Abstract. The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin ) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.

Severe malariaEven with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death.Sevuparin in phase I studyThe drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated.Sevuparin in phase I/II clinical studyA phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study.Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria.Trial registrationClinicalTrials.gov NCT01442168

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria.

SummarySevuparin is a novel drug candidate in phase II development as a treatment for vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin-derived polysaccharide, sevuparin has been designed to retain antiadhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS-RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso-occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso-occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P-and L-selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso-occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti-adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti-adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso-occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

Introduction Sevuparin is a new chemically modified heparin with low anticoagulant activity currently being studied as an adjuvant therapy for severe malaria. Heparins generally have multiple biological properties, including antithrombin III-dependent inhibition of thrombin as well as blockade of P-selectin-mediated adhesion. Selectins have been shown to contribute to both sickle red cell (SS RBC) and neutrophil (PMN) adhesion in vitro and in mice with sickle cell disease (SCD). Sickle mice lacking both P- and E-selectins are relatively resistant to tumor necrosis factor-α (TNFα)-induced vaso-occlusion. We therefore theorized that sevuparin would show activity in inhibiting the selectin-dependent adhesion of red cells and leukocytes seen in the context of SCD, with the potential to decrease vaso-occlusion at a low level of anti-coagulation. Methods We studied both RBC and PMN adhesion to human umbilical vein endothelial cells (ECs) in an in vitro flow chamber. For studies of SS RBC adhesion, we exposed confluent cultures of ECs on gelatin-coated glass slides to interleukin (IL)-13 (50 ng/ml) for 48 h, followed by histamine (100 μM) for 10 min at 37°C immediately prior to performance of adhesion assays. Upregulation of P-selectin expression was confirmed by FACS of mechanically dislodged ECs. RBC adhesion was quantified during continuous flow. For assays of neutrophil adhesion, ECs were treated with human TNFα (10 μg/ml) overnight, followed by incubation for 10 min at 37°C with 100 µM histamine. All slides bearing ECs were washed after incubation with histamine and immediately mounted in the flow chamber for adhesion assays. Inhibition by sevuparin was tested by pre-incubating ECs with different concentrations of sevuparin for 20 min at 37°C prior to adhesion assays. PMNs from SCD patients were tested for adhesion to treated ECs with and without preceding exposure of ECs to sevuparin. In addition, PMNs from normal donors were first exposed to SS RBCs, as previously described, and then tested for adhesion to ECs with and without pre-exposure to sevuparin. PMN adhesion was quantitated after 10 min of no flow, followed by wash out at various shear stresses. The ability of sevuparin to prevent vaso-occlusion in vivo was studied by infusing red fluorescent SS RBCs into nude mice to effect in vivo adhesion and vaso-occlusion visible in implanted window chambers (Zennadi et al. 2007); 500 ng of murine TNFα in 100 μl was injected intraperitoneally (IP) 4 h before infusion of SS RBCs to increase endothelial expression of selectins. Sevuparin or saline control solution was injected SQ at various dose levels prior to infusion of SS RBCs into nude mice. Results SS RBC adhesion to ECs stimulated with IL13 and histamine was greater than adhesion to similarly stimulated ECs pretreated with sevuparin at 100, 200, 400 and 600 µg/ml prior to exposure to flowing SS RBCs (p = 0.047, 0.031, 0.094, 0.065, respectively, using a paired t test, in which each patient sample was only compared to itself), with a robust dose-response (p < 0.001) (Fig 1A). In a similar analysis, 7.5 µg/ml of function blocking monoclonal antibody to CD62P (9E10) also significantly reduced RBC adhesion (p = 0.038). Sevuparin significantly inhibited adhesion of SCD PMNs to ECs treated with TNFα and histamine, and this inhibition exhibited a modest dose-response relationship (Fig 1B). When normal PMNs stimulated by SS RBCs were studied, the differences between adhesion in the absence of sevuparin and in the presence of either 600 or 800 µg/mL sevuparin were also highly significant at both 1 and 2 dyne/cm2 (p = 0.019 and p = 0.011 at 1 dyne/cm2 and p = 0.013 and p = 0.008 at 2 dynes/cm2, respectively) (Fig 1C). In vivo, injection of sevuparin prior to infusion of SS RBCs significantly decreased SS RBC adhesion to vessel walls, as measured by the percent of vessel lengths occupied by adherent cells (Fig 2A). Sevuparin treatment also significantly increased the percent of venules that maintained normal blood flow (Fig 2B). Conclusions Sevuparin is an effective inhibitor of SS RBC adhesion and both SCD and normal PMN adhesion to endothelial cells in vitro. In vivo, sevuparin effectively decreased vaso-occlusion and improved blood flow after TNFα treatment. Therefore, we consider sevuparin a promising anti-adhesion agent with potential to reduce vaso-occlusion in SCD, via reducing RBC adhesion and leukocyte adhesion, possibly through its effect on selectins. Disclosures: Batchvarova: Dilaforette, AB: Research Funding. Shan:Dilaforette, AB: Research Funding. Zennadi:Dilaforette, AB: Research Funding. Lindgren:Dilaforette, AB: Employment. Leitgeb:Dilaforette, AB: Employment. Sulila Tamsen:Dilaforette, AB: Employment. Telen:GlycoMimetics, Inc.: Research Funding; Dilaforette, NA: Research Funding; Pfizer, Inc.: Consultancy.