HK1222873B - Derivatives of n-desulfated glycosaminoglycans and use as drugs - Google Patents

Description

Derivatives of N-desulfated glycosaminoglycans and their use as medicines

SUMMARY OF THE INVENTION

The present invention relates to N-desulfated and optionally 2-O-desulfated glycosaminoglycan derivatives, wherein at least part of the adjacent diols and OH/NH ₂ are converted into the corresponding aldehydes, which are then reduced to the corresponding alcohols. These products have heparanase inhibitory activity and antitumor activity. The glycosaminoglycan derivatives are obtained from natural or synthetic glycosaminoglycans, preferably from unfractionated heparin, low molecular weight heparin (LMWH), heparan sulfate or derivatives thereof. Natural glycosaminoglycans can be obtained from any animal source (different animal species and organs can be used).

The present invention further relates to the method for preparing the above-mentioned, and further relates to their purposes as the active ingredient of the medicine that can be used for pathological condition.Specifically, the pathological condition includes multiple myeloma and other cancers (i.e. sarcoma, cancer, malignant blood disease), including their metastatic form. The glycosaminoglycan derivatives of the present invention can be used as medicines also combined with other therapies (tumor therapy or non-tumor therapy). In addition, the present invention relates to the purposes of the N-desulfated and optionally 2-O-desulfated glycosaminoglycan derivatives (preferably obtained by heparin and low molecular weight heparin (LMWH)) in any treatment indication (i.e. diabetic nephropathy, inflammatory bowel disease, colitis, arthritis, psoriasis, sepsis, atherosclerosis) that obtains benefit by the inhibition of heparanase, and it is also with known determined medicine or treatment combination.

The present invention also relates to a pharmaceutical composition comprising at least one of the N-desulfated and optionally O-desulfated glycosaminoglycan derivatives as an active ingredient, wherein at least part of the adjacent diols and OH/NH ₂ are converted into the corresponding aldehydes and subsequently reduced to the corresponding alcohols. Optionally, the present invention relates to a pharmaceutical composition comprising at least one of the glycosaminoglycan derivatives as an active ingredient in combination with at least one other active ingredient, more preferably a therapeutic compound. Preferably, the glycosaminoglycan derivative is a heparin derivative or a low molecular weight heparin (LMWH).

Background of the Invention

Multiple myeloma is the second most prevalent hematological malignancy and accounts for more than 10% of all blood cancers in the United States, with approximately 20,000 new cases per year and a mortality rate greater than 50% (Graham-Rowe D., 2011, Multiple myeloma outlook. Nature 480, s34-s35).

Over the past few years, promising therapies have been developed, such as the administration of proteasome inhibitors (Velcade), bisphosphonates, thalidomide, etc. The effectiveness of these agents is at least in part due to their effects on the myeloma tumor microenvironment.

While the efficacy of such agents has been demonstrated for myeloma, there is a need for new and improved drugs for the treatment of myeloma and other tumors.

Heparanase is an endo-β-glucuronidase that cleaves the heparan sulfate (HS) chains of proteoglycans (PG-HS), such as syndecan-1, thereby releasing HS-bound growth factors.

In humans, there appears to be a single, dominant functional heparanase that is capable of cleaving HS. Heparanase is expressed by many human tumors, where it significantly increases the angiogenesis and metastatic capacity of tumor cells. In fact, elevated heparanase levels have been associated with late progression and metastasis in many tumor types. For example, high levels of heparanase are associated with shorter postoperative survival in patients. A direct role for heparanase in tumor metastasis has been demonstrated in the laboratories of Professors Vlodavsky and Sanderson, where our novel inhibitor was tested.

In addition to its enzymatic functions, including the release of HS-bound growth factors by invasive cells and the degradation of the extracellular matrix (ECM), heparanase also has non-enzymatic functions that can influence tumor behavior and its microenvironment. Sanderson's group pioneered the study of heparanase and syndecan-1 in myeloma, demonstrating that heparanase plays a major role in the aggressive tumor phenotype. This occurs by promoting the upregulation of VEGF and MMP-9, which together stimulate tumor growth, metastasis, and osteolytic bone destruction. Indeed, it has been shown that heparanase promotes myeloma tumor growth and spontaneous metastasis to bone in vivo, and that heparanase expression by tumor cells drives intense osteolysis, at least in part due to upregulation of RANKL expression. The osteolysis-promoting effect of heparanase may be very important because bone-binding growth factors are released when bone degenerates. In addition, osteoclasts can release tumor growth-promoting factors such as HGF. Together, these factors can help establish a niche within the bone marrow that supports tumor cell targeting and subsequent growth (Fux, L. et al. 2009, Heparanase: busy at the cell surface. Trends Biochem Sci 34(10):511-519; Sanderson R.D. and Yang Y., 2008, Syndecan-1: a dynamic regulator of the myeloma microenvironment. Clin. Exp Metastasis 25:149-59; Ilan N. et al. 2006. Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis. Int. J. Biochem. Cell Biol. 38:2018-2039). Therefore, inhibition of heparanase is a viable target for myeloma therapy, which is supported by the fact that heparanase has a single enzymatic activity and is rarely expressed in normal tissues. Furthermore, heparanase knockout mice have been shown to be viable and to exhibit no observable symptoms, suggesting that strategies derived from heparanase inhibition have minimal or no side effects (Casu B. et al. 2008. Non-anticoagulant heparins and inhibition of cancer. Pathophysiol Haemost Thromb. 36: 195-203; Vlodavsky I. et al. 2007. Heparanase: structure, biological functions, and inhibition by heparin-derived mimetics of heparan sulfate. Curr Pharm Des. 13: 2057-2073; Naggi A. et al. 2005. Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting. J. Biol. Chem. 280: 12103-12113).

Heparin is a linear, polydisperse, sulfated polysaccharide of the glycosaminoglycan family that exhibits anticoagulant and antithrombotic activities. Heparin's carbohydrate chain consists of alternating uronic acid and D-glucosamine residues. The primary repeating units are the disaccharides 2-O-sulfated L-iduronic acid (IdoA2S) α(1→4) and N-,6-O-sulfated D-glucosamine (GlcN6S). The minor components are non-sulfated L-iduronic acid and D-glucuronic acid, as well as N-acetyl D-glucosamine and N-, 3-O-, 6-O-trisulfated D-glucosamine (Casu B., 2005. Structure and active domains of heparin. In: Chemistry and Biology of Heparin and Heparan Sulfate. Amsterdam: Elsevier. 1-28; Casu B. and Lindahl U. 2001, Structure and biological interactions of heparin and heparansulfate. Adv Carbohydr Chem Biochem 57: 159-206). Heparin, which is structurally similar to HS, can effectively inhibit heparanase, but in heparanase inhibition strategies, it is not possible to use it at high doses due to its anticoagulant activity.

Interestingly, low molecular weight heparin (LMWH), which is more bioavailable and less anticoagulant than heparin, appears to prolong the survival of cancer patients through direct effects on tumor growth and metastasis. This may be due, at least in part, to inhibition of heparanase enzymatic activity (Zacharski L.R. and Lee, A.Y. 2008, Heparin as an anticancer therapeutic. Expert Opin Investig Drugs 17: 1029-1037; Yang Y et al. 2007, The syndecan-1 heparan sulfate proteoglycan is a viable target for myeloma therapy. Blood 110: 2041-2048).

In the prior art, effective inhibitors of the enzymatic activity of heparanase have been selected by studying the inhibition of heparanase by non-anticoagulant heparins (most of which contain non-sulfated uronic acid residues modified by opening the glycosidic ring at the 2-3 bond (diol splitting). The inhibitors differ in their degree of O-sulfation, N-acetylation and diol splitting of the non-sulfated uronic acid residues (pre-existing and by graded 2-O-desulfation) (Naggi A., 2005. Glycol-splitting as a device for modulating inhibition of growth factors and heparanase inhibition by heparin and heparin derivative. In: Chemistry and Biology of Heparin and Heparan Sulfate. Amsterdam: Elsevier 461-481).

The term "glycol split" (gs) generally refers to a carbohydrate polymer in which the presence of some monosaccharide residues is opened due to the cleavage of a bond (glycol split) between two adjacent carbons (each having a hydroxyl group). The first generation of glycol-split heparins (so-called "reduced oxidized heparin" (RO-heparin)) is largely composed of unmodified polysulfated blocks (occasionally interrupted by glycol-split residues, corresponding to the non-sulfated glucuronic acid/iduronic acid residues present on the original chain) (Naggi A., 2005. Glycol-splitting as a device for modulating inhibition of growth factors and heparanase inhibition by heparin and heparinderivative. In: Chemistry and Biology of Heparin and Heparan Sulfate. Amsterdam: Elsevier 461-481). This chemical action on heparin (modification of the glucuronic acid residues within the ATIII binding site) reduces or eliminates its anticoagulant activity, enabling it to be used in high doses.

WO 92/17188 discloses the antiproliferative activity of non-anticoagulant types of heparin against smooth muscle cells. The heparin is prepared by N-deacetylation of the N-acetylglucosamine unit (which is the smaller component of the natural heparin chain) with a hydrazine-containing substance, followed by periodate oxidation of the diol or adjacent OH/ _NH2 groups to the corresponding aldehyde. After oxidation, the aldehyde is reduced to the alcohol without substantially fragmenting the glycosaminoglycan. The N-sulfated unit is not affected by redox reactions.

N-desulfated heparin (Chemical Abstracts Accession No. 53260-52-9), also referred to as "desulfonated heparin (heparamine)" in the Merck index (14th edition, 2006), is known to have several effects: reduced anticoagulant activity, some activity against gastric cancer metastasis in mice (by inhibiting VEGF expression and angiogenesis) (Chen-J-L et al. 2007, World J. Gastroenterol 21, 457-461), and prevention of liver/kidney damage caused by ischemia and reperfusion (Chen-J-L et al. 2002, World J. Gastroenterol 8, 897-900). N-desulfated heparin is also known to be an intermediate for the synthesis of various N-acetylated heparins. The extent of N-desulfation can range from 10% to as high as 100% of the N-sulfated glucosamine residues present in heparin (Huang L. and Kerns R. J. 2006, Bioorg. Med Chem, 14, 2300-2313).

WO 01/55221 discloses glycosaminoglycans with a 2-O-desulfation degree of no more than 60% of the total uronic acid units. These glycosaminoglycans have no anticoagulant activity and exhibit antiangiogenic activity based on FGF inhibition. Their inhibitory activity against heparanase was unexpected.

US 2008/0051567 discloses compounds equivalent to 100% N-acetylated and 25% diol-cleaved heparin that produce little or no anticoagulant activity and low release of growth factors from the extracellular matrix, while inhibiting heparanase, tumor growth, angiogenesis, and inflammation in experimental animal models, including Sanderson's myeloma model.

However, there remains a need to provide improved compounds with higher heparanase inhibitory activity, higher selectivity, improved bioavailability and efficacy for the treatment of heparanase-associated pathologies such as myeloma and other tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Generalized structures generated by periodate oxidation and borohydride reduction of glycosaminoglycans.

(1) A disaccharide unit of a glycosaminoglycan polymer comprising a uronic acid (iduronic acid and/or glucuronic acid) and a glucosamine (2-N-acetylated, 2-N-unsubstituted and/or 2-N-sulfated), wherein the hydroxyl group (R ₄ ) may be substituted or unsubstituted with a sulfate group. After N-desulfation, the glycosaminoglycan polymer may comprise a disaccharide unit comprising 2-N-acetylated glucosamine (2) and 2-NH ₂ glucosamine (native and/or N-desulfated) (3). Periodate oxidation and borohydride reduction result in the conversion of adjacent diols (5, 6, 8) of the 2-O-unsulfated uronic acid residues and adjacent OH/NH 2 groups (7, 8) of the 2-N- and 3-O-unsulfated glucosamine residues to the corresponding aldehydes (by oxidation) and then to the corresponding alcohols (by reduction). Note that the disaccharide unit containing two residues, N-desulfated glucosamine and 2-O-nonsulfated uronic acid, is converted to a dialdehyde and then to a diol cleavage residue (8).

Description of the Invention

The present invention relates to novel chemically modified glycosaminoglycans, particularly heparin and LMWH, which strongly inhibit heparanase and its heparan sulfate degrading activity.

The novel compounds of the present invention having heparanase inhibitory activity are derivatives of N-desulfated and optionally 2-O-desulfated glycosaminoglycans, wherein at least a portion of the adjacent diols and OH/NH ₂ are converted to the corresponding aldehyde, which is then reduced to the corresponding alcohol. The conversion to the aldehyde is preferably carried out using periodate under conditions suitable for breaking the bond of the adjacent diol of the uronic acid residue and the C ₂ -C ₃ bond of the glucosamine (having an amine and a hydroxyl substituent, respectively). The starting compound may also contain a native 2-O-non-sulfated uronic acid residue. In particular, N-desulfation occurs on the N-sulfated glucosamine residue, while O-desulfation occurs on the 2-O-sulfated uronic acid residue.

Preferably, the glycosaminoglycan derivatives of the present invention are derived from natural or synthetic glycosaminoglycans, the latter being chemically or enzymatically prepared (Naggi A. et al., 2001, "Toward a biotechnological heparin throughcombined chemical and enzymatic modification of the Escherichia coli K5polysaccharide." Seminars in thrombosis and hemostasis, 27, 5437), such as unfractionated heparin, low molecular weight heparin (LMWH), heparan sulfate or a portion thereof; more preferably, the glycosaminoglycan derivatives are obtained from natural or synthetic heparin or LMWH.

The specific N-desulfation of N-sulfated glucosamine residues essentially facilitates the conversion of these residues into the corresponding aldehydes (and subsequently into the corresponding alcohols), provided that these residues are also 3-O-unsulfated. In Figure 1, all disaccharide units that can be present in a glycosaminoglycan chain and their changes after N-desulfation, oxidation and reduction reactions are shown in a schematic diagram.

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

Each non-sulfated residue of the glycosaminoglycan (non-sulfated residues at carbon 2 and carbon 3) is susceptible to conversion to an aldehyde. Thus, the extensive N-desulfation included in the method of the present invention provides additional residues susceptible to the conversion above the percentage of susceptible units in the natural glycosaminoglycan, wherein the percentage of units in which adjacent OH/NH ₂ is converted to the corresponding aldehyde (and then to the corresponding alcohol) is increased, but the natural content of 2-O-sulfated iduronic acid residues is retained. Optionally, chemically induced 2-O-desulfation of glycosaminoglycans can adjust the ratio of glucosamine and uronic acid susceptible to the oxidation and reduction reactions of the present invention.

The present invention also relates to the N- and optionally 2-O- desulfation of the glycosaminoglycan derivatives, wherein the adjacent diols and OH/NH ₂ are converted into the corresponding aldehydes (with an open ring), which are then reduced to the corresponding alcohols. The present invention also relates to methods for preparing the glycosaminoglycan derivatives and their use as active ingredients in drugs for treating pathological conditions such as multiple myeloma and other cancers (including their metastatic forms). In addition, the present invention relates to the use of the glycosaminoglycan derivatives in any therapeutic indication that benefits from the inhibition of heparanase. The present invention also relates to pharmaceutical compositions comprising the N- desulfated and optionally 2-O- desulfated glycosaminoglycan derivatives, wherein the adjacent diols and OH/NH ₂ are converted into the corresponding aldehydes (with an open ring), which are then reduced to the corresponding alcohols.

The N- and optionally 2-O-desulfated glycosaminoglycan derivatives of the present invention may be obtained by a process comprising:

a) N-desulfation of 25-100%, preferably 30%-90%, more preferably 45%-80% of the N-sulfated glucosamine residues of the glycosaminoglycans; optionally the process further comprises 2-O-desulfation of up to 50%, preferably up to 25%, of the 2-O-sulfated residues of the glycosaminoglycans; the product obtained preferably contains 0%-50% N-acetylated glucosamine residues, 50%-100% N-non-sulfated glucosamine residues;

b) Conversion of the adjacent _OH /NH2 of 2N-,3-O-non-sulfated glucosamine residues and adjacent diols of 2-O-non-sulfated uronic acid residues (chemically desulfated as well as naturally occurring non-sulfated residues on the original chain) to the corresponding aldehydes, preferably by periodate oxidation; the 2N-,3-O-non-sulfated glucosamine residues may be present in 25%-100%.

c) Reduction of the aldehyde to the corresponding alcohol, preferably by sodium borohydride.

Optionally, the method further comprises partial or complete deacetylation of the N-acetylated residues of the glycosaminoglycan.

In a preferred embodiment, the glycosaminoglycan derivatives of the present invention are derived from natural or synthetic (chemically or enzymatically prepared) glycosaminoglycans, preferably from unfractionated heparin, LMWH, heparan sulfate or derivatives thereof. More preferably, the glycosaminoglycan derivatives of the present invention are derived from heparin or LMWH.

In a preferred embodiment, the oxidation (preferably periodate oxidation) is performed under conditions that cleave the vicinal diol of the uronic acid and the bond between _C2 and _C3 of glucosamine (having amino and hydroxy substituents, respectively).

In a preferred embodiment, a modified glycosaminoglycan sample having varying degrees of N-desulfation, preferably a modified heparin or LMWH, is subjected to periodate oxidation and sodium borohydride reduction in an aqueous medium, preferably by known modification methods. The periodate oxidation can be carried out in the presence of NTA (nitrilotriacetic acid), chelating agents and sequestrants to reduce depolymerization, in the presence of NaHCO ₃ or pyridine to alkalize the solution, or in the presence of MnCl ₂ with or without NTA. Preferably, the periodate oxidation is carried out in the presence of NTA. Preferably, the oxidation is carried out at a pH of 5.5-10.0, more preferably 6.0-9.0.

The present invention also relates to a method for cleaving _C2 - _C3 bonds of glucosamine residues of glycosaminoglycans, comprising oxidation of said glycosaminoglycans, preferably by periodate, at a pH comprised between 5.5 and 10, more preferably between 6.0 and 9.0.

Preferably, the glucosamine residues in which adjacent OH/ _NH2 have been converted to the corresponding aldehydes which are then reduced to the corresponding alcohols are 25%-100%, more preferably 50%-100%, most preferably 60%-90% of the glucosamine residues of the glycosaminoglycan.

The molecular weight of the glycosaminoglycan-derived compound obtainable by the above process is 800-30.000 Da, depending on the process conditions and the starting glycosaminoglycan. When unfractionated heparin is used as starting material, the molecular weight of the glycosaminoglycan-derived compound obtainable by the above process is preferably 3000-20000 Da, preferably 4000-12000 Da.

Unexpectedly, the novel glycosaminoglycan derivatives of the present invention have been shown to be potent heparanase inhibitors in vitro and to inhibit myeloma in animal models.

Relative to native glycosaminoglycans and their RO derivatives, products with a greater number of units in which adjacent diols and OH/NH ₂ have been converted to the corresponding aldehydes, which are then reduced to the corresponding alcohols, are also less sulfated. Therefore, they are expected to exhibit less protein interaction and more favorable pharmacokinetics than their analogs with lower levels of modified residues.

The present invention also relates to compounds obtainable by the process described above for use as medicaments.

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

Despite their low degree of sulfation, heparin and low molecular weight heparin derivatives prepared according to the invention show effective inhibition of heparanase activity in experimental models of multiple myeloma in vitro and in vivo.

Furthermore, the derivatives of the present invention, even at low molecular weight (see Examples 5, 6 and 8), exhibit higher heparanase inhibitory activity than RO heparin of similar molecular weight obtained from 2-O-desulfated heparin, as shown in Table 1.

Table 1

The data show a general trend of decreasing heparanase inhibitory activity with decreasing molecular weight of the RO-diol cleaved heparin.

Example

Compound preparation

The N-desulfation of unfractionated heparin (hereinafter referred to as UFH) disclosed in the following examples was carried out by a known modification method (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 ¹³ C-NMR (Naggi A. et al. 2001, "Generation of anti-factor Xaactive, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins." Carbohydr. Res. 336, 4, 283-290).

Samples of modified heparin with varying degrees of N-desulfation were subjected to periodate oxidation to yield a cleavage unit with two aldehyde groups, and then reduced with sodium borohydride ( _NaBH4 ) in aqueous medium to yield the final heparin derivatives; both reactions were performed using known modification methods. Periodate oxidation was performed in the presence of _NaHCO3 , pyridine, _MnCl2 , or _MnCl2 (containing NTA). The hierarchical 2-O-desulfation of UFH was carried out by following known modification methods (Jaseja M. et al. 1989 "Novel regio- and stereo-selective modifications of heparin in alkaline solution. Nuclear magnetic resonance spectroscopic evidence." Canada J Chem, 67, 1449-1455; RN Rej Arthur S. Perlin 1990 "Base-catalyzed conversion of the α-L-iduronic acid 2-sulfate unit of heparin into a unit of α-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). Hereinafter, "RO" indicates the percentage of diol cleavage residues in which the adjacent diol and OH/ _NH2 are converted to the corresponding aldehyde and then to the corresponding alcohol, relative to the total glycosaminoglycan residues.

In vitro testing

Based on previous research by Bisio et al. (Bisio A. et al. 2007 "High-performance liquid chromatographic/mass spectrometric studies on the susceptibility of heparinspecie to cleavage by heparanase." Sem Thromb hemost 33 488-495), the research group of Professor Vlodavsky at the University of Haifa in Israel determined heparanase inhibitory activity in vitro 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). In short, heparanase can cleave the synthetic pentasaccharide fondaparinux (Fondaparinux), an antithrombotic drug, which structurally corresponds to the antithrombin binding site of heparin. After hydrolysis by heparanase, trisaccharides and reducing disaccharides are obtained. The latter can be easily quantified to evaluate heparanase activity. In this embodiment, the assay solution comprises (100 μl) 40 mM sodium acetate buffer pH 5.0 and 100 mM fondaparinux (GlaxoSmithKline), with or without the inhibitor sample. At the start of the assay, heparanase is added to a final concentration of 140 pM. The plate is sealed with adhesive tape and incubated at 37°C for 2-24 hours. The assay is stopped by adding 100 μl of a solution of 1.69 mM 4-[3-(4-iodophenyl)-1H-5 tetrazolyl]-1,3-benzenedisulfonate (WST-1, Aspep, Melbourne, Australia) in 0.1 M NaOH. The plate is resealed with adhesive tape and developed at 60°C for 60 minutes. Absorbance is measured at 584 nm (Fluostar, BMG, Labtech). In each plate, a standard curve constructed with D-galactose as a reducing sugar standard was prepared at a concentration range of 2-100 uM in the same buffer and volume. IC ₅₀ values were determined. The results obtained using the colorimetric assay cited above were verified using a different test (which uses extracellular matrix (ECM) labeled with radioactive sulfate as a substrate). In short, the ECM substrate is deposited by cultured corneal endothelial cells and is therefore very similar to the subendothelial basement membrane in composition, biological function, and barrier properties. Detailed information on the preparation of sulfate-labeled ECM and its use in heparanase assays can be found in: Vlodavsky, I., Current Protocols in Cell Biology, Chapter 10: Unit 10.4, 2001.

In vivo testing

In vivo anti-myeloma activity was tested essentially according to the procedures 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, 5-6 week old CB17 scid/scid mice were obtained from Arlan (Indianopolis, IN) or Charles River Laboratories (USA). The 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. 1x10 ⁶ heparanase-expressing CAG myeloma cells (high or low expression) were subcutaneously injected into the left flank of each mouse. Ten days after tumor cell injection, an Alzet osmotic pump (Durect Corporation, Cupertino, CA) was implanted in the right flank of the mouse. The pump contained a solution of the test compound (neohepariin derivative) or phosphate buffered saline (PBS) as a control. The solution was delivered continuously for 14 days. After 14 days, the animals were sacrificed, and the wet weight of the subcutaneous tumors and the mean serum kappa level were determined and compared among the experimental groups by the log-rank test (p<0.05 was considered statistically significant).

Weekly luciferase bioluminescence imaging provides quantitative data on primary tumors and tracks metastases in bone and soft tissue. Notably, the SCID-hu model is unique in that human tumor cells are injected directly into small segments of human fetal bone that are subcutaneously implanted in SCID mice, thereby closely recapitulating human myeloma.

General operation of NMR analysis

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

N-desulfation of unfractionated heparin

Example 1 (G8220)

UFH (4.01 g, batch number G3378) was dissolved in water (32 ml) and treated with Amberlite IR 120 (H+, 144 ml) with stirring. The filtered acid solution was adjusted to pH 7 with pyridine and then concentrated to dryness under reduced pressure. The resulting pyridinium salt was dissolved in 40 ml of a mixture of DMSO: _H₂O (95:5 by volume) and stirred at 25°C for 48 hours. After dilution with 40 ml of water, the solution was dialyzed against distilled water at 4°C for 16 hours using a 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, degree of N-desulfation determined by ¹³ C-NMR = 74.7% of total glucosamine residues, 2-O sulfated uronic acid determined by ¹³ C-NMR = 18% of total monosaccharides.

Example 2 (G8343)

A sample of UFH (0.25 g, batch number G3378) was converted to the corresponding pyridinium salt and N-desulfated in a mixture of DMSO:MeOH (95:5 by volume) according to the procedure described in Example 1. After stirring for 2 hours at 25° C., the reaction mixture was processed according to the same final procedure described in Example 1 to provide G8343 (0.172 g) in a yield of 69% w/w, MW of 18.000 Da, a degree of N-desulfation of 63.3% of total glucosamine residues as determined by ¹³ C-NMR, and 2-O-sulfated uronic acids of 19% of total monosaccharides as determined by ¹³ C-NMR.

Example 3 (G8516)

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

Example 4 (G8147)

UFH (2.5 g, batch number G3378) was dissolved in water (20 ml) and treated with Amberlite IR 120 (H+, 90 ml) with stirring. The filtered acid solution was adjusted to pH 7 with pyridine and 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) and stirred at 25°C for 18 hours. After dilution with 25 ml of water, the solution was dialyzed against distilled water at 4°C for 16 hours using a membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization yielded G8147 (1.9 g), yield = 76% w/w, MW = 18.200 Da, and degree of N-desulfation = 60% total glucosamine residues as determined by ¹³ C-NMR.

Example 5 (G9416)

UFH (5 g, batch number G3378) was dissolved in water (40 ml) and treated with Amberlite IR 120 (H+, 90 ml) with stirring. The filtered acid solution was adjusted to pH 7 with pyridine and 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) and stirred at 25°C for 18 hours. After dilution with 100 ml of water, the solution was dialyzed against distilled water at 4°C for 16 hours using a membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization yielded G9416 (3.65 g), yield = 73% w/w, MW = 18.800 Da, and degree of N-desulfation = 77% total glucosamine residues as determined by ¹³ C-NMR.

Example 6 (G8079)

UFH (1 g, batch number G3378) was dissolved in water (8 ml) and treated with Amberlite IR120 (H+, 90 ml) with stirring for 30 minutes. The filtered acid solution was adjusted to pH 7 with pyridine and 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) and stirred at 25°C for 16 hours. After dilution with 10 ml of water, the solution was dialyzed against distilled water at 4°C for 3 days using a membrane (cut-off: 3,500 Da). Concentration under reduced pressure and lyophilization yielded G8079 (1 g) with a yield of 100% w/w and a degree of N-desulfation of 60% total glucosamine residues as determined by ¹³ C-NMR.

Periodate oxidation and sodium borohydride reduction of N-desulfated heparin

Example 7 (G8340)

A sample of G8220 from Example 1 (0.25 g, 74.7% 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 _NaIO4 . The pH was adjusted to 6.8 with ₂ M NaHCO3 (approximately 2.1 ml), and 0.08 M nitrilotriacetic acid (NTA, 10 ml) was added to the solution while stirring in the dark at 4°C. The pH was adjusted from 4.0 to 6.6 by adding ₂ M NaHCO3, and the reaction mixture was stirred at 4°C for 8 hours. Excess periodate was quenched by adding ethylene glycol (0.73 ml); after 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da). The desalted solution was treated with _NaBH4 (0.164 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1N HCl to quench excess _NaBH4 . After stirring for 10 minutes, the solution was neutralized with 0.1N NaOH. After dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da), the solution was concentrated under reduced pressure and lyophilized to yield 0.202 g of G8340 (yield = 90% w/w, MW = 8,400 Da). The percentages of RO (53%), IdoA2S (35%), GlcNAc (9%), and _GlcNH2 (12%) in the total monosaccharide residues were determined by ^13C -NMR.

In vitro heparanase inhibition: IC ₅₀ = 20 ng/ml.

In vivo anti-myeloma activity: 60 mg/Kg/day for 14 days: 75% tumor inhibition and 60% serum K inhibition.

Example 8 (G8438)

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

In vitro heparanase inhibition IC ₅₀ : 60 ng/ml.

Example 9 (G8588)

Starting from G8516 of Example 3 (0.171 g of 44% N-desulfated heparin residues) and following the procedure described in Example 7, G8588 (0.136 g) was obtained with a yield of 80%, MW = 11.000 Da, and the percentages of GlcNAc (30%), GlcNH ₂ (13%), IdoA2S (34%), and RO (37%) were determined by ¹³ C-NMR.

Example 10 (G9578)

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

In vitro heparanase inhibition: IC ₅₀ =75 ng/ml;

In vivo anti-myeloma activity (60 mg/kg per day for 14 days): 63% tumor inhibition.

Example 11 (G8188)

A sample of N-desulfated heparin G8147 (0.25 g, 60% N-desulfated heparin residues) from Example 4, dissolved in water (7.3 ml) and cooled to 4°C, was added to an equal volume of 0.2 M _NaIO4 . The pH was adjusted to 6.8 with 2 M _NaHCO3 (approximately 2.1 ml) and stirred in the dark at 4°C for 16 hours. Excess periodate was quenched by adding ethylene glycol (0.73 ml), and after 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da). The desalted solution was treated with _NaBH4 (0.164 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1 N HCl to quench excess _NaBH4 . After stirring for 10 minutes, the solution was neutralized with 0.1 N NaOH. After dialysis against distilled water at 4°C for 16 hours in a membrane (cut-off: 3,500 Da), it was concentrated under reduced pressure and lyophilized to give 0.168 g of G8188, yield = 67% w/w, MW = 3,460 Da. The percentages of RO (43%), IdoA2S (40%), GlcNAc (6%) and _GlcNH2 (4%) were determined by ¹³ C-NMR.

Example 12 (G8189)

A G8147 sample (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 0.2 _{M NaIO4} . The pH was adjusted to 6.8 with pyridine (5% v/v, approximately 730 μl) and stirred at 4°C in the dark for 16 hours. Excess periodate was quenched by the addition of ethylene glycol (0.73 ml). After 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da). The desalted solution was treated with _NaBH4 (0.164 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1 N HCl to quench excess _NaBH4 . After stirring for 10 minutes, the solution was neutralized with 0.1 N NaOH. After dialysis against distilled water at 4°C for 16 hours in a membrane (cut-off: 3,500 Da), it was concentrated under reduced pressure and lyophilized to give 0.181 g of G8189, yield = 72% w/w, MW = 5,150 Da. The percentages of RO (49%), IdoA2S (39%), GlcNAc (7%) and _GlcNH2 (7%) were determined by ¹³ C-NMR.

Example 13 (G8217)

A G8147 sample (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 0.2 M _NaIO4 . The pH was adjusted to 6.8 with 2 M NaHCO3 ₍ approximately 2.1 ml), and 30 ml of 0.05 _M MnCl2 (final concentration in solution 0.00001 M) was added over 16 hours while stirring in the dark at 4°C. Excess periodate was quenched by adding ethylene glycol (0.73 ml). Overnight, the sample precipitated due to a pH change of 8, and its pH was then adjusted to 6 with 1 N HCl. After 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours. The desalted solution was treated with _NaBH4 (0.164 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1N HCl to quench excess _NaBH4 . After stirring for 10 minutes, the solution was neutralized with 0.1N NaOH. After 16 hours of dialysis against distilled water at 4°C using a membrane (cutoff: 3,500 Da), the solution was concentrated under reduced pressure and lyophilized to yield 0.230 g of G8217 (yield = 92% w/w, MW = 7,455 Da). The percentages of RO (31%), IdoA2S (29%), GlcNAc (8%), and _GlcNH2 (6%) in the total glycosaminoglycan residues were determined by ^13C -NMR.

Example 14 (G8219)

A G8147 sample (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 0.2 M _NaIO4 . The pH was adjusted to 6.8 with 2 M _NaHCO3 (approximately 1.2 ml), and 0.08 M nitrilotriacetic acid (NTA, 10 ml) and 31 ml of 0.05 _M MnCl2 were added to the solution while stirring in the dark at 4°C. The pH was adjusted from 4.0 to 6.3 by adding 2 M _NaHCO3 , and the reaction mixture was stirred at 4°C for 8 hours. Excess periodate was quenched by adding ethylene glycol (0.73 ml); after 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da). The dialyzed solution was treated with _NaBH4 (0.164 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1N HCl to quench excess _NaBH4 . After stirring for 10 minutes, the solution was neutralized with 0.1N NaOH. After 16 hours of dialysis against distilled water at 4°C using a membrane (cutoff: 3,500 Da), the solution was concentrated under reduced pressure and lyophilized to yield 0.202 g of G8219 (yield = 90% w/w, MW = 7,330 Da). The percentages of RO (54%), IdoA2S (36%), GlcNAc (9%), and _GlcNH2 (8%) were determined by ^13C -NMR.

Comparative Example 15 (G8092)

To a G8079 sample (1 g, N-desulfated heparin) dissolved in water (29.2 ml) and cooled to 4°C, an equal volume of NaIO ₄ (pH ₅ ) was added, and the reaction mixture was stirred at 4°C for 8 hours. Excess periodate was quenched by adding ethylene glycol (2.9 ml); after 1 hour, the reaction mixture was desalted by dialysis against distilled water at 4°C for 16 hours using a membrane (cutoff: 3,500 Da). The dialyzed solution was treated with NaBH ₄ (0.657 g, 3.4 mmole) and stirred at 25°C for 3 hours. The pH was then adjusted to 4 with 1N HCl to quench excess NaBH ₄ . After stirring for 10 minutes, the solution was neutralized with 0.1N NaOH. After dialysis against distilled water at 4°C for 72 hours using a membrane (cutoff: 3,500 Da), the product was concentrated under reduced pressure and lyophilized to obtain 0.643 g of G8092 (yield = 64% w/w, MW = 6,064 Da). ¹³ C-NMR analysis showed a peak of N-desulfated glucosamine at 95 ppm. Oxidation did not occur at acidic pH.

Claims (23)

1. A method for preparing a glycosaminoglycan derivative that inhibits heparinase, comprising: a) N-desulfatation of 25%-100% of the N-sulfated residues of glycosaminoglycans; b) Oxidation of 25%-100% of the 2-N-,3-O-nonsulfated glucosamine residues and 2-O-nonsulfated uronic acid residues of the glycosaminoglycan under conditions that effectively convert adjacent diols and adjacent OH/ _NH2 into aldehydes; c) The reduction of the oxidized glycosaminoglycan under conditions that effectively convert the aldehyde into an alcohol. 2. The method of claim 1, wherein the oxidation is carried out at a pH of 5.5-10.0 using periodate. 3. The method of claim 1 or 2, wherein the reduction is carried out using sodium borohydride. 4. The method of claim 1 or 2, further comprising: d) 2-O-desulfatation of up to 50% of the 2-O-sulfatated residues of the glycosaminoglycan before or after N-desulfatation. 5. The method of claim 4, comprising 2-O-desulfation of up to 25% of the 2-O-sulfated residues of the glycosaminoglycan. 6. The method of claim 1 or 2, further comprising: e) Before or after N-desulfatation, the N-acetylated residues of the glycosaminoglycan are partially or completely deacetylated. 7. The method of claim 1 or 2, wherein the glycosaminoglycan is a natural or synthetic glycosaminoglycan. 8. The method of claim 7, wherein the natural or synthetic glycosaminoglycan is heparin. 9. The method of claim 7, wherein the natural or synthetic glycosaminoglycan is low molecular weight heparin. 10. The method of claim 7, wherein the natural or synthetic glycosaminoglycan is heparin sulfate. 11. The method of claim 7, wherein the natural or synthetic glycosaminoglycan is unfractionated heparin or heparin with a molecular weight of 3,500-8,000 Da. 12. A method for cleaving the _C2 - _C3 bonds of non-sulfated glucosamine residues in glycosaminoglycans, comprising: Oxidation of the glycosaminoglycan. 13. The method of claim 12, wherein the oxidation of the glycosaminoglycan is carried out at a pH of 6.0-10.0 by periodate. 14. The method of claim 13, wherein the oxidation of the glycosaminoglycan is carried out at a pH of 6.0-9.0 by periodate. 15. A glycosaminoglycan derivative compound obtained by the method of any one of claims 1-14. 16. The derivative compound of claim 15, having a molecular weight of 3,000-20,000 Da. 17. The derivative compound of claim 16, having a molecular weight of 3,500-12,000 Da. 18. An oligosaccharide compound obtained by enzymatic or chemical partial depolymerization of a glycosaminoglycan derivative compound of any one of claims 15-17. 19. Use of the compound of any one of claims 15-18 in the preparation of a medicament having heparinase inhibitory activity. 20. Use of the compound of any one of claims 15-18 in the preparation of an antitumor drug. 21. Use of the compound of any one of claims 15-18 in the preparation of an anti-metastasis medicament. 22. The use of claim 20, wherein the antitumor drug is an antimyeloma drug. 23. The use of claim 21, wherein the anti-metastatic drug is used to treat metastatic forms of multiple myeloma.