In vitro activities of and mechanisms of resistance to antifol antimalarial drugs

Abstract

Certain drugs that interfere with folate metabolism (sulfones, sulfonamides, and inhibitors of dihydrofolate reductase) play an important role in the chemotherapy and prophylaxis of malaria. The activities and mechanisms of action of these drugs are regarded as similar in most respects to their activities against procaryotic microorganisms. Believed incapable of utilizing intact exogenous folates, plasmodia have been regarded as dependent on de novo synthesis of required folate cofactors. The present investigation, conducted in pursuit of a method for testing the in vitro susceptibility of Plasmodium falciparum to antifol antimalarial drugs, produced evidence that earlier assumptions about the folate metabolism of this organism are not correct. Three of four isolates of P. falciparum were successfully maintained in a culture medium depleted of folic acid and p-aminobenzoic acid. The antimalarial activities of sulfonamides and dihydrofolate reductase inhibitors were, furthermore, variably antagonized by the presence of folic acid and p-aminobenzoic acid in the culture medium. Optimum conditions for assessment of antifol antimalarial activity in vitro therefore require precise control of these factors in the culture medium. Our results suggest that resistance to antifol antimalarial drugs involves a complex of factors related to both the de novo synthesis of active folate cofactors and the ability to utilize exogenous intact folates in various forms.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO‐1

“…The susceptibility of different malaria strains to the artemisinin derivatives and other control agents were determined using the tritiated hypoxanthine incorporation assay of Desjardins et al (18) and Chulay et al (19), as modified by Milhous et al (20), except that the drug exposure period was 48 h. Four P. falciparum malaria parasites including clones and isolates were utilized for the drug testing. W2 is a clone of the Indochina I/UNC isolate and is resistant to chloroquine and pyrimethamine, but susceptible to mefloquine.…”

Regioselective Synthesis and Biological Evaluation of 1-Hydroxyl Modified Ailanthinone Derivatives as Antimalarials

“…The assays are based on standard biological screens performed at WRAIR. The detailed procedure is described elsewhere [10,11] and is limited to the assessment of the intrinsic activity against plasmodium in the erythrocyte like asexual life cycle (blood schizontocides). In brief, the cultured parasites were incubated with varied concentration of diluted drugs in 96 well microtiter plate ]-hypoxanthine was added and its uptake in parasite was measured after additional incubation of 24 hrs by first lysing and recovering the DNA on glass fiber filter using Packard Filtermate Cell Harvester.…”

Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth.

“…This conclusion has been disputed in more recent studies. The majority of several tested Plasmodium falciparum lines grow at normal rates in cultivation without detectable levels of PABA or folates (10), indicating that the parasites are synthesizing the PABA they require. Also, Dieckmann and Jung (11) have detected four enzymes involved in PABA synthesis in extracts from P. falciparuminfected red blood cells.…”

“…The number of parasites was determined by evaluation of Giemsa-stained smears and calculation of the total number. The growth rates were determined as described by Milhous et al (10). Briefly, the growth rate equals the difference of the natural logarithms of the number of parasites observed at two time points divided by the duration of time (difference in time points).…”

“…Clone 3D7 and the PABA-requiring clones, B5, D11, E6, and E10, were treated with sulfamethoxazole. These samples were processed according to a standard assay for sulfa drugs (10,17). PRBCs with a parasitemia of0.5% (mostly late-stage trophozoites) and a 4% hematocrit were washed repeatedly with PABA-deficient RPMI 1640 medium.…”