Method of screening for potential anti-metastatic and anti-inflammatory agents using mammalian heparanase as a probe

#The present invention relates to novel qualitative and quantitative glycosidases catalytic activity assays. More particularly, the present invention relates to a method of screening for potential anti-metastatic and anti-inflammatory agents and, most particularly, to a method of screening for potential anti-metastatic and anti-inflammatory agents using mammalian heparanase, preferably purified human recombinant heparanase, as a probe.

#heparan sulfate proteoglycans (HSPGs): HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-5). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (1-5). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair (1-5). The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface (4-6). HSPGs are also prominent components of blood vessels (3). In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (7-9). HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes.

#Involvement of heparanase in tumor cell invasion and metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to escape into the extravascular tissue(s) where they establish metastasis (10). Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase) are thought to be involved in degradation of the BM (10). Among these enzymes is an endo-β-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (7, 9, 11-12). Expression of a HS degrading heparanase was found to correlate with the metastatic potential at mouse lymphoma (11), fibrosarcoma and melanoma (9) cells. The same is true for human breast, bladder and prostate carcinoma cells (see U.S. pat application Ser. No. 09/109,386, which is incorporated by reference as if fully set forth herein). Moreover, elevated levels of heparanase were detected in sera (9) and urine (U.S. patent application Ser. No. 09/109,386,) of metastatic tumor bearing animals and cancer patients and in tumor biopsies (12). Treatment of experimental animals with heparanase alternative substrates and inhibitor (e.g., non-anticoagulant species of low MW heparin, laminarin sulfate) markedly reduced (>90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (8, 9, 13), indicating that heparanase inhibitors may be applied to inhibit tumor cell invasion and metastasis.

#The invention herein described, by way of example only, with reference to the accompanying drawings, wherein:

#FIG. 1 demonstrates polyacrylamide gel analysis of heparanase catalytic activity. Heparin and heparan sulfate were incubated (24 hours, 37° C., pH 5.4) with human recombinant heparanase enzyme. Following incubation, degradation products were fractionated on an acrylamide gel and were stained with methylene blue. Lane A—heparin control; lane B—heparin incubated with the recombinant heparanase; lane C—heparan sulfate control; lane D—heparan sulfate incubated with the recombinant heparanase.

#FIGS. 2a-b demonstrate apparent inhibition of heparanase catalytic activity by heparin. Recombinant heparanase was incubated (24 hours, 37° C., pH 6.6) with sulfate labeled ECM (FIG. 2a) or sulfate labeled ECM-derived soluble HSPG (peak I material, FIG. 2b, ⋄), in the absence (peak II, •) and presence (peak I, □, Δ) of 5 μg/ml heparin. Sulfate labeled degradation products were analyzed by gel filtration over Sepharose 6B.

#Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

#Materials: Sodium heparin from porcine intestinal mucosa (PM-heparin) (Mr. 14,000, Anti-FXa 165 IU/mg, sulfur content 12%) was obtained from Hepar Industries (Franklin, Ohio). A low Mr. fragment (Kabi 2165, Fragmin) of this heparin (Mr. 5,100, Anti-FXa 130 IU/mg, sulfur content 12.4%) was prepared as sodium salt by nitrous acid depolymerization (18). Heparan sulfate from bovine kidney (Cat. No. H 7640), PEG 300 (Cat. No. P 3140), tetrazolium blue chloride (Cat. No. T 4375) and carbazole (Cat. No. C 5132) were purchased from Sigma. Heparin was purchased from Welding. Heparin-sepharose was purchased from Pharmacia. 1,9-Dimethylmethylene blue was purchased from Aldrich (Cat. No. 34108).

#Preparation and characterization of oligosaccharides from nitrous acid depolymerized heparins: heparin fragments consisting of saccharide chains of different sizes were obtained by partial deaminative cleavage of heparin from porcine intestinal mucosa using nitrous acid, as described (18). Sizes ranged from disaccharides to chains with an average length of the starting heparin, i.e., chains of about 40-50 saccharides. Size homogeneous oligosaccharides, similar to the starting heparin in their sulfur/carbon ratio, were prepared also by alkaline treatment of heparin methyl ester (β-elimination) as described elsewhere (19). Similar results were obtained. In both cases, the mixture of heparin fragments was subjected to ion exchange chromatography on DEAE Sepharose using sodium chloride as an eluent. Three fractions were collected: low sulfate fraction, sulfur/carbon ratio (by elemental analysis) 0.14; medium sulfate fraction, sulfur/carbon ratio 0.19; and high sulfate fraction, sulfur/carbon ratio 0.21 (19, 20). Each of the low sulfate, medium sulfate, and high sulfate fractions was further separated into size-homogenous, even-numbered oligosaccharides by gel permeation chromatography on Sephadex G-50 superfine (19, 20). This procedure resulted in a set of oligosaccharides of different sizes and degrees of sulfation. The oligosaccharides having a higher sulfur/carbon ratio than heparin were found by 1H-NMR and 13C-NMR spectroscopy to have their additional sulfate groups mainly in the glucosamines as indicated by a larger proportion of N-sulfate groups over N-acetyl groups and of 6-O-sulfate groups over 6-OH groups. The low sulfate-containing oligosaccharides had a considerably reduced sulfate content in their glucosamines as well as in their iduronic acids (19).