Carboxylated derivatives of glycosaminoglycans and use as drugs

Abstract

A glycosaminoglycan derivative endowed with heparanase inhibitory activity and antitumor activity, bearing carboxylate groups in positions 2 and 3 of at least part of the glycosaminoglycan residues, and to the process for preparing the same. The glycosaminoglycan derivatives of the present invention are generated starting from natural or synthetic glycosaminoglycans, preferably heparin or low molecular weight heparin, optionally 2-O- and 2-N-desulfated by two steps of oxidation. By the first oxidation, adjacent dials and optionally adjacent OH/NH.sub.2 of the glycosaminoglycan residues are converted to aldehydes and by the second oxidation said dialdehydes are converted to carboxylate groups. The first oxidation preferably leads to the cleavage of C2-C3 linkage of the ring of oxidable residues. The invention relates to a process for the preparation of said glycosaminoglycan derivatives and to their use as active ingredients of medicaments.

Claims

1. Carboxylated derivatives of glycosaminoglycan, the carboxylated derivative chain comprising the dimeric sugar moiety (I) but not dimeric sugar moiety (II) or (III): ##STR00001## wherein R.sub.1 is SO.sub.3 or Ac; R.sub.2 is SO.sub.3 or H; R.sub.3 is H or SO.sub.3; wherein the glycosaminoglycan is selected from unfractionated heparin, low molecular weight heparin and heparan sulfate wherein the glycosaminoglycan has a sulfation degree (SO.sub.3.sup.−/COO.sup.−), determined by conductometric titration as defined herein, of from 0.8 to 2.8.

2. Carboxylated derivatives of glycosaminoglycan, the carboxylated derivative chain comprising dimeric sugar moieties (I), (II) and (III): ##STR00002## wherein R.sub.1 is SO.sub.3 or Ac; R.sub.2 is SO.sub.3 or H; and R.sub.3 is SO.sub.3 wherein the glycosaminoglycan has a sulfation degree (SO.sub.3.sup.−/COO.sup.−), determined by conductometric titration as defined herein, of from 0.8 to 2.8.

3. The carboxylated derivatives of glycosaminoglycan according to claim 1, wherein the unfractionated heparin is 2-O-desulfated unfractionated heparin, 2-N-desulfated unfractionated heparin or 2-O-, 2-N-desulfated unfractionated heparin.

4. The carboxylated derivatives of glycosaminoglycan according to claim 1, wherein the low molecular weight heparin is 2-O-desulfated low molecular weight heparin, 2-N-desulfated low molecular weight heparin or 2-O-, 2-N-desulfated low molecular weight heparin.

5. The carboxylated derivatives of glycosaminoglycan according to claim 1, wherein the carboxylated derivatives of glycosaminoglycan have a sulfation degree (SO.sub.3.sup.−/COO.sup.−), determined by conductimetric titration as defined herein, of from 0.82 to 1.92.

6. The carboxylated derivatives of glycosaminoglycan according to claim 1, wherein the carboxylated derivatives have a molecular weight of from 3,000 to 20,000 Da.

7. The carboxylated derivatives of glycosaminoglycan according to claim 1, wherein the carboxylated derivatives have a molecular weight of from 3,500 to 12,000 Da.

8. A method of treating a disease gaining benefit from inhibition of heparanase, comprising administering to a human in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the carboxylated derivatives of glycosaminoglycan of claim 1, wherein the disease is diabetic nephropathy, inflammatory bowel disease, colitis, arthritis, psoriasis, sepsis, atherosclerosis or tumor.

9. A method of treating tumor metastasis or a tumor in a human, the method comprising administering to the human in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the carboxylated derivatives of glycosaminoglycan of claim 1.

10. The method according to claim 9, wherein the tumor is myeloma.

11. The carboxylated derivatives of glycosaminoglycan according to claim 2, wherein the unfractionated heparin is 2-O-desulfated unfractionated heparin, 2-N-desulfated unfractionated heparin or 2-O-, 2-N-desulfated unfractionated heparin.

12. The carboxylated derivatives of glycosaminoglycan according to claim 2, wherein the low molecular weight heparin is 2-O-desulfated low molecular weight heparin, 2-N-desulfated low molecular weight heparin or 2-O-, 2-N-desulfated low molecular weight heparin.

13. The carboxylated derivatives of glycosaminoglycan according to claim 2, wherein the carboxylated derivatives of glycosaminoglycan have a sulfation degree (SO.sub.3.sup.−/COO.sup.−), determined by conductimetric titration as defined herein, of from 0.82 to 1.92.

14. The carboxylated derivatives of glycosaminoglycan according to claim 2, wherein the carboxylated derivatives have a molecular weight of from 3,000 to 20,000 Da.

15. The carboxylated derivatives of glycosaminoglycan according to claim 2, wherein the carboxylated derivatives have a molecular weight of from 3.500 to 12,000 Da.

16. A method of treating an indication gaining benefit from inhibition of heparanase, comprising administering to a human in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the carboxylated derivatives of glycosaminoglycan of claim 2, wherein the indication is diabetic nephropathy, inflammatory bowel disease, colitis, arthritis, psoriasis, sepsis, atherosclerosis or tumor.

17. A method of treating tumor metastasis or a tumor in a human, the method comprising administering to the human in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of the carboxylated derivatives of glycosaminoglycan of claim 2.

18. The method according to claim 17, wherein the tumor is myeloma.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: prevalent structures generated by the process of the present invention. (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 groups (R.sub.3 and R.sub.4) can each or both be substituted by a sulphate group or non-substituted. (2, 3, 4) representative structures (new entities) generated by oxidative cleavage of the ring of 2-non-sulfated heparin residues, followed by further oxidation to tri- or di-carboxylated residues.

(2) FIG. 2: one example of chromatogram obtained with MS detection. The presence of peaks corresponding to oligosaccharides containing the uronic acid di- or tri-carboxylate residues, attributed by the measured values of molecular weight, are highlighted respectively by the code“+carbox2” or “+carbox3” in the graph.

(3) FIG. 3: one example of chromatogram obtained with MS detection. The presence of peaks corresponding to oligosaccharides containing the uronic acid di- or tri-carboxylate residues, attributed by the measured values of molecular weight, are highlighted respectively by the code“+carbox2” or “+carbox3” in the graph.

DETAILED DESCRIPTION OF THE INVENTION

(4) The present invention relates to a novel class of chemically modified glycosaminoglycan derivatives, endowed with heparanase inhibitory activity. In particular it relates to carboxylated glycosaminoglycan derivatives, wherein at least part of the residues are split residues bearing three carboxylate groups (or two, if the split residue is a glucosamine).

(5) The glycosaminoglycan derivatives of the present invention are preferably heparin derivatives, designed as “di/tricarboxylated heparins”, which strongly inhibit the heparan sulfate degrading activity of heparanase. The chemical modifications made to heparin, modifying the residue of glucuronic acid included in the binding site for ATIII, abolish the heparin anticoagulant activity, enabling the use of high doses.

(6) The glycosaminoglycan derivatives of the present invention are obtainable by oxidation, preferably by periodate, of 2-non-sulfated residues of a glycosaminoglycan under conditions effective to convert adjacent diols and optionally adjacent OH/NH.sub.2 of the glycosaminoglycan to aldehydes, followed by oxidation of the oxidized glycosaminoglycan under conditions to convert said dialdehydes to carboxylate groups. In particular, the glycosaminoglycan derivatives of the present invention are preferably obtainable starting from a glycosaminoglycan having a sulfation degree (SO.sub.3—/COO— molar ratio) of from 0.8 to 2.8, preferably of from 0.9 to 2.5. The sulfation degree (SO.sub.3—/COO— molar ratio) is herein intended as determined by conductimetric titration according to Casu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176. The di/tricarboxylated glycosaminoglycan derivatives obtainable by the inventive process disclosed above show a carboxyl increment of from 1.2 to 2.2, wherein said carboxyl increment is calculated as the ratio of the sulfation degree (SO.sub.3—/COO— molar ratio) of the starting material, after the first oxidation step, to the sulfation degree (SO.sub.3—/COO— molar ratio) of the di/tricarboxylated derivative, determined by conductimetric titration as herein defined. More specifically, the sulfation degree of the starting material after the first oxidation step is determined on a sample of the glycosaminoglycan intermediate obtained by the first oxidation step (a), after reduction by NaBH.sub.4.

(7) Preferably, the glycosaminoglycan derivatives of the present invention derive from natural or synthetic (chemically or enzymatically obtained) glycosaminoglycans, more preferably from 2-O- and/or 2-N-desulfated glycosaminoglycans. In a preferred embodiment, said natural or synthetic glycosaminoglycan is unfractionated heparin, LMWH, or heparan sulfate, optionally 2-O- and/or 2-N-desulfated.

(8) More preferably the glycosaminoglycan derivatives derive from natural or synthetic heparins or LMWHs, 2-O- and optionally 2-N-desulfated.

(9) As an example, heparin chains can naturally comprise from about 5% to 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 sulfation degree depends on the heparin source (animal species, organ sources) and on the extraction procedures. Every 2-O- or 2-N-non-sulfated residue of glycosaminoglycans, not bearing 3-0-sulfate substituents, is susceptible of oxidation with ring opening (split) and conversion of vicinal diols and OH/NH.sub.2 to aldehydes. Optionally, graded 2-O-desulfation of the starting glycosaminoglycans allows to modulate the ratio of glucosamine/uronic acid split residues.

(10) The invention further relates to the process for preparing said carboxylated glycosaminoglycan derivatives and further to their use as active ingredients of medicaments for treating pathological conditions, either as sole active ingredient or in combination with other medicaments. Said pathological conditions comprise multiple myeloma and other neoplastic diseases, including their metastatic forms. Furthermore, the invention relates to said carboxylated glycosaminoglycan derivatives for use in any therapeutic indication gaining benefit from inhibition of heparanase. The invention also relates to pharmaceutical compositions containing said carboxylated glycosaminoglycan derivatives, optionally in combination with at least one further active ingredient.

(11) The process for preparing the carboxylated glycosaminoglycan derivatives of the invention comprises: oxidation, preferably by periodate (or by use of oxidizing reagents having similar reactivity), of the susceptible non-sulfated residues of a glycosaminoglycan, under conditions effective to convert adjacent diols and optionally adjacent OH/NH.sub.2 to aldehydes, followed by oxidation of the glycosaminoglycan resulting from the first oxidation, under conditions to obtain two new carboxylate groups from the corresponding aldehyde groups.

(12) The process of the present invention thus comprises the oxidation, preferably by periodate, of 10% to 100%, preferably of from 25% to 100%, of 2-O- and optionally 2-N-, 3-O-non-sulfated residues of a glycosaminoglycan, under conditions effective to convert adjacent diols and optionally adjacent OH/NH.sub.2 to aldehydes; then it comprises the oxidation of the oxidized glycosaminoglycan under conditions effective to convert said dialdehydes to carboxylate groups.

(13) Preferably, the starting glycosaminoglycan is a natural or synthetic glycosaminoglycan; more preferably it is selected from heparin, low molecular weight heparins, or heparan sulfate; most preferably it is selected from heparin and LMWHs, 2-O- and optionally 2-N-desulfated.

(14) Preferably, the starting glycosaminoglycan has a sulfation degree (SO.sub.3—/COO—) of from 0.8 to 2.8, more preferably of from 0.9 to 2.5, by conductimetric titration, as defined herein.

(15) The first oxidation of the inventive process is preferably carried out at a pH of from 3 to 10, more preferably at a pH of from 4.5 to 8. In a preferred embodiment the first oxidation of the inventive process is carried out at a pH of from 3 to 5, in order to cleave only the linkage C.sub.2-C.sub.3 of the non-sulfated uronic acid residues, avoiding side reactions. In another preferred embodiment, the first oxidation of the inventive process is carried out at a pH of from 5.5 to 10, in order to cleave the linkage C.sub.2-C.sub.3 of both the 2-O-non-sulfated uronic acid and N-non-sulfated glucosamine residues.

(16) In a preferred embodiment, the first oxidation is performed under conditions to cleave the bond between the C2 and C3 of both the 2-O-non-sulfated uronic acids and of the 2-N-, 3-O-non-sulfated glucosamines.

(17) Optionally, the inventive process is 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 reaction solution, or in the presence of MnCl.sub.2 with or without NTA. The further dialdehyde oxidation is preferentially performed using NaClO.sub.2, or by the use of agents with comparable oxidizing properties, such as TEMPO (2,2,6,6 Tetra Methyl-1-Piperidinyl-Oxy).

(18) Preferably, carboxylated uronic acid residues in the glycosaminoglycans derivatives of the present invention are from 25% to 100%, more preferably from 50% to 100%, most preferably from 60% to 90%, of the total residues of the total carboxylated residues of the glycosaminoglycan.

(19) The carboxylated glycosaminoglycan derivatives obtainable by the above processes preferably have a molecular weight of from 8000 to 30,000 Da., depending on the process conditions and on the starting glycosaminoglycan employed. In a preferred embodiment, more preferably when unfractionated heparin is employed as the starting glycosaminoglycan, the carboxylated glycosaminoglycan derivative obtainable by the above processes preferably has a molecular weight of from 3,000 to 20,000 Da., preferably from 3,500 to 12,000 Da.

(20) The novel glycosaminoglycan derivatives obtainable by the process of the present invention represent a new class of heparin-like polysaccharides, characterized by the presence of split residues, each bearing two additional carboxylate groups. Note that residues bearing one natural carboxylate group, are converted to tricarboxylated residues by the process of the present invention. Said novel di/tricarboxylated glycosaminoglycan derivatives have unexpectedly shown to be strong heparanase inhibitors in vitro and to inhibit myeloma in animal models.

(21) Glycosaminoglycan derivatives comprising residues bearing two or three carboxylate groups, being also less sulfated than the parent glycosaminoglycan, display more favorable pharmacokinetics than their analogues bearing less carboxylic groups.

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

(23) In particular, the present invention relates to the compounds obtainable by the above processes for use as antitumor, preferably for use as antimyeloma medicaments, either alone or in combination with at least one further active ingredient.

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

EXAMPLES

(25) Compounds Preparation

(26) Samples of unfractionated or fractioned heparins, endowed with different degree of sulfation (SO.sub.3.sup.−/COO.sup.−) by conductimetric titration, were subjected to periodate oxidation (to give split dialdehyde units), performed by modification of known methods. Graded 2-O-desulfation of unfractionated heparins (UFH) was performed following modification of known methods (Jaseja 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 and A. 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). Under basic conditions, 2-O-desulfated (natural or chemically induced) L-iduronic acid units are converted into 2,3-epoxy derivatives and finally to L-galacturonic acid units. The dialdehydes originated from the uronic acid units, and optionally from glucosamines, are preferably, and within a short time, further oxidized to dicarboxylates.

(27) In Vitro Testing

(28) 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. Vlodaysky 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 heparanase cleavage, 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 (100 ul) comprised 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 to start 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 solution of 1.69 mM 4-[3-(4-iodophenyl)-1H-5 tetrazolio]-1,3-benzene disulfonate (WST-1, Aspep, Melbourne, Australia) in 0.11M NaOH. The plates were resealed with adhesive tape and developed at 60° C. for 60 min. 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 range of 2 to 100 μM. The IC.sub.50 value was determined. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics and biochemical parameters of the enzyme can be reliably characterized. Results obtained using the above described colorimetric assay were validated using sulfate labeled extracellular matrix (ECM) 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: Vlodaysky, I., Current Protocols in Cell Biology, Chapter 10: Unit 10.4, 2001. The assay is highly sensitive, better resembles the in vivo conditions, but, due to its biological nature, it is semi-quantitative and limited in terms of biochemical parameters.

(29) In Vivo Testing

(30) 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 at Birmingham. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee. 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 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).

(31) 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.

(32) General Procedure of NMR Analysis

(33) 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 volumes in the spectra was made using standard Bruker TopSpin 2.0 software. The structure of dicarboxylated uronic acid residues was determined by two-dimensional heteronuclear experiments that confirmed the presence of the tricarboxylate residues and permitted the identification of their chemical shift. In the table below the assigned chemical shifts of proton and carbons in position 1, 4, 5 of the dicarboxylated residue, of glucosamine and 2-O-sulfated iduronic acid are reported.

(34) TABLE-US-00001 Signals corresponding to residue position .sup.1H ppm .sup.13C ppm CS-Uronic acid gsox 4.97 81.65 C4-Uronic acid gsox 4.59 81.22 Cl-Uronic acid gsox 5.01 103.71 C4 3.63 79.14 Cl-Glucosamine NS (ANS) 5.40 99.17 Cl-ANS linked to (gsox) 5.03 100.19 Cl-Iduronic acid 2S 5.17 102.11

(35) General Procedure for Calculation of Carboxyl Groups Increment

(36) The increase of carboxyl groups in the uronic acid residues of dicarboxylated heparin derivatives was calculated starting from respective values of molar ratio SO.sub.3—/COO— of the starting material (unfractionated heparin or at least partially desulfated heparins and LMWHs) and of the dicarboxylated derivatives, evaluated by conductimetric titration (Casu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176). In particular, the sulfation degree of the starting material is determined after the first oxidation step, on a reduced sample of the glycosaminoglycan oxidized intermediate (see examples 4-7), while the sulfation degree of the final glycosaminoglycan carboxylated derivative is determined after the second oxidation step (see examples 8-11, 13-14).

(37) SO.sub.3.sup.−/CO.sub.2.sup.−=A (ratio in starting materials) SO.sub.3.sup.−=A/CO.sub.2.sup.−

(38) SO.sub.3.sup.−/CO.sub.2.sup.−=B (ratio in dicarboxylated derivatives) SO.sub.3.sup.−=B/CO.sub.2.sup.−

(39) Given that the number of sulfate groups does not change during the two steps of oxidation, it can be concluded that the increase of the carboxyl groups (CO.sub.2.sup.−.sub.(dicarboxylated derivatives)/CO.sub.2.sup.−.sub.(starting materials)) is equal to the ratio of the individual molar ratios A and B.
Carboxyl increment (C.I.)=AB=CO.sub.2.sup.−.sub.(dicarboxylated derivatives)/CO.sub.2.sup.−.sub.(starting materials).

(40) Enzymatic Cleavage of Heparins and their Carboxylated Derivatives and HPLC/MS Analysis

(41) The substrate (2-3 mg) was dissolved in a 1:1 (v/v) mixture of 100 mM sodium acetate buffer (pH 7.0) and 10 mM calcium acetate to obtain a 7.7 mg/ml solution. To carry out the enzymatic cleavage 144 μI of the mixture of 100 mM sodium acetate and 10 mM calcium acetate (1:1) and 3 μI of heparinases mixture (heparin lyases I, II and III) (1 μI of each lyase, 2 mU/μI enzyme solution) were added to 13 μI of the heparin solution. The reaction mixture was stirred at 37° C. (Thermo-Shaker TS-100, Biosan) for 24 h. The reaction was stopped by adding 3 μI of 3% HCOOH. Each sample was diluted two times with water and analyzed by IPRP-HPLC/ESI-TOF (micrOTOF-Q, Bruker). A C18 kinetex and a gradient (0′-17% B, 15′-20% B, 55′-40% B, 100′ 50% B, 115′ 90% B) that use fase A (pH 6.25) and B (pH 7.95) with 10 mM DBA-CH.sub.3COOH was used with a flow rate 100 μL/min.

Example 1 (G7669)

(42) A sample of UFH (2.5 g of lot. G5842) in 1M NaOH (32 ml) was heated at 60° C. for 30 min. After cooling at room temperature and neutralization with 2N HCl, the solution was dialyzed at room temperature for 3 days against distilled water in membranes (cut-off: 3500 Da). Concentration under reduced pressure and freeze drying gave: 2.15 g (yield=80% w/w) of an intermediate with 13% of the total uronic acid residues bearing epoxide group as determined by .sup.13C-NMR. The sample was then dissolved in water (32 ml) and kept under stirring at 70° C. for 2 days, in order to hydrolyze the epoxy groups. After concentration and freeze-drying, G7669 was obtained.

Example 2 (G8661)

(43) A sample of UFH (2.11 g of lot. G3378) in 27 ml of 1N NaOH was stirred at 60° C. for 30 min. Neutralization, cooling at room temperature and dialysis, concentration and freeze-drying (as described in Example 1) gave the intermediate G8637 (1.5 g). Since its .sup.13C-NMR spectrum indicated the presence of epoxy groups, G8637 (1.5 g) was dissolved in water (32 ml) and kept under stirring at 70° C. for 2 days, in order to hydrolyze the epoxy groups. After concentration and freeze-drying, G8661 was obtained (1.5 g).

Example 3 (G8699)

(44) A sample of UFH (2.01 g of lot. G3378) was processed as described in Example 2 to give the intermediate epoxy-containing derivative G8638. A sample of G8638 (1.4 g) was dissolved in water (32 ml) and heated under stirring at 70° C. for 24 hours, to give, after concentration and freeze-drying, 1.3 g of G8699.

(45) Periodate Oxidation of Unfractionated Heparin and 2-O-Desulfated Derivatives

Example 4 (G7731)

(46) Starting from sample G7669 of Example 1 (1.8 g, 13% 2-O-desulfation) in water (52 ml), the solution was cooled at 4° C., stirred in the dark and 52 ml of 0.2M NaIO.sub.4 were added. After 16 hours, the excess of periodate was quenched by adding ethylene glycol (5.2 ml) and after 1 hr. at 4° C. the reaction mixture was desalted by dialysis at 4° C. for 16 hours. After concentration under reduced pressure and freeze-drying, the dialdehyde-bearing G7731 was obtained (1.5 g), yield=83%. A small portion of sample was reduced with NaBH.sub.4 to measure MW=8,242 Da and SO.sub.3.sup.−/COO.sup.−=2.46, by conductimetric titration.

Example 5 (G8425)

(47) Starting from a sample of UFH (0.25 g, lot. G3378) and following the same procedure described in Example 4, the dialdehyde-bearing G8425 was obtained (0.24 g), yield=96% w/w.

Example 6 (G8678)

(48) Starting from a sample of G8661 of Example 2 (1.5 g) and performing the same procedure described in Example 4, the dialdehyde-bearing G8678 was obtained (1.5 g). A small portion of sample was reduced with NaBH.sub.4 to measure SO.sub.3.sup.−/COO.sup.−=1.96 by conductimetric titration.

Example 7 (G8710)

(49) Starting from a sample of G8699 of Example 3 (0.56 g) and following the same procedure described in Example 4, the dialdehyde-bearing G8710 was obtained (0.56 g). A small portion of sample was reduced with NaBH.sub.4 to measure SO.sub.3.sup.−/COO.sup.−=1.51 by conductimetric titration.

(50) Oxidation of the Dialdehydic Uronic Acid Intermediates to the Corresponding Dicarboxylates

Example 8 (G7927)

(51) A sample of G7731 of Example 4 (0.3 g) was dissolved in water (29 ml), cooled at 0° C. in a double-necked round-bottomed flask and, under stirring in nitrogen atmosphere, it was treated with an aqueous solution (6 ml) containing NaClO.sub.2 (0.362 g). After a drop-wise addition of glacial acetic acid (0.118 ml) up to reach pH 4.0, the reaction mixture was stirred at room temperature for 24 hours. After further 3 hours under stirring at room temperature, by fluxing N.sub.2, a colorless solution was obtained. The reaction mixture was neutralized with 0.5N NaOH and desalted by dialysis as described in Example 4. Concentration and freeze-drying gave G7927 (0.228 g), yield=76% w/w, having:

(52) MW=6,450 Da;

(53) SO.sub.3.sup.−/COO.sup.−=1.23;

(54) Carboxyl increment (C.I.)=2

(55) In vitro heparanase inhibition analysis gave: IC.sub.50=10 ng/ml.

Example 9 (G8437)

(56) Starting from a sample of G8425 of Example 5 (0.25 g), in which only the non-sulfated uronic acids naturally present in the chain of heparin (18%) were oxidized following the same procedure described in example 8, G8437 (0.21 g) was obtained by reducing to 1.5 hours the N.sub.2 fluxing before neutralization of the reaction mixture, having:

(57) MW=12,100 Da;

(58) SO.sub.3.sup.−/COO.sup.−=1.62;

(59) Carboxyl increment (C.I.): 1.43.

(60) In vitro heparanase inhibition analysis gave: IC.sub.50=10 ng/ml.

Example 10 (G8767)

(61) Starting from samples of G8678 of Example 6 (1 g) and following the procedure described in Example 8, G8767 (1.05 g) was obtained, having:

(62) MW=8,800 Da;

(63) SO.sub.3.sup.−/COO.sup.−=1.27;

(64) Carboxyl increment (C.I.)=1.55.

(65) In vivo antimyeloma activity analysis (60 mg/kg day for 14 days) gave: 52% tumor inhibition and 20% serum K inhibition.

Example 11 (G8733)

(66) Starting from sample G8710 of Example 7 (0.56 g) and following the same procedure described in the Example 8, G8733 (0.453 g) was obtained, yield=80% w/w, having:

(67) MW=5,540 Da;

(68) SO.sub.3.sup.−/COO—=0.97;

(69) Carboxyl increment (C.I.)=1.56.

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

Example 12 (G9685)

(71) Starting from a sample of UFH (lot G3378) and following the procedure described in Example 4, the dialdehyde derivative was obtained. The dialdehyde derivative was further oxidized in the presence of sodium chlorite, following the procedure of Example 8, obtaining the carboxylated derivative G9685, MW=11,700 Da.

(72) In vitro heparanase inhibition analysis gave: IC.sub.50=10 ng/ml;

(73) In vivo antimyeloma activity analysis (60 mg/kg day for 14 days) gave: 68% tumor inhibition.

Example 13 (G7897)

(74) A sample of G7731 of Example 4 (0.3 g) dissolved in water (3 ml) and treated with 1.36 ml of NaClO.sub.2 to pH 8.2-8.5 with HCl 4%. Then 1 mg of TEMPO was added and pH was maintained a 7-7.5 with NaOH 1M for 24 hours. The reaction mixture was desalted by dialysis, concentrated and freeze-dried to give G7897 (0.190 g), yield=63% w/w, having:

(75) SO.sub.3.sup.−/COO.sup.−=1.92;

(76) Carboxyl increment (C.I.)=1.28.

Example 14 (G9585)

(77) Starting from a sample of 2-O-desulfated heparin (G9416, SO.sub.3.sup.−/COO.sup.−=1.39), following the procedure described in Example 4, the dialdehyde derivative G9577 was obtained. A sample of G9577 (0.242 g) dissolved in water (21 ml) was cooled at 0° C. in a double-necked round-bottomed flask and, under stirring in nitrogen atmosphere, it was treated with an aqueous solution (1 ml) containing NaClO.sub.2 (0.128 g). After a drop-wise addition of glacial acetic acid (0.084 ml) up to reach pH 4.0, the reaction mixture was stirred at room temperature for 24 hours. After further 3 hours under stirring at room temperature, by fluxing N.sub.2, a colorless solution was obtained. The reaction mixture was neutralized with 0.5N NaOH and desalted by dialysis as described in Example 4. Concentration and freeze-drying gave G9585 (0.148 g), yield=61% w/w, having:

(78) MW=4,700 Da;

(79) SO.sub.3.sup.−/COO.sup.−=0.82;

(80) Carboxyl increment (C.I.)=1.69.

(81) In vitro heparanase inhibition analysis gave: IC.sub.50=44 ng/ml.