Chemoprotective Antimalarial Activity of P218 against Plasmodium falciparum: A Randomized, Placebo-Controlled Volunteer Infection Study.

P218 is a highly selective dihydrofolate reductase inhibitor with potent in vitro activity against pyrimethamine-resistant Plasmodium falciparum. This single-center, randomized, double-blind, placebo-controlled phase Ib study evaluated P218 safety, pharmacokinetics, and chemoprotective efficacy in a P. falciparum sporozoite (PfSPZ) volunteer infection study (VIS). Consecutive dose safety and tolerability were evaluated (cohort 1), with participants receiving two oral doses of P218 1,000 mg 48 hours apart (n = 6), or placebo (n = 2). P218 chemoprotective efficacy was assessed (cohorts 2 and 3) with direct venous inoculation of 3,200 aseptic, cryopreserved PfSPZ (NF54 strain) followed 2 hours later with two P218 doses of 1,000 mg (cohort 2, n = 9) or 100 mg (cohort 3, n = 9) administered 48 hours apart, or placebo (n = 6). Parasitemia was assessed from day 7 using quantitative PCR targeting the var gene acidic terminal sequence (varATS qPCR). By day 28, all participants in cohort 2 (P218 1,000 mg) and 8/9 in cohort 3 (P218 100 mg) were sterilely protected post-PfSPZ VIS, confirming P218 P. falciparum chemoprotective activity. With placebo, all six participants became parasitemic (geometric mean time to positive parasitemia 10.6 days [90% CI: 9.9-11.4]). P218 pharmacokinetics were similar in participants with or without induced infection. Adverse events of any cause occurred in 45.8% (11/24) of participants who received P218 and 50.0% (4/8) following placebo; all were mild/moderate in severity, transient, and self-limiting. There were no clinically relevant changes in laboratory parameters, vital signs, or electrocardiograms. P218 displayed excellent chemoprotective efficacy against P. falciparum with favorable safety and tolerability.

In vitro selection predicts malaria parasite resistance to dihydroorotate dehydrogenase inhibitors in a mouse infection model.

Resistance has developed in Plasmodium malaria parasites to every antimalarial drug in clinical use, prompting the need to characterize the pathways mediating resistance. Here, we report a framework for assessing development of resistance of Plasmodium falciparum to new antimalarial therapeutics. We investigated development of resistance by P. falciparum to the dihydroorotate dehydrogenase (DHODH) inhibitors DSM265 and DSM267 in tissue culture and in a mouse model of P. falciparum infection. We found that resistance to these drugs arose rapidly both in vitro and in vivo. We identified 13 point mutations mediating resistance in the parasite DHODH in vitro that overlapped with the DHODH mutations that arose in the mouse infection model. Mutations in DHODH conferred increased resistance (ranging from 2- to ~400-fold) to DHODH inhibitors in P. falciparum in vitro and in vivo. We further demonstrated that the drug-resistant parasites carrying the C276Y mutation had mitochondrial energetics comparable to the wild-type parasite and also retained their fitness in competitive growth experiments. Our data suggest that in vitro selection of drug-resistant P. falciparum can predict development of resistance in a mouse model of malaria infection.

A Plasmodium berghei sporozoite-based vaccination platform against human malaria.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 µM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.

New molecular settings to support in vivo anti-malarial assays.

BACKGROUND: Quantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods. METHODS: FTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes. RESULTS: The optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed. CONCLUSIONS: This is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.

Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. METHODS: Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. RESULTS: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. CONCLUSION: The quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium-high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.

A Developability-Focused Optimization Approach Allows Identification of in Vivo Fast-Acting Antimalarials: N-[3-[(Benzimidazol-2-yl)amino]propyl]amides.

Malaria continues to be a major global health problem, being particularly devastating in the African population under the age of five. Artemisinin-based combination therapies (ACTs) are the first-line treatment recommended by the WHO to treat Plasmodium falciparum malaria, but clinical resistance against them has already been reported. As a consequence, novel chemotypes are urgently needed. Herein we report a novel, in vivo active, fast-acting antimalarial chemotype based on a benzimidazole core. This discovery is the result of a medicinal chemistry plan focused on improving the developability profile of an antichlamydial chemical class previously reported by our group.

Animal models of efficacy to accelerate drug discovery in malaria.

The emergence of resistance to artemisinins and the renewed efforts to eradicate malaria demand the urgent development of new drugs. In this endeavour, the evaluation of efficacy in animal models is often a go/no go decision assay in drug discovery. This important role relies on the capability of animal models to assess the disposition, toxicology and efficacy of drugs in a single test. Although the relative merits of each efficacy model of malaria as human surrogate have been extensively discussed, there are no critical analyses on the use of such models in current drug discovery. In this article, we intend to analyse how efficacy models are used to discover new antimalarial drugs. Our analysis indicates that testing drug efficacy is often the last assay in each discovery stage and the experimental designs utilized are not optimized to expedite decision-making and inform clinical development. In light of this analysis, we propose new ways to accelerate drug discovery using efficacy models.

A new in vivo screening paradigm to accelerate antimalarial drug discovery.

The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.

Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgammanull mice engrafted with human erythrocytes.

Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2Rgamma(null) mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid beta2microglobulin(null) mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc1.

Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.

Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (> or =0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD(scidbeta2m-/-) engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparum-infected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation.