Similar trends of pyrimethamine resistance-associated mutations in Plasmodium vivax and P. falciparum

The antifolate drugs sulfadoxine and pyrimethamine are commonly used to treat Plasmodium falciparum malaria. However, they can also affect the Plasmodium vivax parasite if it coexists with P. falciparum, as both species have common drug targets. Resistance to the antifolate drugs arises due to point mutations in the target enzymes of the respective parasite. To assess the cross-species impact of antifolate drug treatment, we describe here the dihydrofolate reductase (DHFR) mutations among field isolates of P. vivax and P. falciparum. The overall DHFR mutation rate for P. vivax was lower than that for P. falciparum. However, both species of Plasmodium followed similar trends of DHFR mutations. Similar to P. falciparum, the DHFR mutation rate of P. vivax also varied from region to region. It was lower in P. vivax-dominant regions but higher in the P. falciparum-dominated areas and highest where antifolates are used as the first line of antimalarial treatment. In conclusion, the antifolate treatment of falciparum malaria is proportionately affecting the DHFR mutations of P. vivax, suggesting that the drug should be used with caution to minimize the development of cross-species resistance in the field.     

Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae

Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).     

In Vitro Activity of Pyronaridine against Multidrug-Resistant Plasmodium falciparum and Plasmodium vivax

Pyronaridine, a Mannich base antimalarial, has demonstrated high in vivo and in vitro efficacy against chloroquine-resistant Plasmodium falciparum. Although this drug has the potential to become a prominent artemisinin combination therapy, little is known about its efficacy against drug-resistant Plasmodium vivax. The in vitro antimalarial susceptibility of pyronaridine was assessed in multidrug-resistant P. vivax (n = 99) and P. falciparum (n = 90) isolates from Papua, Indonesia, using a schizont maturation assay. The median 50% inhibitory concentration (IC₅₀) of pyronaridine was 1.92 nM (range, 0.24 to 13.8 nM) against P. falciparum and 2.58 nM (range, 0.13 to 43.6 nM) against P. vivax, with in vitro susceptibility correlating significantly with chloroquine, amodiaquine, and piperaquine (r_s [Spearman''s rank correlation coefficient] = 0.45 to 0.62; P < 0.001). P. falciparum parasites initially at trophozoite stage had higher IC₅₀s of pyronaridine than those exposed at the ring stage (8.9 nM [range, 0.6 to 8.9 nM] versus 1.6 nM [range, 0.6 to 8.9 nM], respectively; P = 0.015), although this did not reach significance for P. vivax (4.7 nM [range, 1.4 to 18.7 nM] versus 2.5 nM [range, 1.4 to 15.6 nM], respectively; P = 0.085). The excellent in vitro efficacy of pyronaridine against both chloroquine-resistant P. vivax and P. falciparum highlights the suitability of the drug as a novel partner for artemisinin-based combination therapy in regions where the two species are coendemic.Almost 40% of the world''s population is at risk for infection by Plasmodium vivax, with an estimated 132 to 391 million clinical infections each year (19). Although chloroquine (CQ) remains the treatment of choice in most of the P. vivax-endemic world, this status is now being undermined by the emergence and spread of chloroquine-resistant (CQR) P. vivax. First reported in the 1980s on the island of New Guinea (2, 23), CQR P. vivax has since spread to other parts of Asia and recently to South America (1). In Papua, Indonesia, CQ resistance in P. vivax has reached levels precluding the use of CQ in most of the province (22, 30). There is an urgency to assess the efficacies of alternative antimalarial agents against drug-resistant P. vivax and to develop new strategies to combat the parasite.Pyronaridine (Pyr), a Mannich base synthesized in China in the 1970s (3, 16), is being developed as a novel antimalarial for multidrug-resistant malaria. It demonstrates potent in vitro activity against erythrocytic stages of Plasmodium falciparum (8, 24, 26, 36), retaining efficacy against CQR isolates (12, 17, 18). Clinical trials have shown excellent efficacy of monotherapy against multidrug-resistant falciparum malaria (14, 24, 25), with the early therapeutic response faster when combined with artesunate (20). Phase III studies with a coformulation of Pyramax (Shin Poong Pharmaceuticals) containing artesunate plus pyronaridine have recently been completed (34).Less is known of the antimalarial properties of pyronaridine against P. vivax, although early clinical studies in China demonstrated a rapid therapeutic response (3). To investigate the activity of pyronaridine against CQR P. vivax, we applied a modified schizont maturation assay on fresh field isolates from Papua, Indonesia, where CQR P. vivax is highly prevalent.     

In vitro activities of novel catecholate siderophores against Plasmodium falciparum.

The activities of novel iron chelators, alone and in combination with chloroquine, quinine, or artemether, were evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum with a semimicroassay system. N4-nonyl,N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound 7) demonstrated the highest level of activity: 170 nM against a chloroquine-susceptible clone and 1 microM against a chloroquine-resistant clone (50% inhibitory concentrations). Compounds 6, 8, and 10 showed antimalarial activity with 50% inhibitory concentrations of about 1 microM. Compound 7 had no effect on the activities of chloroquine, quinine, and artemether against either clone, and compound 8 did not enhance the schizontocidal action of either chloroquine or quinine against the chloroquine-resistant clone. The incubation of compound 7 with FeCI3 suppressed or decreased the in vitro antimalarial activity of compound 7, while no effect was observed with incubation of compound 7 with CuSO4 and ZnSO4. These results suggest that iron deprivation may be the main mechanism of action of compound 7 against the malarial parasites. Chelator compounds 7 and 8 primarily affected trophozoite stages, probably by influencing the activity of ribonucleotide reductase, and thus inhibiting DNA synthesis.     

In vitro activities of furoquinoline and acridone alkaloids against Plasmodium falciparum.

The in vitro activities of furo[2,3b]quinoline and acridone alkaloids against Plasmodium falciparum were evaluated by an isotopic semimicrotest. A pyran ring in the furoquinoline nucleus and 2-O-pyranoglycoside and 2-nitro substituents in the acridone nucleus improved the antimalarial activities of the compounds. These findings provide a clue for further chemical modifications.     

In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron

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 on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P. falciparum, which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl(3)), 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.     

In vitro activities of 2,4-diaminoquinazoline and 2,4-diaminopteridine derivatives against Plasmodium falciparum

The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested. The 50% inhibitory concentrations (IC(50)s) of six compounds were <50 nM, and the most potent compound was 2,4-diamino-5-chloro-6-[N-(2,5-dimethoxybenzyl)amino]quinazoline (compound 1), with an IC(50) of 9 nM. The activity of compound 1 was potentiated by the dihydropteroate synthase inhibitor dapsone, an indication that these compounds are inhibitors of dihydrofolate reductase. Further studies are warranted to assess the therapeutic potential of this combination in vivo.     

Differential in vitro activities of ionophore compounds against Plasmodium falciparum and mammalian cells.

Twenty-two ionophore compounds were screened for their antimalarial activities. They consisted of true ionophores (mobile carriers) and channel-forming quasi-ionophores with different ionic specificities. Eleven of the compounds were found to be extremely efficient inhibitors of Plasmodium falciparum growth in vitro, with 50% inhibitory concentrations of less than 10 ng/ml. Gramicidin D was the most active compound tested, with 50% inhibitory concentration of 0.035 ng/ml. Compounds with identical ionic specificities generally had similar levels of antimalarial activity, and ionophores specific to monovalent cations were the most active. Compounds were further tested to determine their in vitro toxicities against mammalian lymphoblast and macrophage cell lines. Nine of the 22 compounds, i.e., alborixin, lonomycin, nigericin, narasin, monensin and its methylated derivative, lasalocid and its bromo derivative, and gramicidin D, most specific to monovalent cations, were at least 35-fold more active in vitro against P. falciparum than against the two other mammalian cell lines. The enhanced ability to penetrate the erythrocyte membrane after infection could be a factor that determines ionophore selectivity for infected erythrocytes.     

The presence of leukocytes in ex vivo assays significantly increases the 50-percent inhibitory concentrations of artesunate and chloroquine against Plasmodium vivax and Plasmodium falciparum

Plasmodium species ex vivo sensitivity assay protocols differ in the requirement for leukocyte removal before culturing. This study shows that the presence of leukocytes significantly increases the 50% inhibitory concentration (IC₅₀) of P. vivax and P. falciparum to artesunate and chloroquine relative to results with the paired leukocyte-free treatment. Although leukocyte removal is not an essential requirement for the conduct of ex vivo assays, its use has important implications for the interpretation of temporal and spatial antimalarial sensitivity data.Plasmodium falciparum and Plasmodium vivax ex vivo sensitivity assays are an important adjunct to in vivo antimalarial resistance detection. Ex vivo antimalarial sensitivity testing (a specialized form of in vitro sensitivity testing) involves the phenotypic comparison of freshly isolated Plasmodium spp. cultured to a specified endpoint in the presence and absence of antimalarials. Ex vivo assays provide information on the intrinsic sensitivity of malaria parasites, free from confounding variations in patient immune status. Furthermore, in the case of P. vivax, ex vivo assays are also free from the problematic differentiation between relapse, reinfection, or recrudescence which is needed for in vivo testing.Since the development of ex vivo sensitivity antimalarial assays in the 1960s (20), there have been various modifications to the basic method, including changes in the blood medium mixture (BMM) composition (variations in hematocrit, culture medium used, and percent serum added), BMM volume (i.e., 50 μl versus 200 μl), and even incubation gas environment (candle jar versus commercial gas). Another important modification to ex vivo sensitivity antimalarial assays is the depletion of leukocytes from the patient blood sample prior to addition to the culture medium (26). It was supposed that the depletion of patient leukocytes and platelets removes the possibility that variations in host immune status (i.e., variations in leukocyte count) will confound the result of the sensitivity assay. Additionally, the removal of leukocytes aids in the microscopic examination of thick films which are used to assess the antimalarial effect on parasite development (26). Furthermore, thick and thin films made from filtered isolates produce “noise-free” images better suited for digital analysis. Despite the growing trend toward complete leukocyte depletion prior to culture (1, 8, 11, 13, 14, 17, 21-23, 25, 27), some groups still utilize patient samples with no or only partial depletion of leukocytes (removal of the buffy coat only) (5, 6, 15, 16, 28). Despite this major dichotomy in ex vivo assay methodology, it is not known if the removal of leukocytes influences the antimalarial sensitivity of the parasite or the ex vivo growth of P. vivax or P. falciparum. Consequently, the main objective of this study was to investigate whether the presence of leukocytes affects the IC₅₀ (50% inhibitory concentration) of chloroquine and artesunate against P. vivax and P. falciparum. It was also important to determine if chloroquine and artesunate significantly affected leukocyte phagocytosis (parasite removal), which, if it occurred in a dose-dependent manner, would confound the assay test results. For example, if higher concentrations of antimalarial inhibited phagocytosis, the parasitemia at lower concentrations would be reduced relative to that with the higher-concentration treatments, counter to the presumed effect of the antimalarial.This study focuses on a modified version of the original WHO schizont maturation microtest (19), as it remains the most reliable and commonly used methodology for the side-by-side antimalarial sensitivity testing of P. vivax and P. falciparum in field locations (11, 13, 14, 17, 21, 24). Consequently, the methodology used for this study is based on that of Russell et al. (22), the only major change being the use of a gas mix (N₂, 90%; O₂, 5%; and CO₂, 5%) rather than a candle jar. Fifteen P. vivax and 10 P. falciparum isolates were collected from malaria patients attending the clinics of the Shoklo Malaria Research Unit (SMRU) Mae Sod region of Tak Province in the northwest of Thailand from October 2008 to January 2010. Isolates were collected only from patients with no prior antimalarial therapy and with a microscopically determined parasitemia of <10,000 parasites/μl. After written consent was obtained, blood samples were collected by venipuncture in 5-ml-volume lithium-heparin tubes and arrived at the culture lab at SMRU within 5 h of collection. An additional 1 ml of blood collected on EDTA anticoagulant was taken from each patient for automated hematology analysis (model pocH-100i; Sysmex Company). Only samples with >90% of the parasites at the early ring stage (approximately 6 to 8 h postinvasion) were chosen for drug sensitivity testing. Due to this strict criterion, only 8/15 and 8/10 P. vivax and P. falciparum isolates, respectively, were included in the study. All of the patient samples used in this study had a normal white blood cell count with minimal variation around the median of 5.6 × 10³ leukocytes/μl (interquartile range [IQR], 4.5 to 6.8 leukocytes/μl) observed. Each of the chosen samples was divided into two, and half of the sample, referred to as the “leukocyte removed” treatment, was depleted of platelets and leukocytes by CF11 filtration (26). It should be noted that the CF11 filtration methodology does not alter the stage composition or viability of the isolates (24). The remaining half of each sample was not filtered, and this was referred to as the “leukocytes present” treatment. Parasites in these two treatments were tested in parallel against chloroquine diphosphate (CQ) (molecular weight [MW], 515.9; Sigma-Aldrich) and artesunate (AS) (base MW, 282.3; Holly Pharmaceuticals Co Ltd.), as previously described (22). Artesunate was used in preference to its metabolite, dihydroartemisin (DHA), as AS is more commonly used in ex vivo studies and is significantly more stable on predosed dried drug plates (9). All of the microscopy work was carried out by a skilled microscopist, and quality assurance was done by an expert microscopist (with 15 years of experience in field microscopy). It was not possible to blind the microscopic reads, since the thick films of the “leukocytes present” treatment are very distinct (26).The clinical samples examined in this study were collected under the ethical guidelines in the approved protocol OXTREC 027-025 (University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, United Kingdom).Dose-response curves and IC₅₀s were calculated by fitting the data to a sigmoidal inhibitory E_max (maximum effect) pharmacodynamic model using the WinNonlin v. 4.1 (Pharsight Corporation, CA) software program using duplicate well data for each drug concentration. The median IC₅₀s presented in Fig. ​Fig.11 A and 1B and ​and22 B were compared using a Wilcoxon paired-rank test. The median IC₅₀s presented in Fig. 2C and D were compared using a Kruskal-Wallis test and Dunn''s post hoc analysis. Statistical analysis was carried out and graphics produced using the GraphPad Prism v. 5 software program.Open in a separate windowFIG. 1.The effect of leukocyte depletion on the ex vivo sensitivity (solid horizontal lines show median IC₅₀ [nM]) of paired isolates of Plasmodium vivax (n = 8) and Plasmodium falciparum (n = 8) to artesunate (A) or chloroquine (B). Solid symbols indicate the absence of leukocytes.Open in a separate windowFIG. 2.Neutrophil and monocyte phagocytosis of Plasmodium vivax (A) and Plasmodium falciparum significantly reduce the parasitemia in ex vivo cultures over a period of 42 h (B). The presence of artesunate (C) or chloroquine (D) does not significantly affect the ability of leukocytes to phagocytose infected red blood cells or hemozoin relative to results with the drug-free control (phagocytic index). In the image of a Giemsa-stained thin film (A), examples of a neutrophil (N), monocyte (MΦ), phagocytosed infected red blood cell (i), and hemozoin clump (p) are marked on the photomicrograph.In all cases tested, the presence of leukocytes ex vivo increased the antimalarial IC₅₀ relative to that for the paired leukocyte-free treatment (Fig. ​(Fig.1).1). We suggest that the increase in IC₅₀s is simply due to leukocytes reducing the total concentration of antimalarial agent free to act on the parasitized red blood cells (i.e., the leukocytes act as a “biological sink” or “bio-sponge” for the antimalarials). Chloroquine is preferentially accumulated into leukocytes, such as neutrophils (which make up the majority of leukocytes present in our isolates) and monocytes (7, 18). However, it is not known if artesunate is preferentially accumulated into or neutralized by leukocytes. Interestingly, an earlier in vitro study has also shown how the presence of certain cell types with a preferential uptake of artemisinin competes with parasitized erythrocytes for the accumulation of antimalarials (10). In this case study, the presence of α-thalassemic erythrocytes, which have a higher accumulation capacity than infected wild-type red cells, resulted in lower artemisinin concentrations available to kill the parasites and a subsequent increase in artemisinin IC₅₀s. Although we suspect that leukocytes are also reducing the availability of artesunate in our study, we cannot discount the alternative possibility, that leukocytes directly increase the actual susceptibility of the parasite to the antimalarials added.It is notable that although the chloroquine IC₅₀ of P. falciparum was increased in the “leukocyte present” treatment, this change did not reach statistical significance. This is probably due to the fact that chloroquine has little effect on these highly resistant P. falciparum strains. Plasmodium falciparum in this region of Thailand is well known to be highly resistant to chloroquine, where a 300 nM concentration of this antimalarial is not sufficient to inhibit schizont maturation. Therefore, it is less likely that we would be able to detect much of an increase in IC₅₀s over those observed in this study (Fig. ​(Fig.1).1). In future studies, it would be useful to repeat this experiment with chloroquine-sensitive isolates of P. falciparum, which are extremely rare in the study area.To examine the effect of antimalarials on leukocyte phagocytosis over 42 h of culture (“leukocytes present” treatments only), a randomly selected sample of 100 leukocytes (consisting of neutrophils, lymphocytes, monocytes, eosinophils, and basophils) in the thin films of each species and drug concentration was examined by light microscopy under 100× oil emersion. The phagocytic index was defined as the percentage of leukocytes containing at least one parasite or obvious granule of hemozoin normalized to that of the drug-free control (Fig. ​(Fig.2).2). The parasitemia of each drug-free control was determined before and after culture for 42 h.Prior to the start of our study, only 0 to 1.3% (range) of the leukocytes contained any detectable hemozoin or parasites. However, after 42 h of drug-free culture, a median (range) of 38.2% (13.9 to 71.0%) or 38.9% (3.8 to 93.1%) of leukocytes contained P. vivax or P. falciparum, respectively. This resulted in a significant drop in parasitemias of both species over the 42-h incubation (Fig. ​(Fig.2).2). It is notable that in addition to free merozoites, entire infected red blood cells (IRBCs) were found in the neutrophils and monocytes postculture (Fig. ​(Fig.2).2). These findings contradict early reports suggesting that only merozoites are readily phagocytosed by granulocytes and that infected red blood cells are relatively protected from phagocytosis (except if damaged) (2). Although we did not observe IRBCs or merozoites in lymphocytes, it was relatively common to find significant quantities of hemozoin phagocytosed by this cell type. There was no indication that the development of ring stages to healthy schizonts was impeded by the presence of leukocytes; there was only a reduction in overall parasitemia. These results support findings of earlier studies showing no marked inhibition of schizont maturation with peripheral blood mononuclear cells or hyperimmune serum (3, 4). Importantly, the concentrations of chloroquine and artesunate used did not significantly affect the phagocytic index (Fig. ​(Fig.2).2). Although some studies have found that chloroquine and artesunate significantly reduced the phagocytic function of granulocytes, it should be noted that these effects were noted only at concentrations of drug above 100,000 nM chloroquine (12) and 5,000 nM artesunate (29), both of which are well in excess of concentrations used in our study and in vivo therapeutic ranges for the treatment of malaria.The results of our study have important implications for ex vivo sensitivity assays. First, the presence of leukocytes can, at least for chloroquine, significantly confound the sensitivity profile of P. vivax and P. falciparum in ex vivo antimalarial assays by reducing the amount of antimalarial free to act on the parasites. If leukocytes are not removed, sample IC₅₀s may be higher than normally expected, especially in patient samples with particularly high leukocyte counts. Furthermore, the presence of leukocytes prior to sensitivity testing will result in significantly lower parasitemias postculture, making it more difficult to observe the antimalarial effect; this is of particular concern with P. vivax isolates, where parasitemias may be close to the microscopic threshold of detection (10 to 50 parasites/μl).The intention of this article is not to advocate the sole use of protocols using leukocyte depletion for ex vivo studies on P. vivax and P. falciparum. In fact, it would be advisable to retain host leukocytes for studies trying to correlate ex vivo sensitivity with clinical therapeutic response. However, we do wish to draw attention to a major dichotomy in ex vivo sensitivity protocols that if not taken into account may cause confusion when trying to compare spatial and temporal trends in Plasmodium sp. sensitivity profiles. The results of this study are particularly relevant, since there is a trend to move away from the microtest to more automated methods which utilize a differential increase in DNA (stained using dyes such as SYBR green or ethidium bromide) as a measure for parasite growth or inhibition; we predict that the use of leukocyte-depleted isolates (in an effort to remove background signal) will become more frequent.     

Comparison of artesunate and chloroquine activities against Plasmodium vivax gametocytes

The gametocidal activities of chloroquine and artesunate were compared. The relative risk (RR) of having detectable gametocytes appear after treatment initiation was lower in artesunate-treated patients (n = 792) than in chloroquine-treated patients (n = 695) (RR = 0.29; 95% CI = 0.2 to 0.40; P < 0.0001). The duration and magnitude of gametocyte carriage were also lower for artesunate than chloroquine. By reducing the transmission of Plasmodium vivax to the vector, artesunate could therefore reduce the incidence of P. vivax malaria.     

Potentiating effect of pyrimethamine and sulfadoxine against dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi.

Dihydrofolate reductase was partially purified from a pyrimethamine-sensitive Plasmodium chabaudi clone and a pyrimethamine-resistant clone derived from it and used in a study of the inhibitory effect of pyrimethamine and sulfadoxine, both alone and in combination. Kinetic analysis of the inhibitory effect of sulfadoxine against the enzyme from pyrimethamine-sensitive and -resistant parasites revealed that the drug inhibited the former enzyme competitively, with an inhibition constant (Kis) of 0.7 +/- 0.4 mM, but inhibited the latter enzyme noncompetitively, with Kis and Kii of 8.9 +/- 1.2 and 4.1 +/- 1.2 mM, respectively. Previous studies also showed competitive inhibition by pyrimethamine on the former enzyme and noncompetitive inhibition on the latter enzyme, with some 200-fold-lower affinity. Sulfadoxine and pyrimethamine exhibited a mutually potentiating effect on the enzyme activity, as revealed by the concave isoboles and the fractional inhibitions of less than unity. A potentiating effect was observed for the enzymes from both sources and was not dependent on the degree of the purification of the enzyme. Our results can be explained by assuming simultaneous binding of two inhibitors on the enzyme.     

Baseline in vitro activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya

We have analyzed the in vitro activities of pyronaridine and methylene blue against 59 Plasmodium falciparum isolates from Kenya in association with polymorphisms in Pfcrt (codon 76), Pfmdr1 (codon 86), and Pfnhe (full sequence). The median inhibitory concentrations that kill 50% of parasites were 13.5 and 3.3 nM for pyronaridine and methylene blue, respectively. Their activities were not associated with polymorphisms in these genes. The drugs' high in vitro activities indicate that they would be efficacious against Kenyan isolates in vivo.     

Synergism of desbutyl-benflumetol and retinol against Plasmodium falciparum in vitro

This in vitro study was conducted to assess the blood schizontocidal activity of desbutyl-benflumetol (DBB), a new benzindene derivative, retinol and a combination of both compounds. The tests were carried out according to the methodology of the WHO standard test Mark II, measuring the drug-dependent inhibition of schizont maturation, and using 43 fresh isolates of Plasmodium falciparum from northwestern Thailand, an area with established multidrug-resistance. DBB and retinol showed a mean 50% effective concentration (EC-50) of 3.73 nM and 466.86 nM and 90% effective concentration (EC-90) of 19.83 nM and 5531.69 nM respectively. The combination of DBB and 3.50 muM retinol showed strong inhibition of schizont maturation, with an EC-90 for DBB of 0.67 nM. At the therapeutically relevant EC-99, the combination was about 10 times more effective than expected, suggesting that retinol potentiated the effect of DBB. A concentration of 3.50 muM retinol corresponds to the 95th percentile of the physiological serum levels. It is well known that retinol levels are significantly decreased in acute falciparum malaria. Supplementation with retinol during malaria treatment may improve the therapeutic results of blood schizontocides of the fluorene class.     

In vitro activities of piperaquine and other 4-aminoquinolines against clinical isolates of Plasmodium falciparum in Cameroon

The spread of chloroquine-resistant Plasmodium falciparum calls for a constant search for new drugs. The in vitro activity of piperaquine, a new Chinese synthetic drug belonging to the bisquinolines, was evaluated in 103 fresh clinical isolates of P. falciparum in Cameroon, Central Africa, and compared with that of other 4-aminoquinoline and Mannich base derivatives and dihydroartemisinin. Piperaquine was highly active (geometric mean 50% inhibitory concentration, 38.9 nmol/liter; range, 7.76 to 78.3 nmol/liter) and equally active (P > 0.05) against the chloroquine-sensitive and the chloroquine-resistant isolates. There was a significant but low correlation of response between chloroquine and piperaquine (r = 0.257, P < 0.05). These results suggest that further development of piperaquine, in combination with dihydroartemisinin, holds promise for use in chloroquine-resistant regions of endemicity.     

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs.

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

Mode of action of tubulozoles against Plasmodium falciparum in vitro

The mode of action of the tubulozole isomers, recently recognized as a new class of potential antimalarial agents, was investigated. Whereas neither glycolysis, protease activity, or nucleic acid biosynthesis was primarily affected, protein biosynthesis decreased soon after addition of the drug. Inhibitors of protein biosynthesis, however, did not show synergistic activity with tubulozole. Colcemid, on the other hand, had an effect on protein synthesis similar to that seen with the tubulozoles. Furthermore, combinations of the tubulozole isomers with compounds known to interact with tubulin inhibited malaria in a synergistic or antagonistic fashion. Therefore, the inhibition might be elicited by interaction with tubulin or some other component of the microtubules. This is remarkable insofar as only one of the tubulozole isomers affects mammalian cells by binding to tubulin.     

Activity of fluoroquinolone antibiotics against Plasmodium falciparum in vitro.

The fluoroquinolone antibiotics are structurally related to nalidixic acid. Their primary antibacterial action appears to be mainly due to inhibition of DNA gyrase (DNA topoisomerase II). We determined the activity of several fluoroquinolones in vitro against two strains of Plasmodium falciparum, FCC1 (chloroquine susceptible) and VNS (chloroquine resistant). [3H]hypoxanthine incorporation by malarial parasites was determined at 48 and 96 h. The molarity at which each agent caused a 50% decrease in the incorporation of [3H]hypoxanthine compared with that of drug-free controls was defined as the 50% inhibitory concentration. The fluoroquinolones evaluated were amifloxacin, ciprofloxacin, enoxacin, norfloxacin, ofloxacin, and pefloxacin. Other DNA gyrase inhibitors tested were nalidixic acid, oxolinic acid, novobiocin, and coumermycin A1. Among the fluoroquinolones, ciprofloxacin had the lowest 50% inhibitory concentrations at 48 h against both chloroquine-susceptible and -resistant strains of P. falciparum, (0.26 +/- 0.08) x 10(-4) and (0.38 +/- 0.15) x 10(-4) M, respectively (mean +/- standard deviation). Enoxacin had the lowest 50% inhibitory concentrations against FCC1 and VNS at 96 h, 0.23 x 10(-5) and (0.06 +/- 0.04) x 10(-5) M, respectively. With the VNS strain, fractional inhibitory concentration indexes for the combination of ciprofloxacin and tetracycline were calculated at 48 and 96 h to be 0.93 and 0.79, respectively, indicating modest additive effects. The combination of novobiocin with ciprofloxacin showed indifference in the same system. The antimalarial effects of some fluoroquinolones occur at achievable serum concentrations. Whether inhibition of DNA gyrase contributes to the antimalarial activity of the fluoroquinolones is unknown at present.     

Potent Ex Vivo Activity of Naphthoquine and Methylene Blue against Drug-Resistant Clinical Isolates of Plasmodium falciparum and Plasmodium vivax

The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC₅₀]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC₅₀s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic.     

In vitro activities of chloroquine in combination with chlorpromazine or prochlorperazine against isolates of Plasmodium falciparum.

The combinations of chloroquine plus chlorpromazine and chloroquine plus prochlorperazine were evaluated in vitro for potentiation against isolates of Plasmodium falciparum. Potentiation was observed against all chloroquine-resistant isolates. There was no potentiation against 8 of 10 chloroquine-susceptible isolates. The results indicated that the phenothiazines can potentiate chloroquine action against resistant parasites found in natural conditions.     

In vitro activities of DU-1102, a new trioxaquine derivative, against Plasmodium falciparum isolates

The antimalarial trioxaquine derivative DU-1102, synthesized by covalent linkage between aminoquinoline and trioxane moieties, was highly active against Cameroonian isolates (mean 50% inhibitory concentration of 43 nmol/liter) of Plasmodium falciparum. There was no correlation between the responses to DU-1102 and chloroquine and only a low correlation between the responses to DU-1102 and pyrimethamine, suggesting an independent mode of action of the trioxaquine against the parasites.