Antimalarial cabamiquine inhibits blood-stage of different field isolates of Plasmodium species

Abstract

Malaria remains a major public health concern in Sub-Saharan Africa. The widespread and emerging drug resistance calls for urgent development of new drugs with novel modes of action to treat, control, and eliminate malaria. Cabamiquine, a promising antimalarial drug candidate that inhibits protein synthesis, has demonstrated promising potency across multiple life stages of P. falciparum. Here, in Ghana and Mali, we assessed the ex-vivo susceptibility of non-falciparum malaria field isolates of P. ovale and P. malariae to cabamiquine, in comparison to P. falciparum and explored the candidate drugs potential cross-resistance with existing antimalarial drugs. Thus, cabamiquine susceptible and selected resistant P. falciparum laboratory strains and field isolates were tested against a panel of antimalarial drugs. P. ovale isolates displayed a decreased susceptibility to DHA (median IC50 = 3.962 nM; IC50 range of 0.6587 to 9.154 nM) when compared to P. falciparum (p = 0.0371), but no significant difference was noticed for cabamiquine against both species (P. ovale: median IC50 = 0.9441 nM; IC50s ranged of 0.225 to 2.315 nM). Conversely, P. malariae isolates were less susceptible to cabamiquine (median IC50 = 3.915 nM; range 1.410 to 7.010) compared with P. falciparum (p = 0.001). There was no difference in susceptibility to DHA, both for P. malariae (median IC50 = 7.641 nM; range of 2.510 to 1268 nM) and P. falciparum isolates. With regards to the cabamiquine-resistant parasites (wild-type (WT) and mutants), there was limited variation between their susceptibilities to amodiaquine, tafenoquine, sulfadoxine, pyrimethamine, pyronaridine, active amodiaquine, lumefantrine, pyrimethamine, chloroquine, primaquine, and quinine. Overall, P. ovale and P. malariae are susceptibility to cabamiquine.

Introduction

Malaria is a life-threatening disease with widespread prevalence across tropical and subtropical regions. Over recent decades, global control and elimination efforts have significantly reduced the incidence and mortality of malaria. While Plasmodium (P.) falciparum is the most virulent species and accounts for the majority of malaria-related deaths, particularly in Africa, the role of non-falciparum species, such as P. vivax, P. malariae, and P. ovale, must also be considered in the development of integrated strategies targeting malaria eradication [1–4]. In this context, it should be noted that, although less lethal, these non-falciparum species act as persistent reservoirs, facilitating disease transmission, significantly hindering control and elimination efforts [5, 6]. This challenge is further compounded by the absence of specific treatments of these species. Among these, P. malariae infections are associated with anaemia and can lead to chronic infections and significantly drive morbidity, resulting in severe illness and death if left untreated [6–9]. P. ovale, comprising the two subspecies – P. ovale curtisi and P. ovale wallikeri – are able to form dormant liver-stage hypnozoites, like P. vivax [10–12]. These dormant forms can cause relapsing malaria, hence calling for radical cure treatment with the ability to kill hypnozoites. Inadequate treatment of P. ovale infections contributes to sustain disease transmission, particularly in high-burden regions such as sub-Saharan Africa.

Despite their clinical and epidemiological significance, P. malariae and P. ovale infections are treated with first-line antimalarial drugs (e.g. ACTs = Artemisinin-based combination therapies) primarily developed to treat P. falciparum or P. vivax malaria. However, evidence supporting the efficacy at preclinical and clinical level of these treatments against P. malariae and P. ovale is limited [13–18].

This therapeutic uncertainty raises concerns about suboptimal treatment and the possible development of drug resistance in these species. A further complication to these challenges is the reported drug resistance in P. falciparum, which reduces the number of effective treatment options available. Consequently, to monitor and respond to evolving patterns of treatment efficacy, surveillance efforts are critical [19–23]. This will require coordinated efforts involving clinical studies, molecular investigations, and in vitro or ex vivo drug susceptibility assessments. While P. falciparum adapts well to in-vitro culture conditions, enabling long-term studies of drug response and resistance mechanisms, no such protocols currently exist for P. malariae and P. ovale. As a result, ex-vivo assays are particularly important for evaluating the growth inhibitory activities of antimalarials against these species [13–15].

In the last years, in response to the need for novel antimalarial treatments, cabamiquine (M5717, DDD107498 [24]), has emerged as a promising candidate. Discovered through a phenotypic screening, cabamiquine has been proven to be a highly potent inhibitor of P. falciparum eukaryotic elongation factor 2 (PfeEF2) and shows potent activity across multiple stages of the parasite’s life cycle, including pre-erythrocytic forms [24–28]. Additionally, pre-clinical and early clinical investigations have demonstrated its efficacy, favourable pharmacokinetics, and good tolerability [29, 30]. Due to these promising results, cabamiquine is currently in Phase II clinical trials in combination with pyronaridine (NCT05689047).

As a novel compound under development, understanding cabamiquine’s efficacy and potential resistance in non-falciparum species is critical for future deployment.

To further investigate cabamiquine effectiveness across different Plasmodium species and its cross resistance with existing antimalarial drugs, we first evaluated the susceptibility of P. malariae and P. ovale isolates to both dihydroartemisinin (DHA) and cabamiquine. Secondly, to assess cabamiquine cross-resistance with existing antimalarial drugs; we utilized cabamiquine-resistant adapted P. falciparum field isolate strains (isolate 1: parasite strain ID EFF192 with E134V mutation and isolate 2: parasite stain ID EFF209 with Y186C mutation) [31]. These cabamiquine-resistant strains were subjected to drug susceptibility testing against a representative panel of antimalarials. Additionally, this approach not only allowed us to assess how other antimalarial drugs might influence the rise of cabamiquine resistance but also provided insights into the compound’s real-world applicability.

Methods

Ethical considerations

The study protocol was approved by the ethical committee of the Ghana Health Service Ethical Review Committee of Ghana with the reference GHS-ERC (GHSERC005/12/17) and the ethical committee of the Faculties of Medicine-Odonto-Stomatology and Pharmacy, University of Science, Techniques and Technologies of Bamako, Mali with the references N°2022/03/CE/ USTTB, and N°2023/03/CE/USTTB. Written informed consent was obtained from every participant after getting clear information about the objectives, advantages, and disadvantages of the study. All the patients who consented to be involved in the study were included and all participants with positive rapid diagnostic tests (RDTs) were treated with Artemether-Lumefantrine (AL), in accordance with national policies.

Parasites

The different assay used parasites isolated from patients (field isolates) as well as laboratory strain of P. falciparum; 3D7. All field isolated parasites used in this study were collected from individuals microscopically diagnosed with P. falciparum, P. malariae and P. ovale mono-infection. P. malariae and P. ovale mono-infections were confirmed, as reported earlier [13, 15]. P. falciparum cabamiquine-resistant parasites generated from P. falciparum 3D7 as well as field isolates were obtained as described previously [31]. It should be noted that the parasites used in this study were not whole genome sequenced, therefore, genetic diversity among the field isolates was not assessed.

Cabamiquine-resistant P. falciparum

Mutant parasites resistant to cabamiquine were selected as previously described [31]. The field isolates (EEF192 and EEF209) and P. falciparum laboratory strains (3D7-FS and 3D7-MM) lines, were used for the mutant generation. The resistant mutants were tested in the current study to assess their susceptibility and or cross-resistance to the panel of antimalarial drugs.

Susceptibility assessment

The panel of drugs used for these assays included 11 antimalarials (most were purchased from Sigma) i.e. dihydroartemisinin (DHA), lumefantrine, quinine, primaquine, tafenoquine, pyrimethamine, atovaquone, sulfadoxine, chloroquine, amodiaquine, and pyronaridine.

All samples were processed for drug assays that were conducted as per protocol of P. falciparum standard culture conditions. Non-falciparum field isolates parasites were washed three times with incomplete RPMI-1640 media (10.43 g of RPMI-1640, 5.96 g of HEPES, 2.5 g of NaHCO₃, 2.5 mL of gentamicin 50 mg/mL for 1 L in H₂O). The P. falciparum resistant strains were first cultured in biological triplicates before being subjected to testing. All the samples were diluted with packed uninfected RBCs of a single donor of O⁺ to 0.5% parasitaemia for P. malariae, P. ovale, and P. falciparum. Then, the diluted samples were added with the complete RPMI-1640 (10.43 g of RPMI-1640, 5.96 g of HEPES, 2.5 g of NaHCO₃, 1 mL of hypoxanthine, 5 g of Albumax, and 2.5 mL of gentamicin 50 mg/mL, in 1 L of H₂O) and dispensed in 96-plates and adjusted to 2% haematocrit. All drugs and compounds, initially in dimethyl sulfoxide (DMSO) at 10 mM, were serially diluted (3-fold) into ten concentrations, from 10 µM to 0.0005 µM. The assay was conducted in triplicates and repeated twice.

The final set-up containing infected and uninfected RBCs and diluted drugs, in a total volume of 120 µL in each well, was maintained at 37 °C in a gas mixture made up of 2% O2, 5% CO2 and balanced with nitrogen gas. The experiments were stopped after an incubation of 72 h for P. malariae and 48 h for P. ovale and P. falciparum. Thus, 80 µL of the supernatant of each well was removed and replaced with SYBR Green I (Invitrogen, USA) stain in lysis buffer (20 mM Tris (pH 7.5), 5 mM EDTA, 0.008% (W/V) saponin, and 0.08% (V/V) Triton X-100) at 1X final concentration. Then, the plates were re-incubated in the dark for about 30 min, and the fluorescence from each well was determined, using the Varioskan Lux multimode microplate fluorescent plate reader (ThermoFisher Scientific, USA) at an excitation of 485 nm, emission of 520 nm, and with a gain of 100. Overall, 10 P. ovale and 12 P. malariae field isolates from different donors were tested against each drug (DHA and cabamiquine). For comparison purposes, a similar number of isolates (at least 10) of P. falciparum isolates were tested, respectively.

Data analysis

Drug-treated data were first normalized to untreated controls. To assess the quality of the assay, a Z’ factor was calculated from positive controls (8 drug-free wells) and negative controls (8 parasite-free, red blood cell control wells). Then dose-response curves were plotted, the data were curve fit with a variable slope function, and the inhibitory concentrations 50% (IC50) were estimated, using GraphPad Prism software version 9. While Z’ values over 0.5 were considered adequate, each curve was examined by individually to ensure suitability. Some of the assays, with Z’ below 0.5, were considered valid depending on factors such as the standard error of the curve fit IC50. The Student’s t-test was applied in GraphPad Prism software to statistically test the difference between P. malariae/P. ovale and P. falciparum IC50s. A p-value less than 0.05 was considered significant.

Results

P. falciparum fieldisolates are more susceptible to DHA and Cabamiquine than P. ovale and P. malaria field isolates

The field isolates of the parasites were confirmed by microscopy. The different stages of the parasites were confirmed for the assay to be set with at least 60% rings. Since the microscopy of P. malariae and P. ovale is always limited by the parasitemia level, we took images of the Giemsa-stained parasites to help dispel any forms of ambiguity (Fig. 1).

Fig. 1.

Giemsa-stained images of P. malariae and P. ovale: Top panel shows the Giemsa-stained images of P. malariae showing the different stages, lower panel shows the Giemsa-stained P. ovale parasites at the different stages of development

In an ex vivo assay P. ovale isolates tested in Ghana were less susceptible to DHA (median IC₅₀ = 3.962 nM; IC₅₀ range of 0.659 to 9.154 nM) compared with P. falciparum isolates (median IC₅₀= 1.029 nM; IC₅₀ range of 0.257 to 5.032 nM) (p = 0.0371) (Fig. 2A). Cabamiquine test showed susceptibility of P. ovale isolates (median IC₅₀= 0.944; IC_50s range of 0.225 to 2.315 nM) and P. falciparum (median IC₅₀= 0.090 nM; IC_50s range of 0.011 to 4.323 nM) (Fig. 2B). Two isolates of P. malariae showed reduced susceptibilities against DHA where IC_50s ranged from 2.510 to 1268 nM (median IC₅₀ = 7.641 nM). Relatively, the P. falciparum isolates (median IC₅₀ = 4.395 nM; range of 1.480 to 9.730 nM) (Fig. 1C) were susceptible. The P. malariae isolates (from Mali) tested, appeared less susceptibility to cabamiquine (median IC₅₀ = 3.915 nM; range of 1.410 to 7.010 nM) whilst, P. falciparum was susceptible (median IC₅₀ = 1.510 nM; range of 0.420 to 2.640 nM) (p = 0.0010) (Fig. 2D).

Fig. 2.

Ex-vivo susceptibility diverse Plasmodium species (P. ovale, P. malariae and P. falciparum) to dihydroartemisinin (DHA) and cabamiquine. The susceptibility of P. ovale versus P. falciparum (A and B) in Ghana and P. malariae versus P. falciparum (C and D) in Mali to DHA and cabamiquine were tested in an ex vivo growth inhibitory assay. The half-maximal inhibitory concentration (IC₅₀ (nM)) of the tested compound (drug) is plotted against the isolates (parasite species). The median IC₅₀ is indicated with a bar (in the middle). Student t-test comparison between the isolates is indicated at the top of the plots. (blue) P. ovale; (red) P. malariae; (black) P. falciparum. isolates

Differential activity of antimalarial drugs against cabamiquine-sensitive and -resistant P. falciparum parasites

To evaluate the potential to drive cross resistance to other antimalarial the previously selected cabamiquine-resistant adapted P. falciparum field isolate strains (isolate 1: parasite strain ID EFF192 with E134V mutation and isolate 2: parasite stain ID EFF209 with Y186C mutation) were tested [31]. The susceptibility of mutant strains selected after exposure to cabamiquine were diverse as compared to the wild type (WT) strains. Nevertheless, among culture-adapted isolates tested against DHA, amodiaquine, tafenoquine, sulfadoxine, pyrimethamine, pyronaridine, and active amodiaquine, the IC_50s estimated in WT appeared like the results for isolates containing mutant strains. However, for lumefantrine, pyrimethamine, chloroquine, and primaquine, at least one of the mutant strains showed some IC_50s about two times the median IC₅₀ of the corresponding wild-type (Fig. 3A-L). When tested against quinine, mutant EEF209_MT isolates showed high sensitivity with an IC₅₀ 4-fold lower than the other strains, whose IC_50s were higher than 800 nM (Fig. 3D).

Fig. 3.

Comparative susceptibility of cabamiquine-resistance associated mutant strains from field isolates. The susceptibility of the wild type as well as resistant mutant were tested for the following drugs; DHA= Dihydroartemisinin (A), LUM= Lumefantrine (B), AMQ= Amodiaquine (C), QN= Quinine (D), TNF= Tafenoquine (E), AVQ= Atovaquone (F), SFX= Sulfadoxine (G), PyR= Pyrimethamine (H), CQ= Chloroquine (I), PMQ= Primaquine (J), PNrD= Pyronaridine (K), Des_ETAm = Des-Ethyl amodiaquine (L). The half-maximal inhibitory concentration (IC₅₀ (nM)) of the tested compound (drug) is plotted against the isolates. The median IC₅₀ is indicated with a bar (in the middle). Three independent experiments done in duplicate are displayed in this figure

The results of P. falciparum 3D7 and mutants’ susceptibility are shown in Fig. 3. For most the strains, the susceptibility is similar, with not significant slightly higher values for 3D7_FS and 3D7_MM.

Interestingly, in Fig. 2 field isolates and in Fig. 3 lab strain selected cabamiquine resistant parasites, no cross-resistance was observed with the current existing antimalarial drug. Thus, existing antimalarial drugs potently inhibited cabamiquine resistant parasites (Figs. 3 and 4).

Fig. 4.

Comparative susceptibility of cabamiquine-resistance associated mutant strains from 3D7 P. falciparum strain. The susceptibility of the wild type (3D7_WT) as well as resistant mutant (3D7_FS and 3D7_MM) were tested for the following drugs; DHA= Dihydroartemisinin (A), LUM= Lumefantrine (B), AMQ= Amodiaquine (C), QN= Quinine (D), TNF= Tafenoquine (E), AVQ= Atovaquone (F), SFX= Sulfadoxine (G), PyR= Pyrimethamine (H), CQ= Chloroquine (I), PMQ= Primaquine (J), PNrD= Pyronaridine (K), Des_ETAm = Des-Ethyl amodiaquine (L). The half-maximal inhibitory concentration (IC₅₀ (nM)) of the tested compound (drug) is plotted against the isolates (parasite species). The mean IC₅₀ is indicated with a bar (in the middle). Three independent experiments done in duplicate are displayed in this figure

Discussion

Eliminating malaria requires integrative approaches involving all diverse Plasmodium species. This study evaluated the ex-vivo susceptibility of non-falciparum species, P. malariae and P. ovale, comparing to that of P. falciparum using field isolates parasites. The primary focus was on cabamiquine, an investigational drug candidate that has completed Phase IIa clinical trials in combination with pyronaridine, in comparison to the first-line treatment, DHA [13, 15, 32, 33]. This, henceforth, is an important step in antimalarial drug development, as usually, non-falciparum species are neglected and not part of the early discovery programs.

In Ghana, P. ovale showed slightly reduced susceptibility to DHA compared to P. falciparum. However, the values remained within the expected range, indicating P. ovale susceptibility to DHA, as previously reported [13]. In Mali, while P. malariae displayed less susceptibility than P. falciparum in cabamiquine testing; no difference was observed from DHA assays, except for two higher DHA IC_50s, suggesting possible decreased susceptibility. Although previous studies showed similar potency of artemether, another artemisinin derivative, to inhibit the growth of P. malariae [14]. The higher IC_50s may mean cases of resistance to DHA to be further investigated when greater access to its biology becomes possible. Thus, more data are needed to clarify this concern about P. ovale and P. malariae tolerance to DHA. In Ghana, a study had showed delayed clearance of P. ovale and P. malariae infections after a treatment with DHA-piperaquine [13, 34, 35]. Clinical trials of DHA-piperaquine, pyronaridine-artesunate, and artemether-lumefantrine (Coartem^®) showed a low efficacy against both species (P. ovale and P. malariae) [17, 36]. The inherent spread of the data is typical of working with field isolates. These clinical reports are consistent with the current ex vivo data findings. The ex vivo assay is limited by the different stage representation of the parasite’s development, though we focused on isolates that have high number of ring stages (above 70%) as well as the differences in parasites life cycle. Thus, field isolate parasites fitness and welfare matter in ex vivo assay and can definitively bias drug potency as an unwell parasite will be efficiently killed better than a healthy one and that can lead to some of the significant differences in field isolate parasites drug susceptibility.

Interestingly, both P. ovale and P. malariae were susceptible to cabamiquine. This comparable susceptibility of non-falciparum species to P. falciparum is an interesting finding as it suggests cabamiquine as a tool for broad cure. Looking at the mode of action (affecting protein translation) as well as the molecular targets (eukaryotic translation elongation factor 2 (eEF2)) of this compound, there is amino acids sequence conservation across the different Plasmodium species. This, therefore, present an opportunity to target the different stages of the parasite’s life cycle (liver, blood and mosquitos’ stages). The first clinical trials for cabamiquine demonstrated encouraging results for the safety, pharmacokinetics, and antimalarial activity of the drug candidate [26]. The drug displayed a good potency for monotherapy, but combinations with other antimalarials have been tested in-vivo in mouse model. This is made possible by its long half-life as well as the ability to block gametocyte development. Among the partner drugs tested, pyronaridine [37] had been selected as a combination partner for cabamiquine Ph2a trials [24, 26, 28]. Importantly pyronaridine has proven to be clinically effective against P. ovale and P. malaria species. Thus, cabamiquine-pyronaridine may be developed as efficacious treatment for all types of malaria.

The use of drugs is essential in malaria control and elimination. However, one of the main hindrances in using antimalarials is the development of resistance and the differential susceptibilities in the different Plasmodium species. In this study, after the cabamiquine resistant mutant strains were selected, they were subjected to susceptibility assays using a panel of drugs [31]. The IC_50s estimated from the mutant strains were similar to that of the wild-type for the compounds tested (dihydroartemisinin, amodiaquine, tafenoquine, sulfadoxine, pyrimethamine, pyronaridine, and active amodiaquine) suggesting no cross resistance with cabamiquine [28, 32, 34, 38–41]. However, the results were different for lumefantrine, pyrimethamine, chloroquine, and primaquine where some of the mutant IC_50s were higher in P. falciparum field isolates [38, 42]. Meanwhile, the data presented in this manuscript demonstrates that cabamiquine is potent compound against P. falciparum, P. ovale and P. malariae. We have, further, shown that resistance to cabamiquine does not drive cross-resistance to the known antimalarial compounds. This suggest that a combination therapy with the other potent antimalarials will be a good tool in the control of malaria.

Conclusion

Malaria control and elimination in endemic areas remain challenging because of the drug resistance. There is an essential need of preventing the emergence and spread of resistance, as well as finding new treatments. This study was an opportunity to integrate the non-falciparum in the process of the discovery and development of cabamiquine, a candidate antimalarial drug that has been successfully tested for the early clinical trials with promising phase IIa results. The study showed that cabamiquine is active against P. ovale and P. malariae. The drugs tested showed potency against cabamiquine-resistant parasites, as well as against wild-type parasites.

Abbreviations

WT

Wild type

P

Plasmodium

DHA

Dihydroartemisinin

IC50

Half-maximal Inhibitory Concentration

ACT

Artemisinin-based Combination Therapy

PfeEF2

Plasmodium falciparum eukaryotic Elongation Factor 2

RDT

Rapid Diagnostic Test

AL

Artemether Lumefantrine

DMSO

Dimethyl Sulfoxide

CO2

Carbon Dioxide

uL

Micro litres

QN

Quinine

LUM

Lumefatrine

AMQ

Amodiaquine

TNF

Tefanoquine

PMQ

Primaquine

PNrD

Pyronaridine

CQ

Chloroquine

AVQ

Atovaquone

SFX

Sulfadoxine

Author contributions

L.D., Y.A. designed the study with Merck team; CD-G and TS. L.D., A.S., Y.A., F.S., O.D., F.D., and F.D performed the experiments and analyzed the data. L.N.A-E and A.A.D. provided access to some important Laboratory and bioinformatic facilities. L.D. and Y.A wrote the original manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID 10.13039/100009945).

Data availability

All Data is generated and given in the manuscript, either main text or supplementary data in the form of figures and tables.

Declarations

Ethics approval and consent to participate

The study was approved by Ghana Health Service Ethical Review Committee of Ghana with the reference GHS-ERC (GHSERC005/12/17) and the ethical committee of the Faculties of Medicine-Odonto-Stomatology and Pharmacy, University of Science, Techniques and Technologies of Bamako, Mali with the references N°2022/03/CE/ USTTB, and N°2023/03/CE/USTTB.

Consent for publication

All authors reviewed and approved.

Competing interests

The authors declare no competing interests.