Xavier C. Ding

A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

The antimalarial drug DSM265 displays activity against blood and liver stages of Plasmodium falciparum and has a long predicted half-life in humans. Long-acting new treatment for drug-resistant malaria Malaria kills 0.6 million people annually, yet current malaria drugs are no longer fully effective because the parasite that causes malaria is becoming resistant to these agents. Phillips et al. have identified a new drug that kills both drug-sensitive and drug-resistant malaria parasites by targeting the ability of the parasite to synthesize the nucleotide precursors required for synthesis of DNA and RNA. This drug kills parasites in both the blood and liver and is sufficiently long-acting that it is expected to cure malaria after a single dose or to be effective if dosed weekly for chemoprevention. Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.

P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

A framework for assessing the risk of resistance for anti-malarials in development

Resistance is a constant challenge for anti-infective drug development. Since they kill sensitive organisms, anti-infective agents are bound to exert an evolutionary pressure toward the emergence and spread of resistance mechanisms, if such resistance can arise by stochastic mutation events. New classes of medicines under development must be designed or selected to stay ahead in this vicious circle of resistance control. This involves both circumventing existing resistance mechanisms and selecting molecules which are resilient against the development and spread of resistance. Cell-based screening methods have led to a renaissance of new classes of anti-malarial medicines, offering us the potential to select and modify molecules based on their resistance potential. To that end, a standardized in vitro methodology to assess quantitatively these characteristics in Plasmodium falciparum during the early phases of the drug development process has been developed and is presented here. It allows the identification of anti-malarial compounds with overt resistance risks and the prioritization of the most robust ones. The integration of this strategy in later stages of development, registration, and deployment is also discussed.

Defining the biology component of the drug discovery strategy for malaria eradication

Malaria is still considered a deadly scourge in Africa, Asia, and South America despite improved vector control and curative treatments with new antimalarial combinations. The next challenge is to work towards disease eradication. To achieve this goal it is crucial to develop, validate, and integrate biological assays into test cascades that align with the key target product profiles. For anti-relapse, a parent molecule should kill hypnozoites or cause activation of Plasmodium vivax liver stages. For transmission blocking, dual equal-activity antimalarials killing both the asexual and the sexual parasite stages in human blood are favored. Finally, by assessing cross resistance and generating drug resistance in the laboratory, it is expected that new medicines with acceptable resistance profiles will be forthcoming.

Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum

BackgroundRecent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.MethodsThe approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4–7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.ResultsThe speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.ConclusionThe results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria

Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. Results The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7–100), and 99.7% (95% CI, 99.2–100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1–100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1–100) and specificity was 99.7% (95% CI, 99.2–100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3–99.5) and 100% respectively. Conclusions The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.

Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

ABSTRACT Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

ABSTRACT Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC50s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC50s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.

Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar

Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3–2.4) and 0.7% (95%CI 0.4–1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0–55.8) and the specificity was 99.9% (CI95% 99.8–100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/μL, range 0.2–770) and HTP-LAMP negative (1.4 p/μL, range 0.1–7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon.

Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.