RESUMEN
BACKGROUND: Ganaplacide, also known as KAF156, is among the new antimalarial drug candidates that have successfully reached Phase III clinical trials, and is proposed in combination with lumefantrine. This combination could replace the current front-line artemisinin-based combination therapies (ACTs) in case of Plasmodium falciparum resistance to both artemisinins and partner drugs. Indeed, the African continent, where the malaria burden is the highest, is currently experiencing worrying multiple emergences and spread of artemisinin resistance, which urges for the exploration of the antiparasitic properties of KAF156 in this context. OBJECTIVES AND METHODS: The objectives of this work were firstly to evaluate the risk of cross-resistance between artemisinins and KAF156 alone, and in combination with lumefantrine, using a panel of artemisinin-resistant strains carrying different pfk13 mutations and markers of other antiplasmodial drug resistances; secondly to explore in vitro the relevance of combining KAF156 and lumefantrine with artemisinins, based on the model of triple ACTs. RESULTS: Our results highlighted that KAF156 activity was not impaired by mutations in pfk13, pfcrt, pfmdr1, pfmdr2, pfdhps and pfdhfr genes or by pfmdr1 amplification. Moreover, we demonstrated that KAF156 alone and in combination with lumefantrine was active against artemisinin-resistant parasites, including when they are quiescent. CONCLUSIONS: All these in vitro results evidence that multi-drug resistant parasites currently in circulation in the field might not affect KAF156 efficacy, and are encouraging signs for KAF156 use in a triple ACT to preserve the use of artemisinins for as long as possible.
RESUMEN
Over the past thirty years, epigenetic regulation of gene expression has gained increasing interest as it was shown to be implicated in illnesses ranging from cancers to parasitic diseases. In the malaria parasite, epigenetics was shown to be involved in several key steps of the complex life cycle of Plasmodium, among which asexual development and sexual commitment, but also in major biological processes like immune evasion, response to environmental changes or DNA repair. Because epigenetics plays such paramount roles in the Plasmodium parasite, enzymes involved in these regulating pathways represent a reservoir of potential therapeutic targets. This review focuses on epigenetic regulatory processes and their effectors in the malaria parasite, as well as the inhibitors of epigenetic pathways and their potential as new anti-malarial drugs. Such types of drugs could be formidable tools that may contribute to malaria eradication in a context of widespread resistance to conventional anti-malarials.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Parásitos , Plasmodium , Animales , Humanos , Plasmodium falciparum , Malaria Falciparum/parasitología , Epigénesis Genética , Malaria/parasitología , Antimaláricos/farmacología , Antimaláricos/uso terapéuticoRESUMEN
The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.
Asunto(s)
Antimaláricos , Citostáticos , Malaria Falciparum , Malaria , Humanos , Antimaláricos/química , Citostáticos/uso terapéutico , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum , Péptidos/farmacología , Péptidos/uso terapéuticoRESUMEN
Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.
Asunto(s)
Antimaláricos , Artemisininas , Dominio BTB-POZ , Proteínas Protozoarias , Antimaláricos/farmacología , Artemisininas/farmacología , Resistencia a Medicamentos/genética , Mutación , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/genéticaRESUMEN
Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin resistance context, novel drug combinations were evaluated against artemisinin-sensitive and artemisinin-resistant Plasmodium falciparum parasites. Corresponding hybrid molecules were also synthesized and evaluated for comparison with combinations and individual pharmacophores (e.g. atovaquone, mefloquine or triclosan). Combinations and hybrids showed remarkable antimalarial activity (IC50 = 0.6 to 1.1 nM for the best compounds), strong selectivity, and didn't present any cross-resistance with artemisinin. Moreover, the combination triclosan + atovaquone showed high activity against artemisinin-resistant parasites at the quiescent stage but the corresponding hybrid lost this pharmacological property. This result is essential since only few molecules active against quiescent artemisinin-resistant parasites are reported. Our promising results highlight the potential of these combinations and paves the way for pharmacomodulation work on the best hybrids.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/farmacología , Atovacuona/farmacología , Mefloquina/farmacología , Plasmodium falciparum/efectos de los fármacos , Triclosán/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Artemisininas/química , Atovacuona/síntesis química , Atovacuona/química , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos/efectos de los fármacos , Humanos , Malaria Falciparum/tratamiento farmacológico , Mefloquina/síntesis química , Mefloquina/química , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Triclosán/síntesis química , Triclosán/químicaRESUMEN
BACKGROUND: Quiescence is an unconventional mechanism of Plasmodium survival, mediating artemisinin resistance. This phenomenon increases the risk of clinical failures following artemisinin-based combination therapies (ACTs) by slowing parasite clearance and allowing the selection of parasites resistant to partner drugs. OBJECTIVES: To thwart this multiresistance, the quiescent state of artemisinin-resistant parasites must be taken into consideration from the very early stages of the drug discovery process. METHODS: We designed a novel phenotypic assay we have named the quiescent-stage survival assay (QSA) to assess the antiplasmodial activity of drugs on quiescent parasites. This assay was first validated on quiescent forms from different artemisinin-resistant parasite lines (laboratory strain and field isolates), using two reference drugs with different mechanisms of action: chloroquine and atovaquone. Furthermore, the efficacies of different partner drugs of artemisinins used in ACTs were investigated against both laboratory strains and field isolates from Cambodia. RESULTS: Our results highlight that because of the mechanism of quiescence and the respective pharmacological targets of drugs, drug efficacies on artemisinin-resistant parasites may be different between quiescent parasites and their proliferating forms. CONCLUSIONS: These data confirm the high relevance of adding the chemosensitivity evaluation of quiescent parasites by the specific in vitro QSA to the antiplasmodial drug development process in the current worrisome context of artemisinin resistance.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Artemisininas/farmacología , Artemisininas/uso terapéutico , Cambodia , Resistencia a Medicamentos , Malaria Falciparum/tratamiento farmacológico , Parásitos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Proteínas ProtozoariasRESUMEN
The emergence of Plasmodium falciparum parasites, responsible for malaria disease, resistant to antiplasmodial drugs including the artemisinins, represents a major threat to public health. Therefore, the development of new antimalarial drugs or combinations is urgently required. In this context, several hybrid molecules combining a dihydroartemisinin derivative and gold(I) N-heterocyclic carbene (NHC) complexes have been synthesized based on the different modes of action of the two compounds. The antiplasmodial activity of these molecules was assessed in vitro as well as their cytotoxicity against mammalian cells. All the hybrid molecules tested showed efficacy against P. falciparum, in a nanomolar range for the most active, associated with a low cytotoxicity. However, cross-resistance between artemisinin and these hybrid molecules was evidenced. These results underline a fear about the risk of cross-resistance between artemisinins and new antimalarial drugs based on an endoperoxide part. This study thus raises concerns about the use of such molecules in future therapeutic malaria policies.
Asunto(s)
Antimaláricos , Arteméter , Oro , Compuestos Orgánicos de Oro , Plasmodium falciparum/crecimiento & desarrollo , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/farmacología , Arteméter/química , Arteméter/farmacología , Oro/química , Oro/farmacología , Humanos , Compuestos Orgánicos de Oro/síntesis química , Compuestos Orgánicos de Oro/química , Compuestos Orgánicos de Oro/farmacologíaRESUMEN
Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs.
Asunto(s)
Antihelmínticos , Antimaláricos , Plasmodium falciparum/crecimiento & desarrollo , Schistosoma mansoni/crecimiento & desarrollo , Animales , Antihelmínticos/síntesis química , Antihelmínticos/química , Antihelmínticos/farmacología , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/farmacología , HumanosRESUMEN
Background: Owing to the emergence of multiresistant Plasmodium falciparum parasites in Southeast Asia, along with the impressive decrease in the efficacy of the endoperoxide compound artemisinin and of artemisinin-based combination therapies, the development of novel antimalarial drugs or combinations is required. Although several antiplasmodial molecules, such as endoperoxide-based compounds, are in advanced research or development, we do not know whether resistance to artemisinin derivatives might impact the efficacy of these new compounds. Objectives: To address this issue, the antiplasmodial efficacy of trioxaquines, hybrid endoperoxide-based molecules, was explored, along with their ability to select in vitro resistant parasites under discontinuous and dose-escalating drug pressure. Methods: The in vitro susceptibilities of artemisinin- and trioxaquine-resistant laboratory strains and recent Cambodian field isolates were evaluated by different phenotypic and genotypic assays. Results: Trioxaquines tested presented strong cross-resistance with artemisinin both in the artemisinin-resistant laboratory F32-ART5 line and in Cambodian field isolates. Trioxaquine drug pressure over 4 years led to the in vitro selection of the F32-DU line, which is resistant to trioxaquine and artemisinin, similar to the F32-ART lineage. F32-DU whole genome sequencing (WGS) revealed that resistance to trioxaquine was associated with the same non-synonymous mutation in the propeller domain of the K13 protein (M476I) that was found in the F32-ART lineage. Conclusions: These worrisome results indicate the risk of cross-resistance between artemisinins and endoperoxide-based antiplasmodial drugs in the development of the K13 mutant parasites and question the usefulness of these molecules in the future therapeutic arsenal.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/farmacología , Resistencia a Medicamentos , Plasmodium falciparum/efectos de los fármacos , Cambodia , Genotipo , Humanos , Malaria Falciparum/parasitología , Proteínas Mutantes/genética , Pruebas de Sensibilidad Parasitaria , Fenotipo , Proteínas Protozoarias/genética , Selección Genética , Secuenciación Completa del GenomaRESUMEN
Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/farmacología , Resistencia a Medicamentos , Malaria Falciparum/parasitología , Plasmodium falciparum , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Humanos , Modelos Biológicos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genéticaRESUMEN
A series of twenty five molecules, including imidazolium salts functionalized by N-, O- or S-containing groups and their corresponding cationic, neutral or anionic gold(I) complexes were evaluated on Plasmodium falciparum in vitro and then on Vero cells to determine their selectivity. Among them, eight new compounds were synthesized and fully characterized by spectroscopic methods. The X-ray structures of three gold(I) complexes are presented. Except one complex (18), all the cationic gold(I) complexes show potent antiplasmodial activity with IC50 in the micro- and submicromolar range, correlated with their lipophilicity. Structure-activity relationships enable to evidence a lead-complex (21) displaying a good activity (IC50=210nM) close to the value obtained with chloroquine (IC50=514nM) and a weak cytotoxicity.
Asunto(s)
Antimaláricos/farmacología , Oro/farmacología , Metano/análogos & derivados , Compuestos Organometálicos/síntesis química , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Antimaláricos/toxicidad , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cloroquina/química , Cloroquina/farmacología , Cristalografía por Rayos X , Oro/química , Concentración 50 Inhibidora , Metano/química , Metano/farmacología , Estructura Molecular , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/toxicidad , Relación Estructura-Actividad , Células VeroRESUMEN
Malaria treatment with ACTs (Artemisinin-based Combination Therapies), combining a chemical derivative of artemisinin, and a partner drug has, for more than 15 years, produced a notable decrease in the mortality in tropical and subtropical areas. However, since 2008, a serious threat has emerged in western Cambodia, where the clinical efficacy of artemisinins has significantly declined, with a delayed parasite clearance rate and high recrudescence rates in the following weeks. Resistance of Plasmodium to artemisinins is now reported in several countries in South-East Asia. ACTs remain effective as long as the partner drug retains its activity but more and more clinical failures related to parasite resistance to both resistant parasites in sub-Saharan Africa, the continent most affected by malaria, as was the case in the past with other antimalarial treatments. It is therefore essential to better understand, from phenotypic and genotypic points of view, the mechanisms of resistance developed by the parasite Plasmodium falciparum face artemisinin and its derivatives in order to offer new therapeutic tools.
Asunto(s)
Artemisininas/administración & dosificación , Resistencia a Medicamentos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/administración & dosificación , Quimioterapia Combinada , Humanos , Plasmodium falciparum/fisiologíaRESUMEN
The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm's digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals-which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.
RESUMEN
The use of artemisinin and its derivatives has helped reduce the burden of malaria caused by Plasmodium falciparum. However, artemisinin-resistant parasites are able, in the presence of artemisinins, to stop their cell cycles. This quiescent state can alter the activity of artemisinin partner drugs leading to a secondary drug resistance and thus threatens malaria eradication strategies. Drugs targeting epigenetic mechanisms (namely epidrugs) are emerging as potential antimalarial drugs. Here, we set out to evaluate a selection of various epidrugs for their activity against quiescent parasites, to explore the possibility of using these compounds to counter artemisinin resistance. The 32 chosen epidrugs were first screened for their antiplasmodial activity and selectivity. We then demonstrated, thanks to the specific Quiescent-stage Survival Assay, that four epidrugs targeting both histone methylation or deacetylation as well as DNA methylation decrease the ability of artemisinin-resistant parasites to recover after artemisinin exposure. In the quest for novel antiplasmodial drugs with new modes of action, these results reinforce the therapeutic potential of epidrugs as antiplasmodial drugs especially in the context of artemisinin resistance.
RESUMEN
Malaria remains a major public health disease due to its high yearly mortality and morbidity. Resistance to the gold standard drug, artemisinin, is worrisome and needs better understanding in order to be overcome. In this work, we sought to study whether redox processes are involved in artemisinin resistance. As artemisinin is known to act among others via production of reactive species, we first compared the production of reactive oxygen species and concomitant protein oxidation in artemisinin-sensitive and artemisinin-resistant parasites when treated with artemisinin. The results undoubtedly demonstrated, using different original methods, that the level of ROS, including superoxide production, and oxidized protein were lower in the resistant strain. Interestingly, the major in-between strain difference was reported at the earlier ring stages, which are the forms able to enter in a quiescence state according to the ART resistance phenomenon. Moreover, we demonstrated a better homeostasis regulation in relation with higher expression of antioxidants in the artemisinin-resistant parasites than their sensitive counterparts after artemisinin exposure, notably, superoxide dismutase and the glutathione (GSH) system. These findings enrich the body of knowledges about the multifaceted mechanism of artemisinin resistance and will help in the design and development of newer antimalarials strategies active against resistant parasites.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Artemisininas/farmacología , Resistencia a Medicamentos/genética , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Oxidación-Reducción , Plasmodium falciparum/genéticaRESUMEN
Several measures are in place to combat the worldwide spread of malaria, especially in regions of high endemicity. In part, most common antimalarials, such as quinolines and artemisinin and its derivatives, deploy an ROS-mediated approach to kill malaria parasites. Although some antimalarials may share similar targets and mechanisms of action, varying levels of reactive oxygen species (ROS) generation may account for their varying pharmacological activities. Regardless of the numerous approaches employed currently and in development to treat malaria, concerningly, there has been increasing development of resistance by Plasmodium falciparum, which can be connected to the ability of the parasites to manage the oxidative stress from ROS produced under steady or treatment states. ROS generation has remained the mainstay in enforcing the antiparasitic activity of most conventional antimalarials. However, a combination of conventional drugs with ROS-generating ability and newer drugs that exploit vital metabolic pathways, such antioxidant machinery, could be the way forward in effective malaria control.
RESUMEN
Understanding the mode of action of antimalarials is central to optimizing their use and the discovery of new therapeutics. Currently used antimalarials belong to a limited series of chemical structures and their mechanisms of action are coutinuously debated. Whereas the involvement of reactive species that in turn kill the parasites sensitive to oxidative stress, is accepted for artemisinins, little is known about the generation of such species in the case of quinolines or hydroxynaphtoquinone. Moreover, the nature of the reactive species involved has never been characterized in Plasmodium-infected erythrocytes. The aim of this work was to determine and elucidate the production of the primary radical, superoxide in Plasmodium-infected erythrocytes treated with artemisinin, dihydroartemisinin, chloroquine and atovaquone, as representatives of three major classes of antimalarials. The intracellular generation of superoxide was quantified by liquid chromatography coupled to mass spectrometry (LC-MS). We demonstrated that artemisinins, atovaquone and to a lesser extent chloroquine, generate significant levels of superoxide radicals in Plasmodium falciparum sensitive strains. More so, the production of superoxide was lowered in chloroquine-resistant strain of Plasmodium treated with chloroquine. These results consolidate the knowledge about the mechanism of action of these different antimalarials and should be taken into consideration in the design of future drugs to fight drug-resistant parasites.
Asunto(s)
Antimaláricos , Medicamentos Esenciales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Cloroquina/farmacología , Resistencia a Medicamentos , Plasmodium falciparum , SuperóxidosRESUMEN
The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.
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Antimaláricos , Malaria Falciparum , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Endocitosis , Malaria/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparumRESUMEN
Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC-MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E+) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.