Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties

Structural features of heparin potentially important forheparanase-inhibitory activity were examined by measur-ing the ability of heparin derivatives to affect the degrada-tion of pH]acetylated heparan sulphate by tumor cell he-paranases. ICJO values were determined using an assaywhich distinguished degraded from undegraded substrateby precipitation of the latter with cetylpyridinium chloride(CPC). Removal of heparin's 2-O-sulphate and 3-O-sul-phate groups enhanced heparanase-inhibitory activity(50%). Removal of its carboxyl groups slightly loweredthe activity (18%), while combining the treatments abol-ished the activity. At least one negative charge on theiduronic acid/idose moiety, therefore, is necessary for he-paranase-inhibitory activity. Replacing heparin's N-sul-phate groups with N-acetyl groups reduced its activity(37%). Comparing this heparin derivative with 2,3-O-de-sulphated heparin, the placement of sulphate groups ap-pears important for activity since the two structures havesimilar nominal linear charge density. In addition, unsub-stituted uronic acids are nonessenrial for inhibition sincetheir modification (periodate-oxidation/borohydride-re-duction) enhanced rather than reduced heparanase-inhibi-tory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohy-dride-reduced] heparin) were tested in the chick chorioal-lantoic membrane (CAM) bioassay for anti-angiogenic ac-tivity and found to be at least as efficacious as heparin.2,3-O-desulphated heparin also significantly decreased thetumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged thesurvival times of C57BL/6N mice in a B16-F10 melanomaexperimental lung metastasis assay.