Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cationic antimalarials.

Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the emerging multi-drug resistance of P. falciparum malaria, like choline analogues targeting plasmodial phospholipidic metabolism. Here we describe the synthesis of amidoxime derivatives as prodrug candidates of reverse-benzamidines and hybrid compounds able to mimic choline, as well as the design of a new series of asymmetrical bis-cationic compounds. Bioconversion studies were conducted on amidoximes in asymmetrical series and showed that amidoxime prodrug strategy could be applied on C-alkylamidine moieties, like benzamidines and that N-substituents did not alter the bioconversion of amidoximes. The antimalarial activity of the three series of compounds was evaluated in vitro against P. falciparum and in vivo against P. vinckei petteri in mice.

Contribution of the precursors and interplay of the pathways in the phospholipid metabolism of the malaria parasite.

The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.

High Accumulation and In Vivo Recycling of the New Antimalarial Albitiazolium Lead to Rapid Parasite Death.

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

New insight into the mechanism of accumulation and intraerythrocytic compartmentation of albitiazolium, a new type of antimalarial.

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance.

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

A novel live-dead staining methodology to study malaria parasite viability.

BACKGROUND: Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead. METHODS: Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive. RESULTS: Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing. CONCLUSIONS: The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity.

In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.

Multiple roles for Plasmodium berghei phosphoinositide-specific phospholipase C in regulating gametocyte activation and differentiation.

Critical events in the life cycle of malaria parasites are controlled by calcium-dependent signalling cascades, yet the molecular mechanisms of calcium release remain poorly understood. The synchronized development of Plasmodium berghei gametocytes relies on rapid calcium release from internal stores within 10 s of gametocytes being exposed to mosquito-derived xanthurenic acid (XA). Here we addressed the function of phosphoinositide-specific phospholipase C (PI-PLC) for regulating gametocyte activation. XA triggered the hydrolysis of PIP(2) and the production of the secondary messenger IP(3) in gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca(2+) signal. However, microgametocyte differentiation into microgametes was blocked even when the inhibitor was added up to 5 min after activation, suggesting a requirement for PI-PLC beyond the early mobilization of calcium. In contrast, inhibitors of calcium release through ryanodine receptor channels were active only during the first minute of gametocyte activation. Biochemical determination of PI-PLC activity was confirmed using transgenic parasites expressing a fluorescent PIP(2) /IP(3) probe that translocates from the parasite plasmalemma to the cytosol upon cell activation. Our study revealed a complex interdependency of Ca(2+) and PI-PLC activity, with PI-PLC being essential throughout gamete formation, possibly explaining the irreversibility of this process.

Symmetrical choline-derived dications display strong anti-kinetoplastid activity.

OBJECTIVES: to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. METHODS: from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. RESULTS: the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. CONCLUSIONS: the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

Phosphatidylinositol 3-phosphate, an essential lipid in Plasmodium, localizes to the food vacuole membrane and the apicoplast.

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.

Genetic and transcriptional analysis of phosphoinositide-specific phospholipase C in Plasmodium.

Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled.

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways.

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.

Statistical model to evaluate in vivo activities of antimalarial drugs in a Plasmodium cynomolgi-macaque model for Plasmodium vivax malaria.

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation.

Quantitation of SAR97276 in mouse tissues by rapid resolution liquid chromatography-mass spectrometry.

1,12-Bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane dibromide (SAR97276, T3) is a new antimalarial drug, which is currently being evaluated in clinical trials for severe malaria. Drug accumulation inside the parasite and a dual mechanism of action are a major strength of this compound, as it could help delay the development of resistance. The purpose of this article was to develop a rapid resolution LC-MS method for quantifying SAR97276 in mouse tissues. The LC system consisted of Zorbax Eclipse XDB C8 (1.8 microm, 50 x 4.6 mm, 60 degrees C) column. Elution with a gradient mobile phase consisting of ACN-trimethylamine-formate buffer (pH 3) at a flow rate of 1 mL/min yielded sharp, utmost-resolved peaks within 2 min. Tissue samples were powdered under liquid nitrogen. After protein precipitation with citric acid, SPE using WCX cartridges was used for sample preparation. There was no influence of the matrix on the detection of either SAR97276 or the IS. Assay precision was <13% and accuracy was 90-107%. The lower LOQs were 3.3 microg/kg in brain and 33 microg/kg in liver and heart. This newly developed method was used to study the tissue distribution of SAR97276 in mouse as part of the ongoing development of SAR97276.

Characterisation of the phosphatidylinositol synthase gene of Plasmodium species.

Phosphatidylinositol (PI) is a versatile lipid that not only serves as a structural component of cellular membranes, but also plays important roles in membrane anchorage of proteins and in signal transduction through distinct phosphorylated derivatives of the inositol head group. PI is synthesised by PI synthase from CDP-diacylglycerol and myo-inositol. The enzymatic activity in Plasmodium falciparum and P. knowlesi has previously been characterised at the biochemical level. Here we characterise the PI synthase gene of P. falciparum and P. knowlesi. The cDNA sequence identified a highly spliced gene consisting of nine exons and encoding a protein of 209 and 207 amino acids, respectively. High sequence conservation enabled the prediction of the PI synthase genes of P. berghei, P. chabaudi and P. vivax. All Plasmodium PI synthase proteins appear to be highly hydrophobic, although no consensus for the number and location of distinct transmembrane domains could be detected. The P. falciparum PI synthase (PfPIS) gene successfully complemented a Saccharomyces cerevisiae PIS1 deletion mutant, demonstrating its enzymatic function. Complementation efficiency was dramatically improved when hybrid constructs between N-terminal S. cerevisiae and C-terminal P. falciparum sequences were used. Determination of in vitro PIS activities of complemented yeast strains confirmed the enzymatic function of the Plasmodium protein.

SC83288 is a clinical development candidate for the treatment of severe malaria.

Severe malaria is a life-threatening complication of an infection with the protozoan parasite Plasmodium falciparum, which requires immediate treatment. Safety and efficacy concerns with currently used drugs accentuate the need for new chemotherapeutic options against severe malaria. Here we describe a medicinal chemistry program starting from amicarbalide that led to two compounds with optimized pharmacological and antiparasitic properties. SC81458 and the clinical development candidate, SC83288, are fast-acting compounds that can cure a P. falciparum infection in a humanized NOD/SCID mouse model system. Detailed preclinical pharmacokinetic and toxicological studies reveal no observable drawbacks. Ultra-deep sequencing of resistant parasites identifies the sarco/endoplasmic reticulum Ca2+ transporting PfATP6 as a putative determinant of resistance to SC81458 and SC83288. Features, such as fast parasite killing, good safety margin, a potentially novel mode of action and a distinct chemotype support the clinical development of SC83288, as an intravenous application for the treatment of severe malaria.

Chemotherapy against babesiosis.

Babesiosis is caused by a haemotropic protozoal parasite of the genus Babesia, member of the phylum Apicomplexa and transmitted by the bite of an infected tick. There are many Babesia species affecting livestock, dogs, horses and rodents which are of economic significance. Infections can occur without producing symptoms, but babesiosis may also be severe and sometimes fatal caused by the intraerythrocytic parasite development. The disease can cause fever, fatigue and haemolytic anemia lasting from several days to several months. There are a number of effective babesiacides, but imidocarb dipropionate (which consistently clears the parasitaemia; often the only available drug on the market) and diminazene aceturate are the most widely used. Some Babesia spp. can infect humans, particularly Babesia microti and Babesia divergens, and human babesiosis is a significant emerging tick-borne zoonotic disease. Clinical manifestations differ markedly between European and North American diseases. In clinical cases, a combination of clindamycin and quinine is administered as the standard treatment, but also administration of atovaquone-azithromycin is successful. Supportive therapy such as intravenous fluids and blood transfusions are employed when necessary. More specific fast-acting new treatments for babesiosis have now to be developed. This should be facilitated by the knowledge of the Babesia spp. genome and increased interest for this malaria-like parasite.

Dual molecules as new antimalarials.

A new antimalarial pharmacological approach based on inhibition of the plasmodial phospholipid metabolism has been developed. The drugs mimic choline structure and inhibit de novo phosphatidylcholine biosynthesis. Three generations of compounds were rationally designed. Bisquaternary ammonium salts showed powerful antimalarial activity, with IC(50) in the nanomolar range. To remedy their low per os absorption, bioisosteric analogues (bis-amidines) were designed and exhibited similar powerful activities. Finally, the third generation compounds are bis-thiazolium salts and their non-ionic precursors: prodrugs, which in vivo can lead to thiazolium drugs after enzymatic transformation. The compounds are equally effective against multiresistant Plasmodium falciparum malaria. These molecules exert a very rapid cytotoxic effect against malarial parasites in the very low nanomolar range and are active in vivo against P. vinckei-infected mice, with ED(50) lower than 0.2 mg/kg. They are able to cure highly infected mice and, retain full activity after a single injection. They also retain full activity against P. falciparum and P. cynomolgi in primate models with no recrudescence and at lower doses. Compounds are accumulated in P.falciparum-infected erythrocyte, which ensures their potency and specificity. Recently, we discovered that compounds also interact with malarial pigment enhancing the antimalarial effect. It is quite likely that they are dual molecules, exerting their antimalarial activity via two simultaneous toxic effects on the intracellular intraerythrocytic parasites. The current leader compounds are accessible in few steps from commercial products. These crystalline molecules present a remarkable biological activity and low toxicity which is promising for the development of a new antimalarial drug.

Liquid chromatography-electrospray mass spectrometry determination of a bis-thiazolium compound with potent antimalarial activity and its neutral bioprecursor in human plasma, whole blood and red blood cells.

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.

Phospholipids in parasitic protozoa.

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs.