Heparin has been used clinically as an anticoagulant for over 60 years. Typically isolated from porcine intestine, heparin is a mixture of dimeric glycosidic sequences generating complex polysaccharide glycosaminoglycan chains. Recently, certain lots of heparin have been associated with an acute, rapid onset of significant side effects indicative of an allergic-type reaction. To identify potential causes for this serious rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high resolution analytical techniques. Through comparison of these results with those obtained on reference lots, suspect lots were found to contain a highly sulfated chondroitin sulfate contaminant. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-galactosamine. Surprisingly, the disaccharide unit contains an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. The presence of such a contaminant could elicit a biological response as highly sulfated polysaccharides, such as dextran sulfate, are known to be potent mediators of the immune system. Given the nature of the contaminant, traditional screening tests -such as those present as part of the current United States Pharmacopeia heparin monograph -cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be employed to determine whether or not heparin lots contain the contaminants reported here.

Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothelial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (L-iduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis, angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single functional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.

Purpose: Heparanase promotes myeloma growth, dissemination, and angiogenesis through modulation of the tumor microenvironment, thus highlighting the potential of therapeutically targeting this enzyme. SST0001, a nonanticoagulant heparin with antiheparanase activity, was examined for its inhibition of myeloma tumor growth in vivo and for its mechanism of action.Experimental Design: The ability of SST0001 to inhibit growth of myeloma tumors was assessed using multiple animal models and a diverse panel of human and murine myeloma cell lines. To investigate the mechanism of action of SST0001, pharmacodynamic markers of angiogenesis, heparanase activity, and pathways downstream of heparanase were monitored. The potential use of SST0001 as part of a combination therapy was also evaluated in vivo.Results: SST0001 effectively inhibited myeloma growth in vivo, even when confronted with an aggressively growing tumor within human bone. In addition, SST0001 treatment causes changes within tumors consistent with the compound's ability to inhibit heparanase, including downregulation of HGF, VEGF, and MMP-9 expression and suppressed angiogenesis. SST0001 also diminishes heparanase-induced shedding of syndecan-1, a heparan sulfate proteoglycan known to be a potent promoter of myeloma growth. SST0001 inhibited the heparanase-mediated degradation of syndecan-1 heparan sulfate chains, thus confirming the antiheparanase activity of this compound. In combination with dexamethasone, SST0001 blocked tumor growth in vivo presumably through dual targeting of the tumor and its microenvironment.Conclusions: These results provide mechanistic insight into the antitumor action of SST0001 and validate its use as a novel therapeutic tool for treating multiple myeloma.