In Vitro Sensitivities of Plasmodium falciparum to Different Antimalarial Drugs in Uganda

The control of malaria is challenged by resistance of Plasmodium falciparum to multiple drugs. New combination regimens are now advocated for the treatment of uncomplicated falciparum malaria, but the extent of resistance to newer agents is incompletely understood. We measured the in vitro sensitivity of P. falciparum parasites cultured from children enrolled in a drug efficacy trial in Kampala, Uganda, from 2006 to 2008. Sensitivities were measured by comparing levels of histidine-rich protein-2 in parasites incubated with different concentrations of drugs with those in untreated controls. The cultured parasites exhibited a wide range of sensitivities to chloroquine (CQ); monodesethylamodiaquine (MDAQ), the major active metabolite of amodiaquine; and quinine (QN). Mean 50% inhibitory concentration (IC₅₀) results were above standard cutoffs for resistance for CQ and MDAQ. Parasites were generally sensitive to dihydroartemisinin (DHA), lumefantrine (LM), and piperaquine (PQ). For CQ, MDAQ, and QN but not the other drugs, activities against individual strains were highly correlated. We also assessed known resistance-mediating polymorphisms in two putative transporters, pfcrt and pfmdr1. When parasites that were least and most sensitive to each drug were compared, the pfmdr1 86Y mutation was significantly more common in parasites that were most resistant to CQ and MDAQ, and the pfmdr1 D1246Y mutation was significantly more common in parasites that were most resistant to MDAQ and QN. In summary, we demonstrated in parasites from Kampala a range of sensitivities to older drugs; correlation of sensitivities to CQ, MDAQ, and QN; and good activity against nearly all strains for DHA, LM, and PQ.Resistance of Plasmodium falciparum to available drugs remains a major challenge to the control of malaria. Older drugs, including the aminoquinolines chloroquine (CQ) and amodiaquine (AQ) and the antifolate sulfadoxine/pyrimethamine (SP), are already seriously compromised, with unacceptable levels of treatment failure in most of Africa (61). In the setting of increasing drug resistance, the WHO has recommended artemisinin-based combination therapy (ACT) for the treatment of uncomplicated falciparum malaria (42). The commonly used ACTs in Africa are artemether/lumefantrine (AM/LM), artesunate/amodiaquine (AS/AQ), and dihydroartemisinin/piperaquine (DHA/PQ), each containing an artemisinin combined with a longer-acting drug. These ACTs have shown excellent efficacy for the treatment of malaria in Africa. However, there is concern that heavy use of ACTs will offer strong selective pressure for parasites with diminished sensitivity to the drugs. This development may seriously jeopardize the efficacy of ACTs.Multiple recent studies in Africa have demonstrated excellent efficacy of AM/LM, AS/AQ, and DHA/PQ for the treatment of falciparum malaria (8, 18, 29, 30, 34, 46, 63, 64). Clinical trials provide our primary means of assessing antimalarial drug efficacy, but they offer only an indirect measure of the sensitivity of parasites to drugs because outcomes can be affected by multiple factors independent of drug sensitivity, including compliance with treatment regimens, drug absorption, pharmacokinetics, antimalarial immunity, and human genetic polymorphisms. In addition, clinical trials are not a sensitive means of identifying early selection of parasites with diminished drug sensitivity, as moderate decreases in sensitivity may have limited impact on clinical outcomes.The sensitivity of malaria parasites to drugs can be evaluated directly using parasites cultured in vitro. Systems for the culture of P. falciparum are well established, although the adaptation of parasites from an active infection to culture remains somewhat problematic. Thus, information on the in vitro drug sensitivity of cultured parasites is limited. Available studies have shown a wide range of sensitivities to older drugs. Parasites with diminished sensitivity to CQ, AQ, and SP are commonly seen (61). For newer drugs, including the active artemisinin metabolite DHA and the ACT partner drugs LM and PQ, African studies have suggested good sensitivity of most parasites (4, 6, 10, 48, 49). However, there is reason for concern that increasing use of ACTs and monotherapies may select for parasites with resistance to important newer agents. First, one study demonstrated parasites with markedly diminished sensitivity to artemether from French Guiana and Senegal (28). Second, recent data from Cambodia have shown diminished responsiveness to artesunate and prolonged parasite clearance times, suggesting the emergence of parasites with diminished sensitivity to artemisinins (15, 39, 54). Third, poor in vitro sensitivity to AQ and its active metabolite monodesethylamodiaquine (MDAQ) has already been demonstrated in Africa (55), parasite resistance-mediating polymorphisms predicted poor response to treatment with AQ and were selected by prior AQ therapy (12, 13, 23, 25, 27, 43), and use of an AQ-containing regimen selected for parasites with diminished in vitro drug sensitivity in subsequent new infections (36). Fourth, treatment with AM/LM selected in subsequent infections for parasites with polymorphisms that may lead to diminished drug responsiveness (14, 27, 31, 57). Fifth, PQ has a history of widespread resistance after broad use as monotherapy some decades ago in China (9). African studies have generally demonstrated good sensitivity of African parasites to piperaquine, but some parasites with in vitro 50% inhibitory concentrations (IC₅₀s) over 100 nM have been identified (6, 10, 35).In some cases, parasite genetic polymorphisms that mediate decreased drug responsiveness are known. The K76T polymorphism in the putative transporter pfcrt is the key mediator of resistance to CQ (11, 21) and also impacts response to AQ (13, 23). Mutations in another putative transporter, pfmdr1, appear to decrease sensitivity to CQ, AQ, and QN, and some of the same mutations may increase sensitivity to other drugs, including mefloquine and halofantrine, both of which are related to LM, and artemisinin. Increases in pfmdr1 copy number decrease in vitro sensitivity to mefloquine, halofantrine, LM, QN, and artemisinin (56) and have clearly been associated with mefloquine treatment failure (50). However, some pfmdr1 polymorphisms (S1034C and N1042D) and increases in pfmdr1 gene copy number are generally not seen in Africa (14, 25, 59), and our understanding of the importance of the pfmdr1 polymorphisms that are common in Africa (N86Y, Y184F, and D1246Y) is incomplete.In order to better characterize the sensitivity of P. falciparum from Kampala, Uganda, to relevant antimalarial drugs, we collected parasites causing uncomplicated malaria in a cohort of children enrolled in a drug efficacy trial, determined the sensitivity of these parasites to six key antimalarial drugs, and searched for associations between sensitivities to different drugs and between in vitro drug sensitivity and parasite genotypes. We found that parasites in Kampala exhibit a broad range of drug sensitivities, especially to CQ, MDAQ, and QN; that sensitivities to these three drugs, but not other tested drugs, were tightly correlated; and that polymorphisms in pfmdr1 were associated with but did not fully explain resistance to these drugs.     

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 Pyronaridine, Alone and in Combination with Other Antimalarial Drugs, against Plasmodium falciparum

The in vitro activities of pyronaridine, alone and in combination with established antimalarial drugs, were assessed by isotopic microtest. Pyronaridine was highly active against all Cameroonian isolates. A positive correlation was observed between the response to pyronaridine and that to chloroquine. Drug combination studies showed synergy between pyronaridine and primaquine, additive effects with 4-aminoquinolines, and weak antagonism with dihydroartemisinin, antifolates, or amino alcohols.     

Potent Plasmodium falciparum Gametocytocidal Activity of Diaminonaphthoquinones,Lead Antimalarial Chemotypes Identified in an Antimalarial Compound Screen

Forty percent of the world''s population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria.     

Repeat Polymorphisms in the Low-Complexity Regions of Plasmodium falciparum ABC Transporters and Associations with In Vitro Antimalarial Responses

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC₅₀) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.     

In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.     

In Vitro Activity of Fluorescent Dyes against Asexual Blood Stages of Plasmodium falciparum

Many successful antimicrobial drugs originate from synthetic dyes. This paper reports the in vitro activity of 14 fluorescent dyes against Plasmodium falciparum. Five of these dyes (Hoechst 33342, MitoRed, DiOC₆, SYTO 9, and rhodamine B) show activity at a low nanomolar concentration against two P. falciparum strains in the histidine-rich protein 2 drug sensitivity assay, while toxicity in HeLa cells is low. These dyes may be a starting point for developing new drugs against P. falciparum.     

In Vitro Antimalarial Activity of a New Organometallic Analog, Ferrocene-Chloroquine

The in vitro activities of new organometallic chloroquine analogs, based on 4-amino-quinoleine compounds bound to a molecule of ferrocene, were evaluated against chloroquine-susceptible, chloroquine-intermediate, and chloroquine-resistant, culture-adapted Plasmodium falciparum lineages by a proliferation test. One of the ferrocene analogs totally restored the activity of chloroquine against chloroquine-resistant parasites. This compound, associated with tartaric acid for better solubility, was highly effective. The role of the ferrocene in reversing chloroquine resistance is discussed, as is its potential use for human therapy.     

In Vitro Activities of Benflumetol against 158 Senegalese Isolates of Plasmodium falciparum in Comparison with Those of Standard Antimalarial Drugs

The 50% inhibitory concentration (IC₅₀s) of benflumetol (range, 12.5 to 240 nM; mean, 55.1 nM) for 158 Senegalese isolates were evaluated. Ten isolates (6%) showed decreased susceptibility to benflumetol. Benflumetol was slightly more potent against chloroquine-resistant isolates (P < 0.025). No correlation or weak correlations in the responses to benflumetol and pyrimethamine, chloroquine, amodiaquine, artemether, quinine, and pyronaridine were observed, and these correlations are insufficient to suggest cross-resistance. Benflumetol may be an important alternative drug for the treatment of chloroquine-resistant malaria.     

Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.     

In Vitro Antimalarial Activity of Novel Semisynthetic Nocathiacin I Antibiotics

Presently, the arsenal of antimalarial drugs is limited and needs to be replenished. We evaluated the potential antimalarial activity of two water-soluble derivatives of nocathiacin (BMS461996 and BMS411886) against the asexual blood stages of Plasmodium falciparum. Nocathiacins are a thiazolyl peptide group of antibiotics, are structurally related to thiostrepton, have potent activity against a wide spectrum of multidrug-resistant Gram-positive bacteria, and inhibit protein synthesis. The in vitro growth inhibition assay was done using three laboratory strains of P. falciparum displaying various levels of chloroquine (CQ) susceptibility. Our results indicate that BMS461996 has potent antimalarial activity and inhibits parasite growth with mean 50% inhibitory concentrations (IC₅₀s) of 51.55 nM for P. falciparum 3D7 (CQ susceptible), 85.67 nM for P. falciparum Dd2 (accelerated resistance to multiple drugs [ARMD]), and 99.44 nM for P. falciparum K1 (resistant to CQ, pyrimethamine, and sulfadoxine). Similar results at approximately 7-fold higher IC₅₀s were obtained with BMS411886 than with BMS461996. We also tested the effect of BMS491996 on gametocytes; our results show that at a 20-fold excess of the mean IC₅₀, gametocytes were deformed with a pyknotic nucleus and growth of stage I to IV gametocytes was arrested. This preliminary study shows a significant potential for nocathiacin analogues to be developed as antimalarial drug candidates and to warrant further investigation.     

In Vitro Activities of Strychnos Alkaloids and Extracts against Plasmodium falciparum

The in vitro antimalarial activities of 46 alkaloids and extracts from Strychnos species were evaluated. Two types of quasidimeric alkaloids exhibit high and selective activities against Plasmodium. Strychnopentamine and isostrychnopentamine were active against chloroquine-sensitive and -resistant strains (50% inhibitory concentration [IC(50)] approximately 0.15 microM), while dihydrousambarensine exhibited a 30-fold higher activity against the chloroquine-resistant strain (IC(50) = 0.03 microM) than it did against the chloroquine-sensitive strain.     

Linking Murine and Human Plasmodium falciparum Challenge Models in a Translational Path for Antimalarial Drug Development

Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rγ^−/− (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC₅₀s) were estimated at 2.0 μg/ml and 1.8 μg/ml in the NSG and IBSM models, respectively, aligning with 1.8 μg/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations. (This trial was registered at the Australian New Zealand Clinical Trials Registry [http://anzctr.org.au] under registration number ACTRN12612000323820.)     

Potentiation of Artemisinin Activity against Chloroquine-Resistant Plasmodium falciparum Strains by Using Heme Models

The influence of different metalloporphyrin derivatives on the antimalarial activity of artemisinin was studied with two chloroquine-resistant strains of Plasmodium falciparum (FcB1-Colombia and FcM29-Cameroon) cultured in human erythrocytes. This potentiation study indicates that the manganese complex of meso-tetrakis(4-sulfonatophenyl)porphyrin has a significant synergistic effect on the activity of artemisinin against both Plasmodium strains.     

Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC₅₀s) for PM and CCG (P < 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC₅₀s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC₅₀s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC₅₀s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).The spread of chloroquine (CQ)-resistant Plasmodium falciparum populations in areas in Africa where malaria is endemic (5, 23, 48) has required that other drugs be introduced for malaria treatment. Pyrimethamine (PM) and proguanil (Paludrine) are specific competitive inhibitors of dihydrofolate reductase (DHFR), a key enzyme in nucleotide biosynthesis (13). Sulfadoxine (SD) is thought to inhibit dihydropteroate synthase (DHPS), an enzyme that catalyzes a reaction in the synthesis of folate in P. falciparum (13). Proguanil is metabolized to its active form, cycloguanil (CG), and has been used primarily for chemoprophylaxis. A combination of PM and SD (Fansidar) acts synergistically to inhibit the folate pathway (9).Genetic analysis of P. falciparum isolates has demonstrated that antifolate resistance in P. falciparum is caused by point mutations in the gene that encodes the protein target of PM, DHFR, leading to amino acid changes in the active site of the enzyme. The amino acid serine at position 108 (Ser-108) is linked to sensitivity to both PM and CG. The Ser-108-to-Asn-108 mutation confers resistance to PM, and Thr-108 is associated with resistance to CG (paired with a mutation of Ala-16 to Val-16). Subsequent mutations of Asn-51 to Ile-51, Cys-59 to Arg-59, and Ile-164 to Leu-164 enhance the resistance of the isolates to PM. These findings were based on the determination of the complete DNA sequence of the DHFR-coding region in a series of P. falciparum isolates whose sensitivities to PM and CG had been previously determined by in vitro testing (10, 14, 24, 25, 55). Isolates from some sub-Saharan and Southeast Asian countries have been analyzed by DNA sequencing (3, 4). This technique is reliable but expensive and time-consuming, which limits its use in large-scale epidemiological studies in areas of endemicity. Alternatively, PCR can be adapted for the rapid detection of sequences differing by a single base pair (8, 17, 21, 32, 35). Specific primers for allele-specific PCR (ASPCR) have been designed for the detection of mutations at codons 16, 108, and 164 in the P. falciparum DHFR gene (15, 26, 27). This approach has been used to detect mutations at codon 108 in epidemiological investigations of Brazilian (26) and Malian (28) P. falciparum isolates and in a comparative study on isolates from East and West Africa and South America (29). Restriction enzyme digestion of PCR products can also be used to identify some of these point mutations in the P. falciparum DHFR gene (12, 53).PM-SD is cheap and well tolerated and is increasingly being used as a first-line treatment against uncomplicated malaria in Kenya. As a result, there is a significant reduction in in vitro chemosensitivity to PM in Kenyan parasites. In the 1980s, an average of 20% of the samples tested were classed as PM resistant (37, 44, 46), but that average rose to about 90% between 1993 and 1995 (18). We report here the application of ASPCR and enzyme digestion methods for the detection of point mutations at codons 51, 59, 108, and 164 of the DHFR gene in in vitro-adapted Kenyan P. falciparum field isolates. The goal was to determine whether particular point mutations in the DHFR-coding region are correlated in each isolate with the isolates’ chemosensitivities to the DHFR inhibitors PM and CCG (the active metabolite of chlorproguanil [CPG] [Lapudrine]) or to the DHPS inhibitors SD and dapsone (DDS). CCG and DDS were included in the study because this combination has proved to be particularly potent against P. falciparum isolates in vitro (50) and in vivo (1).     

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (-)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (-)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC(50) ratio of (-)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (-) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.     

In Vitro Activity of Mirincamycin (U24729A) against Plasmodium falciparum Isolates from Gabon

We assessed the in vitro activity of mirincamycin, a lincosamide antibiotic, against Plasmodium falciparum clinical isolates from Gabon. Growth was determined by HRP2 enzyme-linked immunosorbent assay using an adapted protocol with a prolonged incubation time (6 days) to account for antibiotic-induced delayed death. Mirincamycin''s cis and trans isomers are more active (median 50% inhibitory concentrations [IC₅₀s], 3.2 nM and 2.6 nM) than the comparator drugs clindamycin (IC₅₀, 12 nM) and doxycycline (IC₅₀, 720 nM), and therefore, further clinical development is promising.Drug resistance in Plasmodium falciparum populations around the world and the short half-life of artemisinins have led to a resurgence of interest in combination therapies for the treatment of malaria (9, 14, 16). It is hypothesized that the rate of resistance development is reduced when antimalarial combinations are given. Most commonly, fast-acting antimalarials with short half-lives are combined with slow-acting and slowly eliminated drugs. A drawback of this approach is the exposure of persisting parasites to subinhibitory levels of the slowly eliminated partner. Due to their short half-life but delayed action, antibiotics are particularly attractive combination partners for fast-acting drugs such as quinine, artemisinins, or other drugs (1, 10, 23, 27). Antibiotics act mainly on plasmodial organelles of prokaryotic origin: the mitochondrium and the apicoplast (8). These compounds inhibit parasite growth in the second cycle after exposure. This so-called “delayed-death” phenomenon is due to apicoplast dysfunction, which explains the seeming paradox of parasite death after a short exposure to the drug in the preceding cycle. Molecular details of this process are not known (4, 5). We report our results on the antimalarial properties of mirincamycin (Fig. ​(Fig.1),1), a lincosamide antibiotic similar to clindamycin which is synthetically produced (13). Mirincamycin has been evaluated as an antimalarial drug in animal models before (18, 19, 25, 26), but investigations were not continued, mainly because there was no perceived need for combination therapy at that time. Interest in mirincamycin has reemerged, and the first clinical trials are expected to start in 2010.Open in a separate windowFIG. 1.Chemical structure of the cis (a) or trans (b) isomer of mirincamycin.We tested the inhibitory activities of doxycycline, clindamycin, and mirincamycin with P. falciparum isolates from patients with malaria in Lambaréné, Gabon, between February and May 2009. Parasites were from patients of ages 15 month to 18 years who presented with P. falciparum monoinfection (parasitemia between 10³ and 6 × 10⁵ parasites/μl blood, assessed by thick blood smear [17]) and had no reported intake of antimalarial drugs for at least 1 month. Informed consent and assent were obtained from the legal representative and the participating child, respectively. The investigation was approved by the ethics committee of the International Foundation for the Albert Schweitzer Hospital in Lambaréné. Doxycycline hyclate (molecular weight [MW], 512.94) and clindamycin hydrochloride (MW, 461.44) were obtained from Sigma Aldrich and dissolved in H₂O at stock solutions of 86 mM and 100 mM, respectively; 4′-trans-mirincamycin hydrochloride (MW, 475.47) and 4′-cis-mirincamycin hydrochloride (MW, 475.47) were provided by Richard Westerman (Maldevco) and dissolved in dimethyl sulfoxide at stock solutions of 57 mM and 78 mM, respectively. Drugs were predosed in 96-well plates in threefold serial dilution. Parasites were added in complete culture medium (RPMI 1640, 25 mM HEPES, 2.4 mM l-glutamine, 50 μg/ml gentamicin, and 0.5% [wt/vol] Albumax) at a hematocrit of 1.5% with an adjusted parasitemia of 0.05% and incubated at 37°C in a candle jar. Pilot experiments to determine the best cultivation period and medium change strategy for slow-acting antibiotics showed best results when parasites were kept in culture for 6 days with medium changes on days 2 and 4 without drug replacement. After 6 days, plates were stored at −20°C until the histidine-rich protein 2 (HRP2) concentration was measured according to standard procedures (15). Only samples positive for growth in microscopy (assessed by thick blood smear on days 4, 5, and 6) and with an at least twofold increase in the HRP2 concentration after 6 days of incubation were included in the analysis.Individual inhibitory concentrations were determined by nonlinear regression analysis of log-concentration-response curves using the drc package, v0.9.0, of R v2.6.1 (22, 24). The laboratory strain 3D7A was used to determine the range of activities of the antibiotics tested after 3 and 6 days. The median 50% inhibitory concentrations (IC₅₀s) for cis- and trans-mirincamycin were 1.5 nM and 3.6 nM in the 6-day assay; after 3 days, mirincamycin''s IC₅₀ was more than 10,000-fold higher. This finding was expected, since the difference in activity between the first and second cycles is similar for clindamycin.Twenty-three of 27 collected P. falciparum isolates fulfilled the criteria for successful culture for clindamycin and cis-mirincamycin, 22 for doxycycline, and 21 for trans-mirincamycin.No significant difference was observed between the trans and cis isomers of mirincamycin. Both showed a low median IC₅₀, with 2.6 nM and 3.2 nM, respectively, and a narrow range between isolates (0.6 to 7.7 nM and 0.5 to 8.2 nM, respectively). The median IC₅₀ of clindamycin was 11.6 nM, with a range between 2.4 and 29.1 nM. Doxycycline''s IC₅₀ was considerably higher (720 nM), with a wide range of activities (104 nM to 12 μM). Notably, two isolates had unexpectedly high IC₅₀s for doxycycline (6 and 12 μM) but not for the lincosamides. The IC₅₀s of trans- and cis-mirincamycin are significantly correlated to each other (P < 0.001) and to that of the other lincosamide drug, clindamycin (P < 0.001). The correlation of doxycyline with clindamycin and cis- and trans-mirincamycin was significant (for all comparisons, P < 0.001), although not as strong as those for the lincosamide antibiotics among each other (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Correlation matrix of doxycycline (DOX), clindamycin (CLI), and the two isomers of mirincamycin (cis-MIR and trans-MIR). The leftmost column shows the log-transformed data for the association of cis-MIR with trans-MIR, CLI, and DOX activities, respectively. The lower two boxes of the second column show the association of trans-MIR with CLI and DOX, and the lowest box of the third column shows that for for CLI and DOX. A smoothed line is drawn to visualize the associations. Mirrored through the diagonal, Spearman''s rho for the different correlations is shown. The font size is proportional to the degree of correlation.This work shows for the first time that mirincamycin has a high in vitro activity against clinical P. falciparum isolates. IC₅₀s of both isomers are substantially lower than those for any other antibiotic tested so far (5, 8), including the lincosamide comparator clindamycin (3, 5). The range of activity in individual isolates is narrow, and no outliers that would indicate frequent naturally occurring variants with decreased sensitivity were identified.Preclinical studies of mice and monkeys have shown in vivo activity of mirincamycin against plasmodia. In Plasmodium cynomolgi infections of rhesus monkeys, mirincamycin was curative as a monotherapeutic regimen (18) and showed an additive effect when given together with primaquine (25). In Plasmodium berghei-infected mice, mirincamycin showed activity after subcutaneous and oral administration (12). Interestingly, a hypnozoitocidal effect was observed in monkeys in another study (26). If the same effect is seen in human malarias, then mirincamycin could become an urgently needed alternative to primaquine. Phase I and ADMET studies of mirincamycin were performed in the late 1960s (R. Westerman, personal communication). Toxicity was reported to be similar to that of clindamycin. In contrast to clindamycin, mirincamycin tends to partition into erythrocytes. Normal adults given repeated doses of mirincamycin exhibit both a dose-dependent and accumulative increase in concentrations in erythrocytes that is greater than the plasma concentration. Similar repeated dosing of clindamycin leads to a plateauing of serum and erythrocyte levels with no changes in relative drug concentrations. The pharmacokinetics of mirincamycin in parasitized human erythrocytes and its impact on antiparasitic activity have not been investigated. Their study should be incorporated into anticipated clinical trials.We observed lower IC₅₀s for doxycycline and clindamycin than in previous publications using clinical isolates tested with standard protocols (2, 11, 21). This is most likely due to our longer incubation time, because for drugs with delayed action, a 10- to 10,000-fold increase in activity between the first and second cycles is present. This is especially true for clindamycin (5). Our results are similar to results for laboratory strains incubated for two cycles (5-7, 20).In conclusion, in vitro activity of mirincamycin in clinical isolates from Gabon is higher than those for doxycycline and clindamycin. Further clinical development of this drug is worth pursuing, since it would add a very interesting combination partner to fast-acting antimalarials.     

In Vitro Chemosensitization of Plasmodium falciparum to Antimalarials by Verapamil and Probenecid

We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.The modulation of chloroquine (CQ) resistance with the calcium channel blocker verapamil (VPM), antipsychotic drugs, histamine receptor antagonists, and antidepressant agents among others was the first case of resistance modulation to be reported (reviewed in references 6, 8, and 28).Clinical evaluation of some of these agents has been carried out in areas where CQ resistance is moderate (22, 24-26). However, these agents have the disadvantage of being pharmacologically active, with systemic effects that may result in a variety of side effects. In addition, the minimum concentrations of these agents needed to chemosensitize parasites to CQ (usually more than 1 μM of free drug) (1, 4, 11-13) are not achievable in vivo when normal doses are used; therefore, high doses have to be used, with all of the attendant risks of toxicity. All of these limitations may explain why the reversal of CQ resistance has never attained widespread application.We have demonstrated that the uricosuric drug probenecid (PBN) chemosensitizes parasites to antifolate and CQ (16, 18). In this study, we tested the effect of PBN against the aminoquinolines CQ, piperaquine (PPRQ), primaquine (PMQ), desethylamodiaquin (DEAQ), and amodiaquin (AQ); the amino alcohols lumefantrine (LM), mefloquine (MFQ), halofantrine (HLF), and quinine (QN); the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), and methotrexate (MTX); the benzonaphthyridine pyronaridine (PRN); and the sesquiterpene dihydroartemisinin (DHA). We used the chemosensitizer VPM as a comparator.CQ, PMQ, AQ, MFQ, QN, MTX, PBN, and VPM were purchased from Sigma (Poole, Dorset, United Kingdom). PPRQ was a gift from Universal Corporation Limited, Kikuyu, Kenya. DEAQ, DHA, LM, PRN, and HLF were gifts from Steve Ward, Liverpool School of Tropical Medicine, Liverpool, United Kingdom. Drugs were dissolved as suggested by manufacturers. For PBN, after dilution in dimethyl sulfoxide (500 mg/ml), a subsequent serial dilution was carried out; this consisted of fivefold dilution in absolute ethanol followed by twofold dilution in 2% sodium bicarbonate and then dilution in RPMI 1640 medium (PBN crystallizes when diluted directly from dimethyl sulfoxide to RPMI 1640 medium).We employed two reference P. falciparum laboratory strains: V1S, a multidrug-resistant strain (resistant to CQ, PM, and QN) and 3D7, a strain fully sensitive to all tested antimalarials, except LM and PMQ. Cultures were carried out in RPMI 1640 (GIBCO BRL, United Kingdom), and antimalarial activities were expressed as the drug concentration required for 50% inhibition of [³H]hypoxanthine incorporation (IC₅₀) during the 66-h assay (19).Chemosensitization consisted of testing the activity of the antimalarials in the presence of PBN and VPM at the following ratios: 0.8:0.2, 0.6:0.4, 0.4:0.6, and 0.2:0.8. Chemosensitization was measured geometrically by construction of isobolograms and algebraically, by calculating the sum of the minimum fractional inhibitory concentrations (FICs) (2). FIC values <0.5 and falling between 0.5 and 1 denote pronounced and moderate chemosensitization, respectively. FIC values >1.0 indicate the absence of chemosensitization, and antagonism is denoted by FIC values >4.Our data show that FIC of VPM/CQ, VPM/PMQ, and VPM/LM for V1S ranged between 0.5 and 0.71, a clear indication of chemosensitization or a VPM effect, though moderate (Table ​(Table1).1). The most pronounced effect was observed with DEAQ and QN, with a FIC of <0.5 (Table ​(Table1).1). No chemosensitization was observed when 3D7 was used.

TABLE 1.

In vitro analysis, using the multidrug-resistant strain V1S, of the combination of VPM and PBN with 14 antimalarial drugs^a

Anthracene-Polyamine Conjugates Inhibit In Vitro Proliferation of Intraerythrocytic Plasmodium falciparum Parasites

Anthracene-polyamine conjugates inhibit the in vitro proliferation of the intraerythrocytic human malaria parasite Plasmodium falciparum, with 50% inhibitory concentrations (IC₅₀s) in the nM to μM range. The compounds are taken up into the intraerythrocytic parasite, where they arrest the parasite cell cycle. Both the anthracene and polyamine components of the conjugates play a role in their antiplasmodial effect.