Heparins and fucoidans : future therapeutic options for ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammation of colonic mucosa and submucosa, characterised by abdominal pain, rectal bleeding and diarrhoea. Although the aetiology of UC remains elusive, the mucosal immune response, microbial features and intestinal epithelial barrier defects are all implicated in the pathogenesis of UC. Current drug therapies are not completely effective for the management of UC and many patients eventually require surgical interventions. Therefore there is a need for effective and safe therapies in UC. Both heparins (unfractionated heparin, UFH and low molecular weight heparins, LMWHs) and heparin-like molecules such as fucoidans are complex mixture of polysaccharides. Heparins are widely used anticoagulants whereas fucoidan extracts are available as health supplements. Heparins and fucoidans have been reported to possess a wide range of biological activities including anti-inflammatory activity. At present, the use of heparins for the treatment of UC remains uncertain and requires further investigation. Moreover, the use of heparins is largely limited by the risk of bleeding in conditions where anticoagulation is not required. Therefore we aimed to investigate the therapeutic potential of sulphated polysaccharides (enoxaparin and fucoidans) in a model of colitis with a goal of identifying specific oligosaccharides that are responsible for their anti-inflammatory activity. We first examined whether UFH, LMWHs (dalteparin and enoxaparin) and individual oligosaccharides of enoxaparin can affect cell growth in vitro. In UC, cell proliferation is required for mucosal healing. Enoxaparin was separated into different oligosaccharides by ion-exchange chromatography (IC). The proliferation and viability of human colonic epithelial cancer cells (HT-29, DLD-1 and HCT-116) were investigated in the presence and absence of different concentrations of UFH, dalteparin, enoxaparin and its oligosaccharides. Protein quantification, the numbers of cell colonies and proportion of cells in different phases of the cell cycle were measured. Cell viability was assessed by a colorimetric method, trypan blue exclusion and by measuring apoptosis. Subsequently, investigation of the potential of fucoidan extracts, enoxaparin and its oligosaccharides in colitis was evaluated in vivo. Colitis was induced in male C57BL/6 mice by the administration of dextran sulphate sodium (DSS) through the drinking water. Mice received different treatment once daily either intraperitoneally or orally. Animals were monitored daily for their body weight, stool consistency and rectal bleeding. The experiment was terminated on day 8 and the colons of mice were weighed and processed for histology and to measure cytokine levels. UFH, dalteparin and enoxaparin inhibited cell proliferation in a dose-dependent manner. This inhibitory effect was well-correlated with the induction of cell cycle arrest in the G1 phase and was not associated with any significant changes in cell viability. The isolated oligosaccharides of intact enoxaparin have different degrees of polymerisation ranging from 2 saccharides (dp2; ~ 600 Da) to 24 saccharides (dp24; ~ 8000 Da). Each IC-derived oligosaccharide of enoxaparin had either no, anti- or pro- proliferative effects, depending on its composition. Disaccharides devoid of anticoagulant activity had the strongest anti-proliferative effect, whereas the hexasaccharides promoted cell growth. In vivo results showed that orally administered but not intraperitoneal injected enoxaparin was effective in suppressing the pathology of colitis. The observed effect of oral enoxaparin corresponded well to the reduction of macrophage-associated responses as well as the suppression of inflammatory cytokine levels. Like enoxaparin, fucoidan extracts also ameliorated colitis by reducing the levels of a number of pro-inflammatory cytokines. Intriguingly, among oligosaccharides of enoxaparin tested, oligosaccharides shorter than dp8 were identified to be responsible for the anti-inflammatory activity of enoxaparin. Tetrasaccharides and hexasacchrides were the two main active components that relieved colitis-associated body weight loss and prevented macroscopic changes to the colon tissue. In conclusion, enoxaparin and fucoidan extracts possess anti-inflammatory activity and are effective in an acute colitis model. The in vitro and in vivo results highlighted that depending on their composition, different oligosaccharides can have distinctive effects. It is important to isolate and test different oligosaccharides from the mixture. This approach potentially leads to the identification of specific oligosaccharides that are responsible for anti-inflammatory effects of the parent molecules and could reduce the unwanted side effect associated with the administration of a mixture of polysaccharides. For example, the tetrasaccharide of enoxaparin, shorter than antithrombin-binding pentasaccharide, does not bind to antithrombin, thus its use is unlikely to be associated with a risk of bleeding even when administered at high doses. The identified non-anticoagulant enoxaparin oligosaccharides that have colitis-suppressing properties could therefore represent a novel therapeutic option for the management of UC.