15 research outputs found
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
Improved synchronous production of Plasmodium falciparum gametocytes in vitro.
The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis
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
Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase
Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle
A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-ribosylation factor in Plasmodium falciparum.
In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-ribosylation factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase beta in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P(2) pathways allowing modulation of PtdIns(4,5)P(2) production in response to changes in intracellular calcium concentrations within the parasite
Activity of piperaquine and other 4-aminoquinoline antiplasmodial drugs against chloroquine-sensitive and resistant blood-stages of Plasmodium falciparum. Role of beta-haematin inhibition and drug concentration in vacuolar water- and lipid-phases.
Chloroquine (CQ), a 4-aminoquinoline, accumulates in acidic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to beta-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and 4 and 5, representing halves of PQ, beta-haematin inhibitory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured pK(a)s and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH 7.4) into vacuolar water (pH 4.8) and lipid accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while 4 and 5 were 100 times less potent. CQ with two basic centres has a VAR of 143,482, while 4 and 5, with two basic centres of lower pK(a)s have VARs of 1287 and 1966. In contrast PQ and OH-PQ have four basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R. 5, PQ and OH-PQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to beta-haematin inhibition in vacuolar lipid
Modified Fixed-Ratio Isobologram Method for Studying In Vitro Interactions between Atovaquone and Proguanil or Dihydroartemisinin against Drug-Resistant Strains of Plasmodium falciparum
A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed
Mutations in the Plasmodium falciparum cytochrome b gene are associated with delayed parasite recrudescence in malaria patients treated with atovaquone-proguanil.
BACKGROUND: Fixed-dose combination antimalarial drugs have played an increasingly important role in the treatment and chemoprophylaxis of falciparum malaria since the worldwide failure of monotherapy with chloroquine. Atovaquone-proguanil is one such combination drug used both for prophylaxis in travellers, and for treatment of acute malaria cases in European hospitals and clinics. METHODS: A series of eight atovaquone-proguanil treatment failures and two prophylaxis breakthroughs from four UK hospitals from 2004-2008 were analysed for evidence of mutations in the pfcyt-b gene, previously found to be associated with failure of the atovaquone component. RESULTS: Parasites carrying pfcyt-b mutations were found in five falciparum malaria patients with recrudescent parasitaemia occurring weeks after apparently successful treatment of a primary infection with atovaquone-proguanil. Four of these cases carried parasites with the Tyr268Cys mutation in pfcyt-b, previously reported in two French patients with malaria. In contrast, mutations in pfcyt-b were not found in three patients treated with atovaquone-proguanil who exhibited delayed clearance of the primary infection, nor in two returning travellers with malaria who had used the combination for prophylaxis. Using current and previously published data, mean time to recrudescence of parasites carrying pfcytb codon 268 mutations was estimated as 28.0 days after treatment (95% C.I. 23.0 - 33.0 days), whereas treatment failures without codon 268 mutations received rescue treatment an average of 4.71 days after initial AP treatment (95% C.I. 1.76 - 7.67 days). CONCLUSION: Genetically-determined parasite resistance to atovaquone is associated with delayed recrudescence of resistant parasites three weeks or more after initial clearance of parasitaemia by atovaquone/proguanil therapy. The 268-Cys allele of pfcyt-b may have been overlooked in previous studies of atovaquone-proguanil treatment failure as it is not detected by current RFLP methods