Sevuparin inhibits merozoite invasion of <i>P</i>. <i>falciparum</i> clones, strains and fresh isolates <i>in vitro</i> at low concentrations, independently of parasite origin or phenotype.

<p>The invasion blocking capacity of sevuparin in 34 <i>in vitro</i> propagated <i>P</i>. <i>falciparum</i> isolates expressed as IC50. The inhibitory capacity of sevuparin was titrated in double dilution steps between 0.125 μg/mL and 1 mg sevuparin/mL culture. Ten laboratory isolates were either sensitive (3D7, 3D7PG12, Dd2, HB3) or resistant (R29, TM180, TM284, F32, 7G8, FCR3S1.2) to chloroquine. Three parasites of the W2mef background carried disrupted genes for EBA 140, EBA 175 or EBA 181 (EBA-KO). W2mef is a cloned line of parasites derived from the Indochina III-CDC strain. Of the fresh primary isolates 11 were from Ugandan children with either severe (dot) or uncomplicated (square) malaria and six isolates were from adults infected in Ethiopia/Eritrea, Kenya or Niger. Four Cambodian isolates were sensitive or resistant to artemisinin (red-circled square; IPC-4884, Pursut, artemisinin resistant (RSA 0-3h: 6,5%) and IPC 4912 artemisinin resistant (red circled square; RSA 0–3 h: 49%). ICP 5188 Rattanakiri and IPC 3663 Pailin were artemisinin sensitive (square).</p

The de-sequestering capacity of sevuparin in <i>P</i>. <i>falciparum</i> infected patients.

<p>A total of 44 patients were included in the efficacy part of the trial and were treated with oral atovaquone/proguanil with or without adjunctive treatment in the form of short i.v. infusions of sevuparin. The numbers of trophozoite and schizont IEs were estimated in the peripheral blood samples on thin and thick films that were taken at time points 0, 1, 2, 3, 4, 6, 8, 10, and 11 h and thereafter every 6 h until two consecutive blood samples were parasite negative. The relative numbers were calculated from the number of trophozoite and schizont IEs at one time point related to the baseline number of trophozoite and schizont IEs at time point 0 h (immediately prior to the first dose of sevuparin), and the mean was measured based on all subjects in one group. The red dotted line represents the sevuparin treated patients, and the blue line represents the control patients. Logarithmic y- axis is used. Significantly (p<0.05) higher numbers of trophozoite and schizont IEs were found in the sevuparin treated patients at time point 1 h (p = 0.0322). In a, the relative numbers (mean ± SD) of trophozoite and schizont parasites in the two study groups up to H30 are presented. In b, the detailed relative changes in the number of trophozoite and schizont parasites (mean ± SD) during the first 12 hours after the first injection of sevuparin are shown. In c, individual effects of sevuparin on the number of trophozoites and schizont parasites per patient is shown. The grey arrows indicate the periodic sevuparin infusions.</p

Sevuparin lowers the relative mean number of ring-stage IEs after a single sevuparin infusion in <i>P</i>. <i>falciparum</i> infected patients.

<p>A total of 44 patients were included in the efficacy part of the trial (part 2) and were treated with oral atovaquone/proguanil with or without adjunctive treatment in the form of i.v. infusions of sevuparin. The relative numbers were calculated from the number of ring IEs at one time point related to the baseline value of ring IEs at time point 0 h (immediately prior to the first dose of sevuparin), and the mean was measured based on all subjects in one group. a, The mean relative numbers (mean ± SD) of ring stage parasites in the two study groups from 0 h to 30 h. The numbers of ring-stage IEs were estimated in peripheral blood samples on thin and thick films that were taken at time points 0, 1, 2, 3, 4, 6, 8, 10, and 11 h and thereafter every 6 h until two consecutive blood samples were parasite negative. The red dotted line represents the patients treated with sevuparin (3 mg/kg) and oral atovaquone/proguanil, and the blue line represents the control patients who were given only oral atovaquone/proguanil, a logarithmic y-axis is used. Significantly lower levels of ring stage IEs were found in the sevuparin treated patients at time points 1 h (p = 0.0223), 2 h (p = 0.0246), 3 h (p = 0.0027), 4 h (p = 0.0278), and 6 h (p = 0.0346). (An outlier appears in the data but does not drive the difference as the statistical significant difference between the two groups remains even if data from this patient is excluded from the analysis since the tests used are non-parametric which are thus very robust against divergent.) b, Detailed mean relative changes in the number (mean ± SD) of ring stage parasites during the first 12 hours after the first injection of sevuparin. c, Numbers of ring-stage IEs levels in the individual patients. Oragne arrow indicate an outlier. Grey arrows indicate the short i.v. sevuparin infusions over five minutes.</p

Study design of phase I/II study in patients with uncomplicated <i>falciparum</i> malaria-TSM02.

<p>a, Overview of study design and dosing regimen for all patients in part 1. b, Overview of study design and dosing regimen for patients in part 2 randomized to the anti-malarial regimen (atovaquone and proguanil) alone or sevuparin plus anti-malarial regimen (atovaquone/proguanil).</p

Anticoagulant activity of sevuparin.

<p>Anticoagulant activity of sevuparin.</p

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes - Fig 1

<p>(A) Effect of sevuparin on rosetting (%) of <i>P</i>. <i>falciparum</i> (n = 47) is dose dependent. The data show the median with interquartile range of rosettes formed at each concentration of sevuparin. (B) Median (interquartile range) % disruption of rosetting at each concentration of sevuparin. Sevuparin significantly disrupted rosette formation, p < 0.001.</p

Median (interquartile range) Pf-iRBCs adhering to HDMECs under shear stress at 0.05 Pa, 0.5% parasitaemia and 1% haematocrit at different concentrations of sevuparin (n = 10).

<p>Median (interquartile range) Pf-iRBCs adhering to HDMECs under shear stress at 0.05 Pa, 0.5% parasitaemia and 1% haematocrit at different concentrations of sevuparin (n = 10).</p

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes

<div><p>In severe <i>falciparum</i> 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 <i>falciparum</i> malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> isolates from Thailand were investigated. Trophozoite stages of <i>P</i>. <i>falciparum</i>-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 <i>P</i>. <i>falciparum</i> 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 <i>P</i>. <i>falciparum</i> 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 <i>falciparum</i> malaria.</p></div

Effects of sevuparin on rosette formation and cytoadherence of <i>Plasmodium falciparum</i> infected erythrocytes - Fig 2

<p>(A) The effects of sevuparin on Pf-iRBC adherence (n = 28). Median (interquartile range) adherence number of Pf-iRBCs binding per 1000 HDMECs at each concentration of sevuparin. (B) The inhibition of sevuparin on cytoadherence of <i>P</i>. <i>falciparum</i> (n = 28). Median (interquartile range) inhibition effect on cytoadhesion. The cytoadherence of patient isolates was significantly inhibited by sevuparin at concentrations ≥ 100 μg/mL (all p < 0.05).</p

The correlation between the adherent number of Pf-iRBCs binding per 1000 HDMECs and rosetting number (%) of <i>P</i>. <i>falciparum</i> (n = 28), (r_s = 0.152, p = 0.439).

<p>The correlation between the adherent number of Pf-iRBCs binding per 1000 HDMECs and rosetting number (%) of <i>P</i>. <i>falciparum</i> (n = 28), (r_s = 0.152, p = 0.439).</p