Joel Mosnier

In Vitro Activities of Antibiotics against Plasmodium falciparum Are Inhibited by Iron

ABSTRACT The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated onPlasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity againstP. falciparum, which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl3), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial action and to the fact that they act better after prolonged contact, they probably need to be administered in conjunction with a rapidly acting antimalarial drug, such as a short course of chloroquine or quinine.

Plasmodium falciparum Na+/H+ Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine

ABSTRACT Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC50s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC50 of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC50 of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC50 of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC50 of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC50 of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC50 of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.

Prevalence of In Vitro Resistance to Eleven Standard or New Antimalarial Drugs among Plasmodium falciparum Isolates from Pointe-Noire, Republic of the Congo

ABSTRACT We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria.

In Vitro Activity of Ferroquine Is Independent of Polymorphisms in Transport Protein Genes Implicated in Quinoline Resistance in Plasmodium falciparum

ABSTRACT The in vitro activity of ferroquine (FQ) (SR97193), a 4-aminoquinoline antimalarial compound that contains a ferrocenic nucleus, against 15 Plasmodium falciparum strains was assessed and compared with those of chloroquine (CQ), quinine (QN), monodesethylamodiaquine (MDAQ), and mefloquine (MQ). These 15 strains were genotyped for polymorphisms in quinoline resistance-associated genes such as Pfcrt, Pfmdr1, Pfmrp, and Pfnhe-1. FQ was highly active against CQ-resistant parasites or in parasites with reduced susceptibility to QN, MDAQ, or MQ. Encouragingly, we did not find a correlation between responses to FQ and those to other quinoline drugs. These results suggest that no cross-resistance exits between FQ and CQ or quinoline antimalarial drugs. Mutations in codons 74, 75, 76, 220, 271, 326, 356, and 371 of the Pfcrt gene; codons 86, 184, 1034, 1042, and 1246 of the Pfmdr1 gene; and codons 191 and 437 of the Pfmrp gene were not significantly associated with P. falciparum susceptibility to FQ. Neither the number of ms4760 DNNND or DDNHNDNHNN repeats in Pfnhe-1 nor the profile of ms4760 was significantly associated with the FQ in vitro response. These data suggest the FQ may not interact with transport proteins in quinoline-resistant parasites. The present results justify further clinical trials of FQ in multidrug resistance areas.

Atorvastatin Is 10-Fold More Active In Vitro than Other Statins against Plasmodium falciparum

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, influence a broad array of pathogenic microorganisms. Lovastatin reduces the intracellular growth of Salmonella enterica serovar Typhimurium in cultured macrophages, while atorvastatin does the same in a mouse model (2). Lovastatin additionally reduces the growth of Candida albicans by inhibiting the sterol pathway (11). Statins interfere severely with the growth of protozoan parasites of the Trypanosomatidae family such as Trypanosoma cruzi and various Leishmania species (8, 12, 13). HMG-CoA reductase has been detected in Trypanosoma and Leishmania (3, 8). The presence of an HMG-CoA homolog was not revealed by BLASTX analysis of the Plasmodium falciparum sequence with other protozoal HMG-CoA protein sequences. However, as reported previously, treatment in vitro of Plasmodium falciparum with 120 or 240 μM mevastatin inhibited parasite growth (4, 9). The susceptibilities to simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt (Calbiochem, Merck, Germany), and atorvastatin calcium salt (Molekula, United Kingdom) were assessed in vitro against chloroquine-susceptible P. falciparum strains 3D7 (Africa), D6 (Sierra Leone), and IMT031 (Gabon) and chloroquine-resistant strains W2 (Indochina), Bre1 (Brazil), and FCR3 (The Gambia). Lovastatin and mevastatin were converted to the active form by dissolving the lactone form in 100 μl of 100% ethanol, adding 200 μl of 0.2 M KOH, and then adding 0.2 M HCl for neutralization to pH 7.2 (5). Simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt, and atorvastatin calcium salt were dissolved in dimethyl sulfoxide 1% (vol/vol) in RPMI. Twofold serial dilutions, with final concentrations ranging from 1.5 μM to 200 μM, were prepared in dimethyl sulfoxide 1% in RPMI and distributed into Falcon 96-well plates just before use. The isotopic microdrug susceptibility test used was described previously (10). Table ​Table11 presents the 50% inhibitory concentrations (IC50) of the different statins for P. falciparum. Simvastatin, fluvastatin, lovastatin, and atorvastatin, in the salt active forms, are more active than simvastatin, mevastatin, and lovastatin, in the lactone form. Pravastatin and mevastatin sodium or potassium salts are inactive against P. falciparum (>200 μM). The results indicate that susceptibility to the salts of simvastatin, fluvastatin, lovastatin, and mevastatin is not dependent on the status of chloroquine resistance. The results observed with the simvastatin salt were similar to those reported by other authors (5). Atorvastatin salt, in the range of 5 to 12 μM, is 10-fold more active against P. falciparum than the other salts. Atorvastatin IC90s ranged from 14.8 to 39 μM. The activity of atorvastatin is independent of the status of chloroquine resistance (4.8 to 5.8 μM against chloroquine-resistant strains versus 5.3 to 11.8 μM for the susceptible strains). TABLE 1. In vitro activities of statins against chloroquine-susceptible (3D7, D6, and IMT031) and chloroquine-resistant (W2, Bre1, and FCR3) P. falciparum strains The chemical structures of simvastatin, lovastatin, mevastatin, and pravastatin are closely related. Those of fluvastatin and atorvastatin are very different from the others. The structural differences between atorvastatin and the other statins could explain differential activity. However, we cannot rule out the action of calcium in the differential activity of atorvastatin. Multiple daily doses of 2.5 to 80 mg of atorvastatin produced steady-state maximum plasma concentrations of 1.95 to 252 μg liter−1 (in the range of 0.2 to 0.3 μM for the maximum) (1). In L6 cells (rat skeletal muscle cell line), atorvastatin at 100 μM induced death in 27% of the cells (7). Although the atorvastatin IC50 for P. falciparum exceeds these reported plasma concentrations, it may be below toxic concentrations. Parasites treated with mevastatin show depressed biosynthesis of dolichol and isoprenoid pyrophosphate (4). In addition, mevastatin decreases the viability of cells by inhibiting proteasome activity. Atorvastatin is an inhibitor for phosphoglycoprotein, an efflux protein, and may be a substrate for this transporter as well (6). A phosphoglycoprotein in P. falciparum, Pgh1, is implicated in quinoline resistance. In conclusion, the present observation suggests that atorvastatin is a good candidate for further studies on the use of statins in malaria treatment.

In Vitro Activity of Tafenoquine against the Asexual Blood Stages of Plasmodium falciparum Isolates from Gabon, Senegal, and Djibouti

Antimalarial drugs, when used as monotherapies, are rapidly losing their effectiveness. One promising new drug is the antimalarial 8-aminoquinoline tafenoquine (SB-252263 [formerly WR-238605]), a new synthetic primaquine analogue codeveloped by the U.S. Army and GlaxoSmithKline, which has been shown effective not only against the liver stages, gametocytes, and sporozoites of Plasmodium falciparum (4), but also against the blood stages of the parasite (13). Tafenoquine demonstrated significant protection against P. falciparum infection in Gabon, Ghana, and Kenya (6, 7, 12). Tafenoquine has been reported to be well tolerated, with only mild gastrointestinal effects (8). Isolates were collected in 1999 from malaria patients from Libreville (Gabon, Central Africa), Dielmo and Ndiop (Senegal, West Africa), and Djibouti (East Africa). The isotopic, microdrug susceptibility test used was described previously (10). The 50% inhibitory concentration (IC50) values for tafenoquine were in the range 0.9 to 9.7 μM in Djibouti, 0.6 to 33.1 μM in Gabon, and 0.5 to 20.7 μM in Senegal. The geometric mean IC50 was 2.68 μM in Djibouti, versus 4.62 μM in Gabon and 5.06 μM in Senegal (Table ​(Table1).1). Tafenoquine was found to possess marked blood schizonticidal activity in P. falciparum in areas with high percentages of multidrug-resistant parasite populations. There was no difference in the tafenoquine mean IC50 values between Dielmo-Ndiop and Libreville, even though the levels of reduced susceptibility for chloroquine, mefloquine, cycloguanil, and pyrimethamine were different. Conversely, tafenoquine was significantly more active in Djibouti than in Gabon or Senegal (P = 0.016). The results of these in vitro tests were comparable with those reported by other authors in culture-adaptated P. falciparum clones and strains (2, 11). TABLE 1. In vitro susceptibilities and prevalence of resistance or reduced susceptibility of wild isolates of Plasmodium falciparum from Djibouti, Gabon, and Senegal to the drugs shown Published in vitro data for the blood schizonticidal activity of primaquine in P. falciparum show a range of IC50 values between 0.3 μM and 14 μM (1, 2, 13). In this study, there was no difference in the tafenoquine mean IC50 values between the three areas (P = 0.111). Tafenoquine is more active in vitro than primaquine, wherever the area. Tafenoquine exerts a blood schizonticidal activity 4 to 100 times higher than that of primaquine in the Plasmodium berghei and Plasmodium yoelii mouse model (9). Tafenoquine had a half-life that is more than 50 times longer than that of primaquine (3, 5). The difference in kinetics results in more prolonged, high concentrations of tafenoquine in the blood. These properties permit weekly dosing for prophylaxis and short-term or single-dose therapy for radical cure. Only 3.5% of the variation of response to tafenoquine is explained by response variation to primaquine. The coefficients of determination, r2, ranging from 0.001 to 0.113, are too weak to consider that cross-resistance may exist between tafenoquine and standard antimalarial drugs. Since correlation analysis provides an insight into the mode of action and cross-susceptibilities between different drugs, these data may be seen as an indication of the relative independence of tafenoquine from the susceptibility of P. falciparum to standard antimalarial drugs. In conclusion, these data permit definition of the baseline of in vitro susceptibility to tafenoquine before its use and will allow the monitoring of its resistance or its reduced susceptibility when tafenoquine will be commonly used. Given its greater schizonticidal activity, tafenoquine is a promising candidate as a short treatment for P. falciparum and Plasmodium vivax malaria. However, the potential side effects of tafenoquine, such as the production of methemoglobin and the risk of hemolysis in glucose-6-phosphate dehydrogenase-deficient patients, have to be taken into consideration (14).

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

ObjectiveThe main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine.MethodsThe study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake.ResultsFrom 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2.ConclusionBased on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.

Dihydroethanoanthracene Derivatives as In Vitro Malarial Chloroquine Resistance Reversal Agents

ABSTRACT The ability of four 9,10-dihydroethanoanthracene derivatives (BG920, BG932, BG958, and BG996), as well as verapamil and promethazine, to reverse chloroquine resistance was assessed against 24 chloroquine-resistant and 10 chloroquine-susceptible strains of Plasmodium falciparum from different countries. The 9,10-dihydroethanoanthracene derivatives clearly increase chloroquine susceptibility only in chloroquine-resistant isolates.

Atorvastatin treatment is effective when used in combination with mefloquine in an experimental cerebral malaria murine model

BackgroundOne of the major complications of Plasmodium falciparum infection is cerebral malaria (CM), which causes one million deaths worldwide each year, results in long-term neurological sequelae and the treatment for which is only partially effective. Statins are recognized to have an immunomodulatory action, attenuate sepsis and have a neuroprotective effect. Atorvastatin (AVA) has shown in vitro anti-malarial activity and has improved the activity of mefloquine (MQ) and quinine.MethodsThe efficiency of 40 mg/kg intraperitoneal AVA, alone or in association with MQ, was assessed in an experimental Plasmodium berghei ANKA rodent parasite model of CM and performed according to different therapeutic schemes. The effects on experimental CM were assessed through the evaluation of brain histopathological changes and neuronal apoptosis by TUNEL staining.ResultsAVA alone in the therapeutic scheme show no effect on survival, but the prophylactic scheme employing AVA associated with MQ, rather than MQ alone, led to a significant delay in mouse death and had an effect on the onset of CM symptoms and on the level of parasitaemia. Histopathological findings show a correlation between brain lesions and CM onset. A neuronal anti-apoptotic effect of AVA in the AVA + MQ combination was not shown.ConclusionsThe combination of AVA and MQ therapy led to a significant delay in mouse mortality. There were differences in the incidence, time to cerebral malaria and the level of parasitaemia when the drug combination was administered to mice. When used in combination with MQ, AVA had a relevant effect on the in vivo growth inhibition and clinical outcome of P. berghei ANKA-infected mice.

In Vitro Potentiation of Antibiotic Activities by a Catecholate Iron Chelator against Chloroquine-Resistant Plasmodium falciparum

ABSTRACT FR160, a catechol iron chelator, and tetracyclines or norfloxacin exert in vitro additive or synergistic activity against a chloroquine-resistant Plasmodium falciparum clone. FR160 shows antagonistic effects in association with macrolides, ofloxacin, and rifampin.