Highly active ozonides selected against drug resistant malaria.
ABSTRACT: Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.
Project description:BACKGROUND:Artesunate the most potent antimalarial is widely used for the treatment of multidrug-resistant malaria. The antimalarial cytotoxicity of artesunate has been mainly attributed to its selective, irreversible and iron- radical-mediated damage of parasite biomolecules. In the present research, iron oxide nanoparticle fortified artesunate was tested in P. falciparum and in an experimental malaria mouse model for enhancement in the selectivity and toxicity of artesunate towards parasite. Artesunate was fortified with nontoxic biocompatible surface modified iron oxide nanoparticle which is specially designed and synthesized for the sustained pH-dependent release of Fe2+ within the parasitic food vacuole for enhanced ROS spurt. METHODS:Antimalarial efficacy of Iron oxide nanoparticle fortified artesunate was evaluated in wild type and artemisinin-resistant Plasmodium falciparum (R539T) grown in O + ve human blood and in Plasmodium berghei ANKA infected swiss albino mice. Internalization of nanoparticles, the pH-dependent release of Fe2+, production of reactive oxygen species and parasite biomolecule damage by iron oxide nanoparticle fortified artesunate was studied using various biochemical, biophysical, ultra-structural and fluorescence microscopy. For determining the efficacy of ATA-IONP+ART on resistant parasite ring survival assay was performed. RESULTS:The nanoparticle fortified artesunate was highly efficient in the 1/8th concentration of artesunate IC50 and led to retarded growth of P. falciparum with significant damage to macromolecules mediated via enhanced ROS production. Similarly, preclinical In vivo studies also signified a radical reduction in parasitemia with ~8-10-fold reduced dosage of artesunate when fortified with iron oxide nanoparticles. Importantly, the ATA-IONP combination was efficacious against artemisinin-resistant parasites. INTERPRETATION:Surface coated iron-oxide nanoparticle fortified artesunate can be developed into a potent therapeutic agent towards multidrug-resistant and artemisinin-resistant malaria in humans. FUND: This study is supported by the Centre for Study of Complex Malaria in India funded by the National Institute of Health, USA.
Project description:BACKGROUND:Drug efficacy against kelch 13 mutant malaria parasites can be determined in vitro with the ring-stage survival assay (RSA). The conventional assay protocol reflects the exposure profile of dihydroartemisinin. METHODS:Taking into account that other anti-malarial peroxides, such as the synthetic ozonides OZ439 (artefenomel) and OZ609, have different pharmacokinetics, the RSA was adjusted to the concentration-time profile of these ozonides in humans and a novel, semi-automated readout was introduced. RESULTS:When tested at clinically relevant parameters, it was shown that OZ439 and OZ609 are active against the Plasmodium falciparum clinical isolate Cam3.IR539T. CONCLUSION:If the in vitro RSA does indeed predict the potency of compounds against parasites with increased tolerance to artemisinin and its derivatives, then the herein presented data suggest that following drug-pulses of at least 48 h, OZ439 and OZ609 will be highly potent against kelch 13 mutant isolates, such as P. falciparum Cam3.IR539T.
Project description:The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.
Project description:Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.
Project description:BACKGROUND:The current first line drugs for treating uncomplicated malaria are artemisinin (ART) combination therapies. However, Plasmodium falciparum parasites resistant to ART and partner drugs are spreading, which threatens malaria control efforts. Rodent malaria species are useful models for understanding antimalarial resistance, in particular genetic variants responsible for cross resistance to different compounds. METHODS:The Plasmodium berghei RC strain (PbRC) is described as resistant to different antimalarials, including chloroquine (CQ) and ART. In an attempt to identify the genetic basis for the antimalarial resistance trait in PbRC, its genome was sequenced and compared with five other previously sequenced P. berghei strains. RESULTS:We found that PbRC is eight-fold less sensitive to the ART derivative artesunate than the reference strain PbANKA. The genome of PbRC is markedly different from other strains, and 6,974 single nucleotide variants private to PbRC were identified. Among these PbRC private variants, non-synonymous changes were identified in genes known to modulate antimalarial sensitivity in rodent malaria species, including notably the ubiquitin carboxyl-terminal hydrolase 1 gene. However, no variants were found in some genes with strong evidence of association with ART resistance in P. falciparum such as K13 propeller protein. DISCUSSION:The variants identified in PbRC provide insight into P. berghei genome diversity and genetic factors that could modulate CQ and ART resistance in Plasmodium spp.
Project description:Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED 90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED 90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca 2+/H + antiporter. Conclusions: Selection of amodiaquine resistance yielded stable "multidrug-resistant'' parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.
Project description:BACKGROUND: Early diagnosis and treatment with artesunate-mefloquine combination therapy (MAS) have reduced the transmission of falciparum malaria dramatically and halted the progression of mefloquine resistance in camps for displaced persons along the Thai-Burmese border, an area of low and seasonal transmission of multidrug-resistant Plasmodium falciparum. We extended the same combination drug strategy to all other communities (estimated population 450,000) living in five border districts of Tak province in northwestern Thailand. METHODS AND FINDINGS: Existing health structures were reinforced. Village volunteers were trained to use rapid diagnostic tests and to treat positive cases with MAS. Cases of malaria, hospitalizations, and malaria-related deaths were recorded in the 6 y before, during, and after the Tak Malaria Initiative (TMI) intervention. Cross-sectional surveys were conducted before and during the TMI period. P. falciparum malaria cases fell by 34% (95% confidence interval [CI], 33.5-34.4) and hospitalisations for falciparum malaria fell by 39% (95% CI, 37.0-39.9) during the TMI period, while hospitalisations for P. vivax malaria remained constant. There were 32 deaths attributed to malaria during, and 22 after the TMI, a 51.5% (95% CI, 39.0-63.9) reduction compared to the average of the previous 3 y. Cross-sectional surveys indicated that P. vivax had become the predominant species in Thai villages, but not in populations living on the Myanmar side of the border. In the displaced persons population, where the original deployment took place 7 y before the TMI, the transmission of P. falciparum continued to be suppressed, the incidence of falciparum malaria remained low, and the in vivo efficacy of the 3-d MAS remained high. CONCLUSIONS: In the remote malarious north western border area of Thailand, the early detection of malaria by trained village volunteers, using rapid diagnostic tests and treatment with mefloquine-artesunate was feasible and reduced the morbidity and mortality of multidrug-resistant P. falciparum.
Project description:The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0-3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia.IMPORTANCE ACTs have helped halve the malaria disease burden in recent years; however, emerging resistance to ART derivatives threatens to reverse this substantial progress. Resistance is driven primarily by mutations in the P. falciparum K13 gene. These mutations pose a threat to ozonides, touted as promising alternatives to ARTs that share a similar mode of action. We report that DHA was considerably more potent than OZ439 and OZ277 against ART-sensitive asexual blood-stage parasites cultured in vitro We also document that mutant K13 significantly compromised the activity of the registered drug OZ277. In contrast, OZ439 remained effective against most parasite lines expressing mutant K13, with the exception of I543T that merits further monitoring in field-based OZ439 efficacy studies. K13 mutations differed considerably in their impact on parasite growth rates, in a strain-dependent context, with the most prevalent C580Y mutation being fitness neutral in recently culture-adapted strains from Cambodia, the epicenter of emerging ART resistance.
Project description:BACKGROUND:Humanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen. METHODS:P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2R?-/-immunocompromised mice, with progressive dose increments as parasites recovered. The parasite's response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro. RESULTS:Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of >?3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2-11.2) vs. ART-R30: 11.5 nM (6.6-16.9), F?=?0.525, p?=?0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9-118.4), F?=?304.8, p?<?0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro. CONCLUSION:P. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.
Project description:In vitro drug treatment with artemisinin derivatives, such as dihydroartemisinin (DHA), results in a temporary growth arrest (i.e., dormancy) at an early ring stage in Plasmodium falciparum This response has been proposed to play a role in the recrudescence of P. falciparum infections following monotherapy with artesunate and may contribute to the development of artemisinin resistance in P. falciparum malaria. We demonstrate here that artemether does induce dormant rings, a finding which further supports the class effect of artemisinin derivatives in inducing the temporary growth arrest of P. falciparum parasites. In contrast and similarly to lumefantrine, the novel and fast-acting spiroindolone compound KAE609 does not induce growth arrest at the early ring stage of P. falciparum and prevents the recrudescence of DHA-arrested rings at a low concentration (50 nM). Our findings, together with previous clinical data showing that KAE609 is active against artemisinin-resistant K13 mutant parasites, suggest that KAE609 could be an effective partner drug with a broad range of antimalarials, including artemisinin derivatives, in the treatment of multidrug-resistant P. falciparum malaria.