Search

Background: Emergence of anti-malarial drug resistance and perpetual increase in malaria incidence necessitates the development of novel anti-malarials. Histone deacetylases (HDAC) has been shown to be a promising target for malaria, despite this, there are no HDAC inhibitors in clinical trials for malaria treatment. This can be attributed to the poor pharmacokinetics, bioavailability and selectivity of the HDAC inhibitors., Methods: A collection of HDAC inhibitors were screened for anti-malarial activity, and the best candidate was profiled in parasite-killing kinetics, growth inhibition of sensitive and multi-drug resistant (MDR) strains and against gametocytes. Absorption, distribution, metabolism and excretion pharmacokinetics (ADME-PK) parameters of FNDR-20123 were determined, and in vivo efficacy was studied in a mouse model for Plasmodium falciparum infection., Results: A compound library of HDAC inhibitors (180 in number) was screened for anti-malarial activity, of which FNDR-20123 was the most potent candidate. The compound had been shown to inhibit Plasmodium HDAC with IC 50 of 31 nM and human HDAC with IC 50 of 3 nM. The IC 50 obtained for P. falciparum in asexual blood-stage assay was 42 nM. When compared to atovaquone and pyrimethamine, the killing profiles of FNDR-20123 were better than atovaquone and comparable to pyrimethamine. The IC 50 values for the growth inhibition of sensitive and MDR strains were similar, indicating that there is no cross-resistance and a low risk of resistance development. The selected compound was also active against gametocytes, indicating a potential for transmission control: IC 50 values being 190 nM for male and > 5 µM for female gametocytes. FNDR-20123 is a stable candidate in human/mouse/rat liver microsomes (> 75% remaining post 2-h incubation), exhibits low plasma protein binding (57% in humans) with no human Ether-à-go-go-Related Gene (hERG) liability (> 100 µM), and does not inhibit any of the cytochrome P450 (CYP) isoforms tested (IC 50  > 25 µM). It also shows negligible cytotoxicity to HepG-2 and THP-1 cell lines. The oral pharmacokinetics in rats at 100 mg/kg body weight shows good exposures (C max  = 1.1 µM) and half-life (T 1/2  = 5.5 h). Furthermore, a 14-day toxicokinetic study at 100 mg/kg daily dose did not show any abnormality in body weight or gross organ pathology. FNDR-20123 is also able to reduce parasitaemia significantly in a mouse model for P. falciparum infection when dosed orally and subcutaneously., Conclusion: FNDR-20123 may be a suitable candidate for the treatment of malaria, which can be further developed.

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.I R539T ., 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.I R539T .

Background: Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.I R539T )., Methods: The previously described in vitro ring-stage survival assay (RSA 0-3h ) was employed and a simplified variation of the original protocol was developed., Results: At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.I R539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA 0-3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides., Conclusion: The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.I R539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.

Background: Recent 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., Methods: The 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., Results: The 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., Conclusion: The 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.

Background: Semi-synthetic artemisinin derivatives are powerful peroxidic drugs in artemisinin-based combination therapy (ACT) recommended as first-line treatment of Plasmodium falciparum malaria in disease-endemic countries. Studies by Eckstein-Ludwig and co-workers showed both thapsigargin and artemisinin specifically inhibit the sarcoplasmic reticulum Ca²⁺-ATPase of Plasmodium falciparum (PfATP6). In the present study the type of interaction between thapsigargin and artemisinin derivatives as well as the ozonide OZ277 (RBx11160 or arterolane) was evaluated in parasite cultures. The latter compound is an adamantane-based peroxide and the first fully synthetic clinical candidate recently registered in India by Ranbaxy Laboratories Ltd. for anti-malarial combination therapy., Methods: Drug interaction studies were performed using a previously described fixed ratio method and anti-malarial activity measured using the [3H] hypoxanthine incorporation assay., Results: The sum 50% and 90% fractional inhibitory concentration (∑FIC₅₀, ₉₀) of the interaction of thapsigargin with OZ277, artemether or artesunate, against NF54 and K1 strains of P. falciparum ranged from 0.9 to 1.4., Conclusion: The interaction of thapsigargin with OZ277, artesunate or artemether was additive, data consistent with previous observations indicating that activity of anti-malarial peroxides does not derive from reversible interactions with parasite targets.

Background: The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The method is highly reproducible, however, the handling of radioactive material is costly, hazardous and requires the availability of appropriate technology and trained staff. Several other ways to evaluate in vitro anti-malarial activity do exist, all with their own assets and limitations., Methods: The newly developed double-antibody sandwich ELISA described here is based on the properties of a non-overlapping pair of monoclonal antibodies directed against Plasmodium falciparum aldolase. This glycolytic enzyme possesses some unique nucleotide sequences compared to the human isoenzymes and has been highly conserved through evolution. Out of twenty possibilities, the most sensitive antibody pair was selected and used to quantitatively detect parasite aldolase in infected blood lysates., Results: A total of 34 compounds with anti-malarial activity were tested side-by-side by ELISA and the [3H]hypoxanthine incorporation assay. The novel ELISA provided IC 50s closely paralleling those from the radioactivity-based assay (R = 0.99, p < 0.001). At the investigated assay conditions (72 h incubation time, parasitaemia = 0.3%), the assay was found to be reproducible and easy to perform., Conclusion: The newly developed ELISA presents several advantages over the comparative method, the [3H]hypoxanthine incorporation assay. The assay is highly reproducible, less hazardous (involves no radioactivity) and requires little and cheap technical equipment. Relatively unskilled personnel can conduct this user-friendly assay. All this makes it attractive to be employed in resource-poor laboratories.