Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine

BACKGROUND: Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own. METHODS: Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined. RESULTS AND CONCLUSIONS: The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT

Effects of piperaquine, chloroquine, and amodiaquine on drug uptake and of these in combination with dihydroartemisinin against drug-sensitive and -resistant Plasmodium falciparum strains.

Piperaquine is being developed as a long-acting component in artemisinin combination therapies. It was highly active in vitro and drug interaction studies showed that dihydroartemisinin combinations with piperaquine, chloroquine, and amodiaquine were indifferent tending toward antagonism. Competitive uptake of radiolabeled chloroquine and dihydroartemisinin in combination with other antimalarials was observed

The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids

BACKGROUND: The 8-amino and 9-hydroxy substituents of antimalarial cinchona alkaloids have the erythro orientation while their inactive 9-epimers are threo. From the X-ray structures a 90° difference in torsion angle between the N1-H1 and C9-O12 bonds in the two series is believed to be important. In order to kill the malaria parasite, alkaloids must cross the erythrocyte and parasite membranes to accumulate in the acid digestive vacuole where they prevent detoxication of haematin produced during haemoglobin breakdown. METHODS: Ionization constants, octanol/water distribution and haematin interaction are examined for eight alkaloids to explain the influence of small structural differences on activity. RESULTS: Erythro isomers have a high distribution ratio of 55:1 from plasma to the erythrocyte membrane, while for the more basic threo epimers this is only 4.5:1. This gives an increased transfer rate of the erythro drugs into the erythrocyte and thence into the parasite vacuole where their favourable conformation allows interaction with haematin, inhibiting its dimerization strongly (90 ± 7%) and thereby killing the parasite. The threo compounds not only enter more slowly but are then severely restricted from binding to haematin by the gauche alignment of their N1-H1 and C9-O12 bonds. Confirmatory molecular models allowed measurement of angles and bond lengths and computation of the electronic spectrum of a quinine-haematin complex. CONCLUSION: Differences in the antiplasmodial activity of the erythro and threo cinchona alkaloids may therefore be attributed to the cumulative effects of lipid/aqueous distribution ratio and drug-haematin interaction. Possible insights into the mechanism of chloroquine-resistance are discussed

Influence of LAR and VAR on Para-Aminopyridine Antimalarials Targetting Haematin in Chloroquine-Resistance

Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aro- matic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol sub- stituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logRe- sistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria

We report the first in vitro and genetic confirmation of Malarone(®) (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone(®). Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously

Dynamics of pfcrt alleles CVMNK and CVIET in chloroquine-treated Sudanese patients infected with Plasmodium falciparum

BACKGROUND: Parasite resistance to the anti-malarial drug chloroquine is common in eastern Sudan. Dynamic within-host changes in the relative abundance of both sensitive and resistant Plasmodium falciparum parasites were examined in a cohort of chloroquine-treated patients presenting with uncomplicated falciparum malaria, using a novel allele-specific quantitative approach. METHODS: Treatment outcomes were determined for 93 patients of all ages in a per protocol cohort using a modified 14-day WHO protocol. Parasite DNA samples at days 0, 1, 2, 3, 7 and 14 following treatment were analysed using real-time quantitative PCR methods that distinguished resistant and sensitive genotypes at amino acids 72-76 of the pfcrt locus. RESULTS: Chloroquine treatment was not efficacious, and of 93 assessable patients, only 10 individuals (10.7%; 95% C.I. 4.34-17.2%) enjoyed an adequate clinical and parasitological response. Resistant parasites with the haplotype CVIET at codons 72-76 of the pfcrt locus were dominant in the starting population. Chloroquine sensitive parasites with the haplotype CVMNK were detected in 19 individuals prior to treatment (20.43%; 95% C.I. 5.14-18.5%). In these patients, CQ treatment rapidly selected CVIET parasites, and this haplotype overwhelmingly dominated the parasite population in each individual by day 2 after treatment. CONCLUSIONS: Such rapid intra-host selection of particular genotypes after the introduction of drug will cause frequent misidentification of parasite genotypes present in the starting population. This will have a potentially serious confounding effect on clinical trials which employ PCR-corrected estimates of treatment failure, as resistant parasites below the detection threshold in the pre-treatment sample can be erroneously classified as "new" infections during follow-up, over-estimating drug efficacy

The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance

Oral activated charcoal prevents experimental cerebral malaria in mice and in a randomized controlled clinical trial in man did not interfere with the pharmacokinetics of parenteral artesunate.

BACKGROUND: Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. METHODS/PRINCIPAL FINDINGS: We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF(+) CD4(+) T cells and multifunctional IFNgamma(+)TNF(+) CD4(+) and CD8(+) T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. CONCLUSIONS/SIGNIFICANCE: oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN64793756

Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 μL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt

Superhumps in Cataclysmic Binaries. XXV. q_crit, epsilon(q), and Mass-Radius

We report on successes and failures in searching for positive superhumps in cataclysmic variables, and show the superhumping fraction as a function of orbital period. Basically, all short-period systems do, all long-period systems don't, and a 50% success rate is found at P_orb=3.1+-0.2 hr. We can use this to measure the critical mass ratio for the creation of superhumps. With a mass-radius relation appropriate for cataclysmic variables, and an assumed mean white-dwarf mass of 0.75 M_sol, we find a mass ratio q_crit=0.35+-0.02. We also report superhump studies of several stars of independently known mass ratio: OU Virginis, XZ Eridani, UU Aquarii, and KV UMa (= XTE J1118+480). The latter two are of special interest, because they represent the most extreme mass ratios for which accurate superhump measurements have been made. We use these to improve the epsilon(q) calibration, by which we can infer the elusive q from the easy-to-measure epsilon (the fractional period excess of P_superhump over P_orb). This relation allows mass and radius estimates for the secondary star in any CV showing superhumps. The consequent mass-radius law shows an apparent discontinuity in radius near 0.2 M_sol, as predicted by the disrupted magnetic braking model for the 2.1-2.7 hour period gap. This is effectively the "empirical main sequence" for CV secondaries.Comment: PDF, 45 pages, 9 tables, 12 figures; accepted, in press, to appear November 2005, PASP; more info at http://cba.phys.columbia.edu