Inhibition of merozoite invasion and transient de-sequestration by sevuparin in humans with Plasmodium falciparum malaria

Abstract

Severe malaria: Even 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 study: The 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 study: A 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 registration: ClinicalTrials.gov NCT01442168.

Conflict of interest statement

Competing Interests: MW holds shares in and is a director of the board of Modus Therapeutics AB, a company of the Karolinska Development AB involved in the development of adjunct treatment for severe malaria. The authors declare no other competing financial interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Sevuparin is an acidic, negatively charged, antiadhesive polysaccharide drug.

The predominant structure of sevuparin with the main monosaccharide constituents. The weight average molecular weight of sevuparin is 6.5–9.5 kDa.

Fig 2. Study design of phase I/II study in patients with uncomplicated falciparum malaria-TSM02.

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).

Fig 3. CONSORT flow diagram.

Flow diagram of the progress through the phases of TSM02 randomized trial with two groups for Part and Part 2. a, Part 1 enrolment, intervention allocation, follow-up, and data analysis. b, Part 2 enrolment, intervention allocation, follow-up, and data analysis.

Fig 4. Sevuparin lowers the relative mean number of ring-stage IEs after a single sevuparin infusion in P. falciparum infected patients.

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.

Fig 5. The de-sequestering capacity of sevuparin in P. falciparum infected patients.

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

Fig 6. Sevuparin inhibits merozoite invasion of P. falciparum clones, strains and fresh isolates in vitro at low concentrations, independently of parasite origin or phenotype.

The invasion blocking capacity of sevuparin in 34 in vitro propagated P. falciparum 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).