a process comprising:

(12) a) N-desulfation of from 25 to 100%, preferably of from 30% to 90%, more preferably of from 45% to 80% of N-sulfated glucosamine residues of a glycosaminoglycan; optionally the process further comprises 2-O-desulfation up to 50%, preferably up to 25%, of 2-0 sulfated residues of a glycosaminoglycan; the obtained product preferably comprises from 0% to 50% of N-acetylated glucosamine residues, from 50% to 100% of N-non-sulfated glucosamine residues;
b) conversion into the corresponding aldehydes, preferably by periodate oxidation, of the adjacent OH/NH.sub.2 of 2N-, 3-O-non sulfated glucosamine residues and of adjacent diols of 2-O-non-sulfated uronic acid residues (chemically desulfated as well as the naturally present non-sulfated ones along the original chain); the 2N-, 3-O-non sulfated glucosamine residues can be present as from 25% to 100%; and
c) reduction of said aldehydes, preferably by sodium borohydride, into the corresponding alcohols.

(13) Optionally, the process further comprises partial or total deacetylation of N-acetylated residues of the glycosaminoglycan.

(14) In preferred embodiments, the glycosaminoglycan derivatives of the present invention are obtained from natural or synthetic (chemically or enzymatically prepared) glycosaminoglycans, preferably from unfractionated heparins, LMWHs, heparan sulfate or derivatives thereof. More preferably, the glycosaminoglycan derivatives of the present invention are obtained from heparins or LMWHs.

(15) In a preferred embodiment, the oxidation, preferably periodate oxidation, is performed under conditions to cleave both the vicinal diols of uronic acids and the bond between the C.sub.2 and C.sub.3 of glucosamine, bearing respectively amino and hydroxyl substituents.

(16) In a preferred embodiment, modified glycosaminoglycan samples, preferably modified heparin or LMWH, endowed with different degrees of N-desulfation, are subjected to periodate oxidation and sodium borohydride reduction in aqueous media, performed by modification of known methods. Periodate oxidation may be performed in the presence of NTA (nitrilotriacetic acid), a chelating and sequestering agent used to reduce depolymerization, in the presence of NaHCO.sub.3 or pyridine to alkalinize the solution, or in the presence of MnCl.sub.2 with or without NTA. Preferably, periodate oxidation is performed in the presence of NTA. Preferably, oxidation is performed at pH comprised between 5.5 and 10.0, more preferably comprised between 6.0 and 9.0.

(17) The present invention further relates to a process for breaking the C.sub.2-C.sub.3 linkage of glucosamine residues of a glycosaminoglycan, comprising: oxidation, preferably by periodate, at a pH comprised between 5.5 and 10, more preferably between 6.0 and 9.0, of said glycosaminoglycan.

(18) Preferably, the glucosamine residues in which adjacent OH/NH.sub.2 have been converted into the corresponding aldehyde, which aldehydes are then reduced to the corresponding alcohols, are from 25% to 100%, more preferably from 50% to 100%, most preferably from 60% to 90%, of the glucosamine residues of the glycosaminoglycan.

(19) The glycosaminoglycan derived compounds obtainable by the process above preferably have a molecular weight of from 800 to 30,000 Da, depending on the process conditions and on the starting glycosaminoglycan. When unfractionated heparin is employed as starting material, the glycosaminoglycan derived compounds obtainable by the process above preferably have a molecular weight of from 3,000 to 20,000 Da, preferably from 4,000 to 12,000 Da.

(20) The novel glycosaminoglycan derivatives of the present invention have been unexpectedly shown to be strong heparanase inhibitors in vitro and to inhibit myeloma in animal models.

(21) The products with the higher number of units wherein the adjacent diols and OH/NH.sub.2 have been converted to the corresponding aldehydes, which aldehydes have been then reduced to the corresponding alcohols, are also less sulfated with respect to the natural glycosaminoglycans and to their RO derivatives. Therefore, it is expected that they display less protein interactions and more favorable pharmacokinetics than their analogues with lower contents of modified residues.

(22) The present invention further relates to the compounds obtainable by the processes described above for use as medicaments.

(23) In particular, the present invention relates to the compounds obtainable by the processes described above for use as antimetastatic agents, as antitumor, preferably as antimyeloma.

(24) Heparin and low molecular weight heparin derivatives prepared according to the present invention, in spite of their low sulfation degree, have shown effective inhibition of heparanase activity, both in vitro and in vivo, in a multiple myeloma experimental model.

(25) Furthermore, the derivatives of the present invention have shown, even at low molecular weights (see examples 5, 6 and 8), heparanase inhibitory activity higher than that of RO heparins obtained from 2-O-desulfated heparins of similar molecular weight. The latters are show in table 1.

(26) TABLE-US-00001 TABLE 1 Average Heparanase % of glycol split molecular weight, inhibition Sample uronic acids MW (kDa) IC.sub.50 (ng/ml) RO 1 Heparin 28 17 8 RO 2 Heparin 26 10 50 RO 3 Heparin 23 7 500 RO 4 Heparin 23 5 750

(27) Data show a general trend to reduced heparanase inhibiting activity with the lowering of the molecular weight of glycol split heparin of RO type.

EXAMPLES

(28) Compounds Preparation

(29) N-desulfation of unfractionated heparin (hereinafter UFH) disclosed in the following examples was performed by modification of known methods (Inoue, Y. and Nagasawa, K. 1976, Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol, Carbohydr Res 46 (1) 87-95). The degree of N-desulfation was determined by .sup.13C-NMR (Naggi, A., et al. 2001, Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins, Carbohydr. Res. 336, 4, 283-290).

(30) Samples of modified heparins, endowed with different degree of N-desulfation, were subjected to periodate oxidation to give split units with two aldehyde groups and sodium borohydride (NaBH.sub.4) reduction in aqueous medium to give final heparin derivatives; both reactions were performed by modification of known methods. Periodate oxidation was preferably performed in the presence of NaHCO.sub.3, pyridine, MnCl.sub.2, or MnCl.sub.2 with NTA. Graded 2-O-desulfation of UFH was performed following modification of known methods (Jasej a, M. et al. 1989, Novel regio-and stereo-selective modifications of heparin in alkaline solution. Nuclear magnetic resonance spectroscopic evidence, Canad J Chem, 67, 1449-1455; R. N. Rej Arthur S. Perlin 1990, Base-catalyzed conversion of the a-L-iduronic acid 2-sulfate unit of heparin into a unit of a-L-galacturonic acid, and related reactions, Carbohydr. Res. 200, 25, 437-447; Casu, B. et al. 2004, Undersulfated and Glycol-Split Heparins Endowed with Antiangiogenic Activity, J. Med. Chem., 47, 838-848). Hereafter “RO” indicates the % of glycol split residues in which adjacent diols and OH/NH.sub.2 have been converted into the corresponding aldehyde and then into the corresponding alcohols, over the total glycosaminoglycan residues.

(31) In Vitro Testing

(32) Based on previous studies of Bisio et al. (Bisio, A. et al. 2007, High-performance liquid chromatographic/mass spectrometric studies on the susceptibility of heparin specie to cleavage by heparanase, Sem Thromb Hemost 33 488-495), heparanase inhibiting activity was determined in vitro by the group of Prof Vlodavsky at the University of Haifa, Israel, according to the method described by Hammond et al. (Hammond et al. 2010, Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening, Anal Biochem. 396, 112-6). Briefly, heparanase can cleave the synthetic pentasaccharide, Fondaparinux, which is an antithrombotic drug, structurally corresponding to the antithrombin binding site of heparin. After hydrolysis by heparanase, a trisaccharide and a reducing disaccharide are obtained. The latter can be easily quantified in order to assess heparanase activity. In the present examples, the assay solution comprises (100 μl) 40 mM sodium acetate buffer pH 5.0 and 100 mM Fondaparinux (GlaxoSmithKline), with or without inhibitor sample. Heparanase was added to a final concentration of 140 pM at the beginning of the assay. The plates were sealed with adhesive tape and incubated at 37° C. for 2-24 hours. The assay was stopped by addition of 100 μL of a 1.69 mM 4-[3-(4-iodophenyl)-1H-5 tetrazolio]-1,3-benzene disulfonate (WST-1, Aspep, Melbourne, Australia) solution in 0.1M NaOH. The plates were resealed with adhesive tape and developed at 60° C. for 60 minutes. The absorbance was measured at 584 nm (Fluostar, BMG, Labtech). In each plate, a standard curve constructed with D-galactose as the reducing sugar standard was prepared in the same buffer and volume over the concentration range of 2 to 100 μM. The IC.sub.50 value was determined. Results obtained using the above cited colorimetric assay were validated using a different test that employs an extracellular matrix (ECM) labeled with radioactive sulfate as substrate. Briefly, the ECM substrate is deposited by cultured corneal endothelial cells and hence closely resembles the subendothelial basement membrane in its composition, biological function and barrier properties. Detailed information about the preparation of sulfate labeled ECM and its use for the heparanase assay can be found in: Vlodavsky, I., Current Protocols in Cell Biology, Chapter 10: Unit 10.4, 2001.

(33) In Vivo Testing

(34) The antimyeloma activity in vivo was tested substantially following the procedure described in Yang Y et al. (Yang, Y. et al. 2007, The syndecan-1 heparan sulfate proteoglycan is a viable target for myeloma therapy, Blood 110: 2041-2048). Briefly, CB17 scid/scid mice aged 5 to 6 weeks were obtained from Arlan (Indianapolis, Ind.) or Charles River Laboratories (USA). Mice were housed and monitored in the animal facility of the University of Alabama in Birmingham. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee. Some 1×10.sup.6 heparanase-expressing CAG myeloma cells (high or low expressing) were injected subcutaneously into the left flank of each mouse. Ten days after injection of tumor cells, mice were implanted with Alzet osmotic pumps (Durect Corporation, Cupertino, Calif.) on the right flank. Pumps contained either solution of test compounds (new heparin derivatives) or phosphate buffer (PBS) as control. The solution was delivered continuously for 14 days. After 14 days, the animals were killed and the wet weight of the subcutaneous tumors and the mean sera kappa level were assayed and compared among the experimental groups by log-rank test (p<0.05 was considered statistically significant).

(35) Weekly luciferase bioluminescence imaging provides quantitative data on primary tumors and tracks metastasis within bone as well as soft tissues. Notably, the SCID-hu model is unique in that human tumor cells are injected directly into small pieces of human fetal bone implanted subcutaneously in SCID mice, thus closely recapitulating human myeloma.

(36) General Procedure of NMR Analysis

(37) Spectra were recorded at 25° C. on a Bruker Avance 500 spectrometer (Karlsruhe, Germany) equipped with a 5-mm TCI cryoprobe or with a 10 mm BBO probe. Integration of peak area or volumes in the spectra was made using standard Bruker TopSpin 2.0 software.

N-Desulfation of Unfractionated Heparin

Example 1 (G8220)

(38) UFH (4.01 g, lot. G3378) was dissolved in water (32 ml) and treated under stirring with Amberlite IR 120 (H+, 144 ml). The filtered acid solution was brought to pH 7 with pyridine, then concentrated to dryness under reduced pressure. The resulting pyridinium salt was dissolved in 40 ml of a mixture of DMSO:H.sub.2O (95:5 by volume), then stirred at 25° C. for 48 hours. After dilution with 40 ml of water, the solution was dialyzed at 4° C. for 16 hours against distilled water in membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization gave: G8220 (2.7 g), yield=67% w/w, MW=19,100 Da, N-desulfation degree determined by .sup.13C-NMR=74.7% of the total glucosamine residues, 2-0 sulfated uronic acid determined by .sup.13C-NMR=18% of the total monosaccharides.

Example 2 (G8343)

(39) Following the procedure described in Example 1, a sample of UFH (0.25 g, lot. G3378) was converted in the corresponding pyridinium salt, which was N-desulfated in a mixture of DMSO:MeOH (95:5 by volume). After 2 hours under stirring at 25° C., reaction mixture was processed, following the same final procedure described in Example 1, to give G8343 (0.172 g), yield=69% w/w, MW=18,000 Da, N-desulfation degree determined by .sup.13C-NMR=63.3% of the total glucosamine residues, 2-O sulfated uronic acid determined by .sup.13C-NMR=19% of the total monosaccharides.

Example 3 (G8516)

(40) Starting from a sample of UFH (0.25 g, lot G3378) and following the procedure described in Example 2, but reducing to 40 minutes the N-desulfation reaction time, G8516 was obtained (0.17 g), yield=68%, N-desulfation degree by .sup.13C-NMR=49.7% of the total glucosamine residues, 2-0 sulfated uronic acid determined by .sup.13C-NMR=17% of the total monosaccharides.

Example 4 (G8147)

(41) UFH (2.5 g, lot. G3378) was dissolved in water (20 ml) and treated under stirring with Amberlite IR 120 (H+, 90 ml). The filtered acid solution was brought to pH 7 with pyridine, then concentrated to dryness under reduced pressure. The resulting pyridinium salt was dissolved in 25 ml of a mixture of DMSO:MeOH (90:10 by volume), then stirred at 25° C. for 18 hours. After dilution with 25 ml of water, the solution was dialyzed at 4° C. for 16 hours against distilled water in membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization gave: G8147 (1.9 g), yield=76% w/w, MW=18,200 Da, N-desulfation degree by .sup.13C-NMR=60% of the total glucosamine residues.

Example 5 (G9416)

(42) UFH (5 g, lot. G3378) was dissolved in water (40 ml) and treated under stirring with Amberlite IR 120 (H+, 90 ml). The filtered acid solution was brought to pH 7 with pyridine, then concentrated to dryness under reduced pressure. The resulting pyridinium salt was dissolved in 100 ml of a mixture of DMSO:MeOH (95:5 by volume), then stirred at 25° C. for 18 hours. After dilution with 100 ml of water, the solution was dialyzed at 4° C. for 16 hours against distilled water in membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization gave: G9416 (3.65 g), yield=73% w/w, MW=18,800 Da, N-desulfation degree by .sup.13C-NMR=77% of the total glucosamine residues.

Example 6 (G8079)

(43) UFH (1 g, lot. G3378) was dissolved in water (8 ml) and treated under stirring with Amberlite IR 120 (H+, 90 ml) for 30 minutes. The filtered acid solution was brought to pH 7 with pyridine, then concentrated to dryness under reduced pressure. The resulting pyridinium salt was dissolved in 10 ml of a mixture of DMSO:MeOH (95:5 by volume), then stirred at 25° C. for 16 hours. After dilution with 10 ml of water, the solution was dialyzed at 4° C. for 3 days against distilled water in membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization gave: G8079 (1 g), yield=100% w/w, N-desulfation degree by .sup.13C-NMR=60% of the total glucosamine residues.

(44) Periodate Oxidation and Sodium Borohydride Reduction of N-Desulfated Heparins

Example 7 (G8340)

(45) A sample of G8220 of example 1 (0.25 g, 74.7% of N-desulfated heparin residues), dissolved in water (7.3 ml) and cooled to 4° C., was added to an equal volume of 0.2 M NaIO.sub.4. The pH value was adjusted to 6.8 with 2M NaHCO.sub.3 (about 2.1 ml) and, under stirring in the dark at 4° C., 0.08M nitrilotriacetic acid (NTA, 10 ml) was added to the solution. The pH value from 4.0 was brought to 6.6 by adding 2M NaHCO.sub.3 and the reaction mixture was kept under stirring at 4° C. for 8 hours. The excess of periodate was quenched by adding ethylene glycol (0.73 ml); after 1 hour the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The desalted solution was treated with NaBH.sub.4 (0.164 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess, and after a 10 minutes stirring, neutralized with 0.1N NaOH. After dialysis against distilled water at 4° C. for 16 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.202 g of G8340 was obtained, yield=90% w/w, MW=8,400 Da. The percentages, over the total of monosaccharide residues, of RO (53%), IdoA2S (35%), GlcNAc (9%) and GlcNH.sub.2 (12%) were determined by .sup.13C-NMR.

(46) In vitro heparanase inhibition: IC.sub.50=20 ng/ml.

(47) In vivo antimyeloma activity: 60 mg/Kg/day for 14 days: 75% tumor inhibition and 60% serum K inhibition.

Example 8 (G8438)

(48) Starting from G8343 of Example 2 (0.171 g of 63.3% of N-desulfated heparin residues) and following the same procedure described in Example 7, G8438 was obtained (91.4 mg), yield=82%, MW=6,800 Da. The percentages of RO (38%), IdoA2S (32%), GlcNAc (8%), GlcNH.sub.2 (4%), were determined by .sup.13C-NMR.

(49) In vitro heparanase inhibition IC.sub.50=60 ng/ml.

Example 9 (G8588)

(50) Starting from G8516 of Example 3 (0.171 g of 44% of N-desulfated heparin residues) and following the procedure described in Example 7, G8588 (0.136 g) was obtained, yield=80%, MW=11,000 Da. The percentages of GlcNAc (30%), GlcNH.sub.2 (13%), IdoA2S (34%), RO (37%) were determined by .sup.13C-NMR.

Example 10 (G9578)

(51) Starting from G9416 of Example 5, following the procedure described in Example 7, G9578 was obtained, yield=89%, MW=6,300 Da, N-desulfation degree by .sup.13C-NMR=48% of the total glucosamine residues. The percentages of RO (45%) and IdoA2S (35%) over the total residues of glycosaminoglycan were determined by .sup.13C-NMR. In vitro and in vivo testing on the inventive product yielded the following results:

(52) In vitro heparanase inhibition: IC.sub.50=75 ng/ml;

(53) In vivo antimyeloma activity (60 mg/kg day for 14 days): 63% tumor inhibition.

Example 11 (G8188)

(54) A sample of N-desulfated heparin G8147 of Example 4 (0.25 g, 60% N-desulfated heparin residues), dissolved in water (7.3 ml) and cooled to 4° C., was added to an equal volume of 0.2M NaIO.sub.4. The pH value was adjusted to 6.8 with 2M NaHCO.sub.3 (about 2.1 ml), under stirring in the dark at 4° C. for 16 hours. The excess of periodate was quenched by adding ethylene glycol (0.73 ml) and, after 1 hour, the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The desalted solution was treated with NaBH.sub.4 (0.164 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess and, after a 10 minutes stirring, it was neutralized with 0.1N NaOH. After dialysis against distilled water at 4° C. for 16 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.168 g of G8188 was obtained, yield=67% w/w, MW=3,460 Da. The percentages of RO (43%), IdoA2S (40%), GlcNAc (6%) and GlcNH.sub.2 (4%) were determined by .sup.13C-NMR.

Example 12 (G8189)

(55) A sample of G8147 (0.25 g, 60% of N-desulfated heparin residues), dissolved in water (7.3 ml) and cooled to 4° C., was added to an equal volume of 0.2M NaIO.sub.4. The pH value was adjusted to 6.8 with pyridine (5% v/v, about 730 μl), under stirring in the dark at 4° C. for 16 hours. The excess of periodate was quenched by adding ethylene glycol (0.73 ml) and, after 1 hour, the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The desalted solution was treated with NaBH.sub.4 (0.164 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess and, after 10 minutes of stirring, neutralized with 0.1N NaOH. After dialysis against distilled water at 4° C. for 16 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.181 g of G8189 was obtained, yield=7 2% w/w, MW=5,150 Da. The percentages of RO (49%), IdoA2S (39%), GlcNAc (7%) and GlcNH.sub.2 (7%) were determined by .sup.13C-NMR.

Example 13 (G8217)

(56) A sample of G8147 (0.25 g, 60% of N-desulfated heparin residues), dissolved in water (7 0.3 ml) and cooled to 4° C., was added to an equal volume of 0.2 M NaIO.sub.4. The pH value was adjusted to 6.8 with 2M NaHCO.sub.3 (about 2.1 ml) and 30 ml of MnCl.sub.2 0.05M (the final concentration in the solution was 0.00001M) were added under stirring in the dark at 4° C. for 16 hours. The excess of periodate was quenched by adding ethylene glycol (0.73 ml). During the night, the sample precipitated because the pH became 8 then its pH value was brought to 6 with 1N HCl. After 1 hour, the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The desalted solution was treated with NaBH.sub.4 (0.164 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess and after 10 minutes of stirring, neutralized with 0.1N NaOH. After dialysis against distilled water at 4° C. for 16 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.230 g of G8217 was obtained, yield=92% w/w, MW=7,455 Da. The percentages of RO (31%), IdoA2S (29%), GlcNAc (8%), and GlcNH.sub.2 (6%) on the total of glycosaminoglycan residues were determined by .sup.13C-NMR.

Example 14 (G8219)

(57) A sample of G8147 (0.25 g, 60% of N-desulfated heparin residues), dissolved in water (7 0.3 ml) and cooled to 4° C., was added to an equal volume of 0.2 M NaIO.sub.4. The pH value was adjusted to 6.8 with 2M NaHCO.sub.3 (about 1.2 ml) and, under stirring in the dark at 4° C., 0.08 M nitrilotriacetic acid (NTA, 10 ml) and 31 ml of MnCl.sub.2 0.05M were added to the solution. The pH value was brought from 4.0 to 6.3 by adding 2M NaHCO.sub.3 and the reaction mixture was kept under stirring at 4° C. for 8 hours. The excess of periodate was quenched by adding ethylene glycol (0.73 ml) and, after 1 hour, the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The dialyzed solution was treated with NaBH.sub.4 (0.164 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess, and after 10 minutes of stirring, neutralized with 0.1N NaOH. After dialysis against distilled water at 4° C. for 16 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.202 g of G8219 was obtained, yield=90% w/w, MW=7,330 Da. The percentages of RO (54%), IdoA2S (36%), GlcNAc (9%) and GlcNH.sub.2 (8%) were determined by .sup.13C-NMR.

Comparative Example 15 (G8092)

(58) To a sample of G8079 (1 g, N-desulfated heparin), dissolved in water (29.2 ml) and cooled to 4° C., an equal volume of NaIO.sub.4 (pHs) was added, and the reaction mixture was kept under stirring at 4° C. for 8 hours. The excess of periodate was quenched by adding ethylene glycol (2.9 ml) and, after 1 hour, the reaction mixture was desalted by dialysis against distilled water in membrane (cut-off: 3,500 Da) at 4° C. for 16 hours. The dialyzed solution was treated with NaBH.sub.4 (0.657 g, 3.4 mmoles), stirred for 3 hours at 25° C., then its pH value was brought to 4 with 1N HCl for quenching the NaBH.sub.4 excess, and after 10 minutes of stirring, neutralized with 0.1 N NaOH. After dialysis against distilled water at 4° C. for 72 hours in membrane (cut-off: 3,500 Da), concentration under reduced pressure and freeze drying, 0.643 g of G8092 was obtained, yield=64% w/w, MW=6,064 Da. The .sup.13C-NMR analysis shows a peak at 95 ppm of N-desulfated glucosamine. The oxidation reaction at acidic pH does not occur.

(59) All references, including publications, patent applications, and patents cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

(60) The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(61) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.