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BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic by the World Health Organisation and urgent treatment and prevention strategies are needed. Many clinical trials have been initiated with existing medications, but assessments of the expected plasma and lung exposures at the selected doses have not featured in the prioritisation process. Although no antiviral data is currently available for the major phenolic circulating metabolite of nitazoxanide (known as tizoxanide), the parent ester drug has been shown to exhibitin vitroactivity against SARS-CoV-2. Nitazoxanide is an anthelmintic drug and its metabolite tizoxanide has been described to have broad antiviral activity against influenza and other coronaviruses. The present study used physiologically-based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported nitazoxanide 90% effective concentration (EC90) against SARS-CoV-2.MethodsA whole-body PBPK model was constructed for oral administration of nitazoxanide and validated against available tizoxanide pharmacokinetic data for healthy individuals receiving single doses between 500 mg – 4000 mg with and without food. Additional validation against multiple-dose pharmacokinetic data when given with food was conducted. The validated model was then used to predict alternative doses expected to maintain tizoxanide plasma and lung concentrations over the reported nitazoxanide EC90in >90% of the simulated population. Optimal design software PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials.ResultsThe PBPK model was validated with AAFE values between 1.01 – 1.58 and a difference less than 2-fold between observed and simulated values for all the reported clinical doses. The model predicted optimal doses of 1200 mg QID, 1600 mg TID, 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food, to provide tizoxanide plasma and lung concentrations over the reportedin vitroEC90of nitazoxanide against SARS-CoV-2. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated.ConclusionThe PBPK model predicted that it was possible to achieve plasma and lung tizoxanide concentrations, using proven safe doses of nitazoxanide, that exceed the EC90for SARS-CoV-2. The PBPK model describing tizoxanide plasma pharmacokinetics after oral administration of nitazoxanide was successfully validated against clinical data. This dose prediction assumes that the tizoxanide metabolite has activity against SARS-CoV-2 similar to that reported for nitazoxanide, as has been reported for other viruses. The model and the reported dosing strategies provide a rational basis for the design (optimising plasma and lung exposures) of future clinical trials of nitazoxanide in the treatment or prevention of SARS-CoV-2 infection.

There is a rapidly expanding literature on thein vitroantiviral activity of drugs that may be repurposed for therapy or chemoprophylaxis against SARS-CoV-2. However, this has not been accompanied by a comprehensive evaluation of the ability of these drugs to achieve target plasma and lung concentrations following approved dosing in humans. Moreover, most publications have focussed on 50% maximum effective concentrations (EC50), which may be an insufficiently robust indicator of antiviral activity because of marked differences in the slope of the concentration-response curve between drugs. Accordingly,in vitroanti-SARS-CoV-2 activity data was digitised from all available publications up to 13thApril 2020 and used to recalculate an EC90value for each drug. EC90values were then expressed as a ratio to the achievable maximum plasma concentrations (Cmax) reported for each drug after administration of the approved dose to humans (Cmax/EC90ratio). Only 14 of the 56 analysed drugs achieved a Cmax/EC90ratio above 1 meaning that plasma Cmax concentrations exceeded those necessary to inhibit 90% of SARS-CoV-2 replication. A more in-depth assessment of the putative agents tested demonstrated that only nitazoxanide, nelfinavir, tipranavir (boosted with ritonavir) and sulfadoxine achieved plasma concentrations above their reported anti-SARS-CoV-2 activity across their entire approved dosing interval at their approved human dose. For all drugs reported, the unbound lung to plasma tissue partition coefficient (KpUlung) was also simulated and used along with reported Cmax and fraction unbound in plasma to derive a lung Cmax/EC50as a better indicator of potential human efficacy (lung Cmax/EC90ratio was also calculable for a limited number of drugs). Using this parameter hydroxychloroquine, chloroquine, mefloquine, atazanavir (boosted with ritonavir), tipranavir (boosted with ritonavir), ivermectin, azithromycin and lopinavir (boosted with ritonavir) were all predicted to achieve lung concentrations over 10-fold higher than their reported EC50. This analysis was not possible for nelfinavir because insufficient data were available to calculate KpUlungbut nitozoxanide and sulfadoxine were also predicted to exceed their reported EC50by 3.1- and 1.5-fold in lung, respectively. The antiviral activity data reported to date have been acquired under different laboratory conditions across multiple groups, applying variable levels of stringency. However, this analysis may be used to select potential candidates for further clinical testing, while deprioritising compounds which are unlikely to attain target concentrations for antiviral activity. Future studies should focus on EC90values and discuss findings in the context of achievable exposures in humans, especially within target compartments such as the lung, in order to maximise the potential for success of proposed human clinical trials.

BACKGROUND The intracellular and plasma concentrations of HIV protease inhibitors (HPIs) vary widely in vivo. It is unclear whether there is a concentration-dependent effect of HPIs such that at increasing concentration they may either block their own efflux (leading to 'autoboosting') or influx (leading to saturability/decreased intracellular accumulation). METHOD The effects of various concentrations (0-30 microM) of lopinavir, saquinavir, ritonavir and atazanavir on the accumulation of [(14)C]lopinavir, [(3)H]saquinavir, [(3)H]ritonavir and [(3)H]atazanavir, respectively, were investigated in CEM(parental), CEM(VBL) [P-glycoprotein (ABCB1) overexpressing], CEM(E1000) (MRP1 overexpressing) and in peripheral blood mononuclear cells (PBMCs). We also investigated the effects of inhibitors of ABCB1/ABCG2 (tariquidar), ABCC (MK571) and ABCC1/2 (frusemide), singly and in combination with HPIs, on cellular accumulation. RESULTS In all the cell lines, with increasing concentration of lopinavir, saquinavir and ritonavir, there was a significant increase in the cellular accumulation of [(14)C]lopinavir, [(3)H]saquinavir and [(3)H]ritonavir. Tariquidar, MK571 and frusemide (alone and in combination with lopinavir, saquinavir and ritonavir) significantly increased the accumulation of [(14)C]lopinavir, [(3)H]saquinavir and [(3)H]ritonavir. Ritonavir (alone or in combination with tariquidar) decreased the intracellular accumulation of [(3)H]ritonavir in PBMCs. Atazanavir decreased the accumulation of [(3)H]atazanavir in a concentration-dependent manner in all of the cells tested. CONCLUSIONS There are complex and variable drug-specific rather than class-specific effects of the HPIs on their own accumulation.

In Arabidopsis thaliana , biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, Pf CRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Background and purpose: Cultured pre-adipocytes accumulate and metabolize zidovudine (ZDV), but its mode of accumulation into these cells is unclear. We investigated the mode of accumulation of [3H]-ZDV, and the impact of changes in external pH and modulators of drug transporters on its accumulation and metabolism. Experimental approach: The initial rate and steady-state accumulation of [3H]-ZDV were measured in 3T3-F442A cells. P-glycoprotein (P-gp) expression was detected by Western blotting. External pH was varied, and modulators of intracellular pH and drug transporters were used to study the mode of accumulation of ZDV. Phosphorylated ZDV metabolites were detected by high-performance liquid chromatography. Key results: Intracellular accumulation of ZDV was rapid, reaching equilibrium within 20 min; nigericin increased accumulation by 1.9-fold, but this did not alter the generation of ZDV mono-, di- and triphosphate. The accumulation and metabolism were pH dependent, being maximal at pH 7.4 and least at pH 5.1. Monensin, carbonyl cyanide p-trifluoromethoxy) phenyl hydrazone, brefeldin A, bafilomycin A1 and concanamycin A increased accumulation; 2-deoxyglucose, dipyridamole, thymidine and tetraphenylphosphonium inhibited accumulation. The accumulation was saturable; the derived Kd and capacity of binding were 250 nmol per 106 cells and 265 nM respectively. 3T3-F442A cells express P-gp; inhibitors of P-gp (XR9576 and verapamil), P-gp/BCRP (GF120918), multidrug resistance protein (MRP) (MK571) and MRP/OATP (probenecid) increased the accumulation of ZDV. Saquinavir, ritonavir, amprenavir and lopinavir increased accumulation. Conclusions and implications: The accumulation of ZDV in 3T3-F442A cells was rapid, energy dependent, saturable and pH sensitive. Western blot analysis showed that 3T3-F442A cells express P-gp, and direct inhibition assays suggest that ZDV is a substrate of P-gp and MRP.

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle. (C) 2009 Elsevier Ltd. All rights reserved.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Acquired resistance to therapeutic agents is a major clinical concern in the prevention/treatment of malaria. The parasite has developed resistance to specific drugs through two mechanisms: mutations in target proteins such as dihydrofolate reductase and the bc1 complex for antifolates and nathoquinones, respectively, and alterations in predicted parasite transporter molecules such as P-glycoprotein homologue 1 (Pgh1) and Plasmodium falciparum CRT (PfCRT). Alterations in the expression of Pgh1 have been associated with modified susceptibility to a range of unrelated drugs. The molecular mechanism(s) that is responsible for this phenotype is unknown. We have shown previously (A. M. Ndifor, R. E. Howells, P. G. Bray, J. L. Ngu, and S. A. Ward, Antimicrob. Agents Chemother. 37:1318-1323, 2003) that the anticonvulsant phenobarbitone (PB) can induce reduced susceptibility to chloroquine (CQ) in P. falciparum , and in the current study, we provide the first evidence for a molecular mechanism underlying this phenomenon. We demonstrate that pretreatment with PB can elicit decreased susceptibility to CQ in both CQ-resistant and CQ-sensitive parasite lines and that this is associated with the increased expression of the drug transporter Pgh1 but not PfCRT. Furthermore, we have investigated the proximal promoter regions from both pfmdr1 and pfcrt and identified a number of putative binding sites for nuclear receptors with sequence similarities to regions known to be activated by PB in mammals. Whole-genome analysis has revealed a putative nuclear receptor gene, providing the first evidence that nuclear receptor-mediated responses to drug exposure may be a mechanism of gene regulation in P. falciparum .

The development of drug resistance to affordable drugs has contributed to a global increase in the number of deaths from malaria. This unacceptable situation has stimulated research for new drugs active against multidrug-resistant Plasmodium falciparum parasites. In this regard, we show here that deshydroxy-1-imino derivatives of acridine (i.e., dihydroacridinediones) are selective antimalarial drugs acting as potent (nanomolar K(i)) inhibitors of parasite mitochondrial bc(1) complex. Inhibition of the bc(1) complex led to a collapse of the mitochondrial membrane potential, resulting in cell death (IC(50) approximately 15 nM). The selectivity of one of the dihydroacridinediones against the parasite enzyme was some 5000-fold higher than for the human bc(1) complex, significantly higher ( approximately 200 fold) than that observed with atovaquone, a licensed bc(1)-specific antimalarial drug. Experiments performed with yeast manifesting mutations in the bc(1) complex reveal that binding is directed to the quinol oxidation site (Q(o)) of the bc(1) complex. This is supported by favorable binding energies for in silico docking of dihydroacridinediones to P. falciparum bc(1) Q(o). Dihydroacridinediones represent an entirely new class of bc(1) inhibitors and the potential of these compounds as novel antimalarial drugs is discussed.

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

Widespread parasitic resistance has led to an urgent need for the development and implementation of new drugs for the treatment of Plasmodium falciparum malaria. Artemisinin and its derivatives are becoming increasingly important, used preferably in combination with a second antimalarial agent to increase the efficacy and slow the development of resistance. However, cost, production and pharmacological issues associated with artemisinin derivatives and potential partner drugs are hindering the implementation of combination therapies. This article reviews the molecular basis of the action of, and resistance to, different antimalarials and examines the prospects for the next generation of drugs to combat this potentially lethal human pathogen.

In response to the emergence of parasite drug resistance to currently deployed antimalarials, the scientific community, in partnership with the pharmaceutical industry and public organizations, has fashioned an antimalarial drug development portfolio for the sustained development and registration of safe, effective and cheap antimalarial medicines. The management of this portfolio is being driven by MMV (Medicines for Malaria Venture), with a number of projects recently reaching the clinical end of this drug development pipeline.

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug–protein interactions involving this mutant residue.

Highly active antiretroviral therapy has substantially improved prognosis in human immunodeficiency virus (HIV). However, the integration of proviral DNA, development of viral resistance, and lack of permeability of drugs into sanctuary sites (e.g., brain and lymphocyte) are major limitations to current regimens. Previous studies have indicated that the antimalarial drug chloroquine (CQ) has antiviral efficacy and a synergism with HIV protease inhibitors. We have screened a panel of antimalarial compounds for activity against HIV-1 in vitro. A limited efficacy was observed for CQ, mefloquine (MQ), and mepacrine (MC). However, marked synergy was observed between MQ and saquinavir (SQV), but not CQ in U937 cells. Furthermore, enhancement of the antiviral activity of SQV and four other protease inhibitors (PIs) by MQ was observed in MT4 cells, indicating a class specific rather than a drug-specific phenomenon. We demonstrate that these observations are a result of inhibition of multiple drug efflux proteins by MQ and that MQ also displaces SQV from orosomucoid in vitro. Finally, coadministration of MQ and SQV in CD-1 mice dramatically altered the tissue distribution of SQV, resulting in a >3-fold and >2-fold increase in the tissue/blood ratio for brain and testis, respectively. This pharmacological enhancement of in vitro antiviral activity of PIs by MQ now warrants further examination in vivo.

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of cross-resistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available1, rapidly killing all asexual stages of Plasmodium falciparum2. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria1, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience3, 4, 5 their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins6. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.

A series of short chain chloroquine (CQ) derivatives have been synthesized in one step from readily available starting materials. The diethylamine function of CQ is replaced by shorter alkylamine groups (4-9) containing secondary or tertiary terminal nitrogens. Some of these derivatives are significantly more potent than CQ against a CQ resistant strain of Plasmodium falciparum in vitro. We conclude that the ability to accumulate at higher concentrations within the food vacuole of the parasite is an important parameter that dictates their potency against CQ sensitive and the chloroquine resistant K1 P. falciparum.

The maintenance of acidic pH in the digestive vacuole of the malaria parasite is thought to be crucial to the digestion of host cell haemoglobin and the subsequent process of heme detoxification. It may also be important in the mode of action of chloroquine and in the mechanism of resistance to the drug. Obtaining a definitive measurement of digestive vacuole pH has been surprisingly difficult. Some of the techniques for the measurement of pH in acid vesicles are outlined here along with some key aspects that are specific to malaria parasites. The use of acridine orange and dextran-tagged dyes as probes for the measurement of digestive vacuole pH has proved problematic, yet some surprising findings have emerged from work with these compounds.

The potent antimalarial activity of chloroquine against chloroquine-sensitive strains can be attributed, in part, to its high accumulation in the acidic environment of the heme-rich parasite food vacuole. A key component of this intraparasitic chloroquine accumulation mechanism is a weak base "ion-trapping" effect whereupon the basic drug is concentrated in the acidic food vacuole in its membrane-impermeable diprotonated form. By the incorporation of amino functionality into target artemisinin analogues, we hoped to prepare a new series of analogues that, by virtue of increased accumulation into the ferrous-rich vacuole, would display enhanced antimalarial potency. The initial part of the project focused on the preparation of piperazine-linked analogues (series 1 (7-16)). Antimalarial evaluation of these derivatives demonstrated potent activity versus both chloroquine-sensitive and chloroquine-resistant parasites. On the basis of these observations, we then set about preparing a series of C-10 carba-linked amino derivatives. Optimization of the key synthetic step using a newly developed coupling protocol provided a key intermediate, allyldeoxoartemisinin (17) in 90% yield. Further elaboration, in three steps, provided nine target C-10 carba analogues (series 2 (21-29)) in good overall yields. Antimalarial assessment demonstrated that these compounds were 4-fold more potent than artemisinin and about twice as active as artemether in vitro versus chloroquine-resistant parasites. On the basis of the products obtained from biomimetic Fe(II) degradation of the C-10 carba analogue (23), we propose that these analogues may have a mode of action subtly different from that of the parent drug artemisinin (series 1 (7-16)) and other C-10 ether derivatives such as artemether. Preliminary in vivo testing by the WHO demonstrated that four of these compounds are active orally at doses of less than 10 mg/kg. Since these analogues are available as water-soluble salts and cannot form dihydroartemisinin by P450-catalyzed oxidation, they represent useful leads that might prove to be superior to the currently used derivatives, artemether and artesunate.

Background: The multidrug transporters P-glycoprotein (P-gp) and MRP1 are functionally expressed in several subclasses of lymphocytes. HIV-1 protease inhibitors interact with both; consequently the transporters could reduce the local concentration of HIV-1 protease inhibitors and, thus, influence the selection of viral mutants. Objectives: To study the effect of the expression of P-gp and MRP1 on the transport and accumulation of HIV-1 protease inhibitors in human lymphocytes and to study the effects of specific P-gp and MRP1 inhibitors. Methods: The initial rate and the steady-state intracellular accumulation of radiolabelled ritonavir, indinavir, saquinavir and nelfinavir was measured in three human lymphocyte cell lines: control CEM cells, CEM-MDR cells, which express 30-fold more P-gp than CEM cells, and CEM-MRPcells, which express fivefold more MRP1 protein than CEM cells. The effect of specific inhibitors of P-gp (GF 120918) and MRP1 (MK 571) was also examined. Results: Compared with CEM cells, the initial rates of uptake and the steady-state intracellular concentrations of all protease inhibitors are significantly reduced in CEM-MDR cells. The intracellular concentrations of the protease inhibitors are increased upon co-administration with GF 120918, in some cases to levels approaching those in CEM cells. The intracellular concentrations of the protease inhibitors are also significantly reduced in CEM-MRP cells. Co-administration with MK -571 can partially overcome these effects. Conclusions: The overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors at this major site of virus replication, which, potentially, could accelerate the acquisition of viral resistance. Targeted inhibition of P-gp may represent an important strategy by which this problem can be overcome.

Extensive drug resistance in Plasmodium falciparum emphasizes the urgent requirement for novel antimalarial agents. Here we report potent antimalarial activity of a number of diamidine compounds. The lead compound pentamidine is concentrated 500-fold by erythrocytes infected with P. falciparum. Pentamidine accumulation can be blocked by inhibitors of hemoglobin digestion, suggesting that the drug binds to ferriprotoporphyrin IX (FPIX). All of the compounds bound to FPIX in vitro and inhibited the formation of hemozoin. Furthermore, inhibitors of hemoglobin digestion markedly antagonized the antimalarial activity of the diamidines, indicating that binding to FPIX is crucial for the activity of diamidine drugs. Pentamidine was not accumulated into uninfected erythrocytes. Pentamidine transport into infected cells exhibits an initial rapid phase, nonsaturable in the micromolar range and sensitive to inhibition by furosemide and glibenclamide. Changing the counter-ion in the order Cl(-) < Br(-) < NO(2)(-) < I(-)

Strong support for the NHE hypothesis is the use of the progeny from the genetic cross produced by Wellems et al.10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10 Lanzer's group demonstrated that saturable CQ uptake was reduced and cytosolic pH increased in the resistant progeny. Both of these observations can be explained by alternative mechanisms (see above). The main phenotypic characteristics that define CQ sensitivity and resistance, in both the genetic cross and in unrelated isolates, is the ability of verapamil to enhance CQ's accumulation and activity8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References, 10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References. Alignment of these observations with Lanzer's proposal dictates that verapamil, a known inhibitor of drug transporters and ion channels, must somehow restore the ability of the NHE of CQR parasites (which apparently is already working at maximal capacity) to be stimulated by drugs. This contrasts with recent experimental data demonstrating that the verapamil effect is independent of NHE activity in that the ability of verapamil to raise CQ accumulation in CQR parasites is retained in sodium-free medium8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8. The verapamil effect is perfectly linked with the CQ-resistant phenotype in a genetic cross10xWellems, T.E. et al. Nature. 1990; 345: 253–255Crossref | PubMed | Scopus (354)See all References10. Hence, if mutations of the parasite NHE are involved in CQ resistance, resistant parasites would have to evolve an independent mechanism for verapamil-stimulated drug uptake, simultaneously. This unlikely scenario suggests that mutations of the NHE are not responsible for the CQ-resistant phenotype. This reasoning is supported experimentally by the finding that, in sodium-free medium, the steady-state accumulation of CQ into CQS parasites is fourfold higher than that of CQR parasites8xBray, P.G. et al. Mol. Pharmacol. 1998; 54: 170–179PubMedSee all References8.In a further attempt to validate the NHE paradigm, Lanzer and his colleagues contended19xSanchez, C.P., Horrocks, P., and Lanzer, M. Cell. 1998; 92: 601–602Abstract | Full Text | Full Text PDF | PubMedSee all References19 that NHE is homologous to the recently identified cg2 parasite protein20xSu, X. et al. Cell. 1997; 91: 593–603Abstract | Full Text | Full Text PDF | PubMedSee all References20. Mutations in cg2 were shown to be associated with CQ resistance phenotypes. However, a deeper and more critical scrutiny failed to confirm any homology between the two proteins21xWellems, T.E. et al. Cell. 1998; 94: 285–286Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References21.Lanzer's group have taken care to present an intriguing alternative hypothesis to explain CQ resistance, and most importantly they have re-focused attention on CQ resistance and stimulated many people's interest. We feel that this is the correct time to highlight some of the experimental and conceptual inconsistencies that emerge from these investigations and to position this hypothesis alongside alternative explanations. It is our opinion that the NHE hypothesis has significant shortcomings. We hope that this commentary, rather than merely criticizing their data, will be the stimulus for the robust testing of all of the scientific issues raised.

The antimalarial specificity of chloroquine (CQ) stems from the saturable uptake of the drug into malaria parasites. Strains of Plasmodium falciparum that are resistant to CQ have evolved a mechanism to reduce the saturable uptake of CQ and several biochemical models have been proposed to explain this. These include an efflux process analogous to multi-drug resistance (MDR) in cancer cells, reduced proton trapping due to elevated vacuolar pH, reduced binding to an intracellular receptor and reduced activity of a permease or drug importer. Here, we attempt to reconcile many of the apparently conflicting data used to support these models. Previous data are analysed in the context of our own model in which CQ uptake is determined by access of the drug to ferriprotoporphyrin IX (FPIX), the intracellular receptor. Copyright 1999 Harcourt Publishers Ltd.

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ–FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ–FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.

Despite considerable therapeutic success with the antimalarial 4-aminoquinolines such as chloroquine, there is serious doubt about the future of this drug class due mainly to the development and spread of parasite resistance throughout endemic areas. In this article we review the possible biochemical and molecular basis of resistance. Based on our current understanding we have considered the possibility of developing strategies which may allow the aminoquinolines to once again be used effectively against P. falciparum. Our conclusions are that drug resistance is the result of a reduced rate of drug uptake which in turn reduces the amount of drug available to bind the target. The basis for this reduced accumulation could be an altered pH gradient making the food vacuole more alkaline or the parasite cytosol more acidic, an efflux pump removing drug directly from the membrane or any other process which will reduce the rate of drug uptake. Central to the effectiveness of this resistance mechanism is the transient availability of a high affinity, low capacity drug binding site (possibly haem) within the parasite. Resistance reversers such as verapamil influence the apparent Ka for this drug binding phenomenon via an increased drug uptake rate. We demonstrate that by chemical modification of the aminoquinolines, producing predictable alterations in their physicochemical properties, that it is possible to minimise the verapamil sensitive component of resistance and reduce significantly cross-resistance patterns without loss in absolute activity. Based on these views we suggest that the aminoquinoline antimalarials still have a role to play in the cheap, safe and effective chemotherapy of falciparum malaria.

Aims: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Results: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. Innovation: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. Conclusion: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV. Antioxid. Redox Signal. 19, 683–695.

We have investigated a series of novel 4-aminoquinoline analogues related to amodiaquine, that possess side chain modifications designed to influence both drug pKa and lipophilicity. These compounds have been used to determine the influence of physicochemical properties on antimalarial activity against, and accumulation by, both chloroquine-susceptible and chloroquine-resistant isolates of Plasmodium falciparum. The compounds tested exhibited a 500-fold range of absolute antimalarial potency. Absolute drug potency and drug accumulation were found to be significantly correlated in each of the four isolates of Plasmodium falciparum studied. The level of accumulation was unrelated to lipophilicity and was significantly greater than the predicted levels of accumulation based on drug pKa, compartmental pH, and Henderson-Hasselbach considerations. Further analysis of the relationship between 4-aminoquinoline accumulation and activity implicated the involvement of additional forces in the accumulation process.

The intraerythrocytic malaria parasite is susceptible to oxidative stress and this may play a role in the mechanism of action of some antimalarial agents. Here we show that exposure of the intraerythrocytic malaria parasite to the oxidising agent hydrogen peroxide results in a fall in the intracellular ATP level and inhibition of the parasite's V-type H(+)-ATPase, causing a loss of pH control in both the parasite cytosol and the internal digestive vacuole. In contrast to the V-type H(+)-ATPase, the parasite's digestive vacuole H(+)-pyrophosphatase is insensitive to hydrogen peroxide-induced oxidative stress. This work provides insights into the effects of oxidative stress on the intraerythrocytic parasite, as well as providing an alternative possible explanation for a previous report that light-induced oxidative stress causes selective lysis of the parasite's digestive vacuole.

Control of falciparum malaria has become almost impossible in many areas due to the development of resistance to chloroquine and other antimalarial drugs. Verapamil and a number of unrelated compounds which chemosensitise multi-drug resistant cancer cells also enhance chloroquine susceptibility in Plasmodium falciparum . Chloroquine is accumulated to lower levels in resistant plasmodia, hence the reversal of chloroquine resistance has been attributed to the ability of chemosensitising agents to increase the amount of chloroquine accumulated by the resistant parasite. We have conducted a detailed examination of the effect of verapamil on chloroquine sensitivity and its relationship to chloroquine accumulation. The ability of verapamil to increase steady-state chloroquine accumulation was found to be totally insufficient to explain the increase in chloroquine sensitivity caused by the drug. In contrast, when chloroquine accumulation was increased by raising the pH gradient, the corresponding shifts in sensitivity to chloroquine could be accurately predicted. These results were confirmed with other classes of chemosensitisers and we conclude that an alternative mechanistic explanation is required to completely explain the reversal of chloroquine resistance in P. falciparum

Background Plasmodium falciparum chloroquine resistance (CQR) transporter protein (PfCRT) is known to be the important key of CQR. Recent studies have definitively demonstrated a link between mutations in the gene pfcrt and resistance to chloroquine in P. falciparum. Although these mutations are predictive of chloroquine resistance, they are not quantitatively predictive of the degree of resistance. Methods In this study, a total of 95 recently adapted P. falciparum isolates from Thailand were included in the analysis. Parasites were characterized for their drug susceptibility phenotypes and genotypes with respect to pfcrt. From the original 95 isolates, 20 were selected for complete pfcrt sequence analysis. Results Almost all of the parasites characterized carried the previously reported mutations K76T, A220S, Q271E, N326S, I356T and R371I. On complete sequencing, isolates were identified with novel mutations at K76A and E198K. There was a suggestion that parasites carrying E198K were less resistant than those that did not. In addition, pfcrt and pfmdr1 gene expression were investigated by real-time PCR. No relationship between the expression level of either of these genes and response to drug was observed. Conclusion Data from the present study suggest that other genes must contribute to the degree of resistance once the resistance phenotype is established through mutations in pfcrt.

Tebuquine (5) is a 4-aminoquinoline that is significantly more active than amodiaquine (2) and chloroquine (1) both in vitro and in vivo. We have developed a novel more efficient synthetic route to tebuquine analogues which involves the use of a palladium-catalyzed Suzuki reaction to introduce the 4-chlorophenyl moiety into the 4-hydroxyaniline side chain. Using similar methodology, novel synthetic routes to fluorinated (7a, b) and a dehydroxylated (7c) analogue of tebuquine have also been developed. The novel analogues were subjected to testing against the chloroquine sensitive HB3 strain and the chloroquine resistant K1 strain of Plasmodium falciparum. Tebuquine was the most active compound tested against both strains of Plasmodia. Replacement of the 4-hydroxy function with either fluorine or hydrogen led to a decrease in antimalarial activity. Molecular modeling of the tebuquine analogues alongside amodiaquine and chloroquine reveals that the inter-nitrogen separation in this class of drugs ranges between 9.36 and 9.86 A in their isolated diprotonated form and between 7.52 and 10.21 A in the heme-drug complex. Further modeling studies on the interaction of 4-aminoquinolines with the proposed cellular receptor heme revealed favorable interaction energies for chloroquine, amodiaquine, and tebuquine analogues. Tebuquine, the most potent antimalarial in the series, had the most favorable interaction energy calculated in both the in vacuo and solvent-based simulation studies. Although fluorotebuquine (7a) had a similar interaction energy to tebuquine, this compound had significantly reduced potency when compared with (5). This disparity is possibly the result of the reduced cellular accumulation (CAR) of fluorotebuquine when compared with tebuquine within the parasite. Measurement of the cellular accumulation of the tebuquine analogues and seven related 4-aminoquinolines shows a significant relationship (r = 0.98) between the CAR of 4-aminoquinoline drugs and the reciprocal of drugs IC50.

Recent reports suggest that lower levels of chloroquine accumulation in chloroquine-resistant isolates of Plasmodium falciparum are achieved by energy-dependent chloroquine efflux from resistant parasites. In support of this argument, a rapid chloroquine efflux phenotype has been observed in some chloroquine-resistant isolates of P. falciparum. In this study, no relationship was found between chloroquine sensitivity and the rate of [3H]chloroquine efflux from four isolates of P. falciparum with a greater than 10-fold range in sensitivity to chloroquine. All the isolates tested displayed the rapid efflux phenotype, irrespective of sensitivity. However, chloroquine sensitivity of these isolates was correlated with energy-dependent rate of drug accumulation into these parasites. Verapamil and a variety of other compounds reverse chloroquine resistance. The reversal mechanism is assumed to result from competition between verapamil and chloroquine for efflux protein translocation sites, thus causing an increase in steady-state accumulation of chloroquine and hence a return to sensitivity. Verapamil accumulation at a steady-state is increased by chloroquine, possibly indicating competition for efflux of the two substrates. Increases in steady-state verapamil concentrations caused by chloroquine were identical in sensitive and resistant strains, suggesting that similar capacity efflux pumps may exist in these isolates. These data suggest that differences in steady-state chloroquine accumulation seen in these isolates can be attributed to changes in the chloroquine concentrating mechanism rather than the efflux pump. It seems likely that chloroquine resistance generally in P. falciparum, results at least in part from a change in the drug concentrating mechanism and that changes in efflux rates per se are insufficient to explain chloroquine resistance.

Cepharanthin is a proprietary extract of Stephania cepharantha , widely used in Japan for the treatment of inflammatory diseases. Cephranthin, its component alkaloids, and the standard resistance modulator verapamil were tested against Plasmodium falciparum for capacity to modulate sensitivity to chloroquine. Cepharanthin enhanced the activity of chloroquine against resistant clones by a factor of 15 at a concentration of only 200 nM (1.2 ng/ml). It is 50 times more potent than verapamil and 3 times more potent than the sum of its individual alkaloids. Combinations of component alkaloids acted synergistically to sensitize the parasite to chloroquine, possibly explaining the enhanced potency of Cepharanthin. Cepharanthin differed from verapamil in that it further sensitized clones that are considered to be fully susceptible, improving the baseline activity of chloroquine. Potent sensitization of parasites to chloroquine in vitro coupled with low toxicity suggests that coadministration of Cepharanthin might extend the clinical utility of chloroquine.

Background Malaria is a global health emergency, and yet our understanding of the energy metabolism of the principle causative agent of this devastating disease, Plasmodium falciparum, remains rather basic. Glucose was shown to be an essential nutritional requirement nearly 100 years ago and since this original observation, much of the current knowledge of Plasmodium energy metabolism is based on early biochemical work, performed using basic analytical techniques (e.g. paper chromatography), carried out almost exclusively on avian and rodent malaria. Data derived from malaria parasite genome and transcriptome studies suggest that the energy metabolism of the parasite may be more complex than hitherto anticipated. This study was undertaken in order to further characterize the fate of glucose catabolism in the human malaria parasite, P. falciparum. Methods Products of glucose catabolism were determined by incubating erythrocyte-freed parasites with D-[1-13C] glucose under controlled conditions and metabolites were identified using 13C-NMR spectroscopy. Results Following a 2 h incubation of freed-P. falciparum parasites with 25 mM D-[1-13C] glucose (n = 4), the major metabolites identified included; [3-13C] lactate, [1,3-13C] glycerol, [3-13C] pyruvate, [3-13C] alanine and [3-13C] glycerol-3-phosphate. Control experiments performed with uninfected erythrocytes incubated under identical conditions did not show any metabolism of D-[1-13C] glucose to glycerol or glycerol-3-phosphate. Discussion The identification of glycerol as a major glucose metabolite confirms the view that energy metabolism in this parasite is more complex than previously proposed. It is hypothesized here that glycerol production by the malaria parasite is the result of a metabolic adaptation to growth in O2-limited (and CO2 elevated) conditions by the operation of a glycerol-3-phosphate shuttle for the re-oxidation of assimilatory NADH. Similar metabolic adaptations have been reported previously for other microaerobic/anaerobic organisms, such as yeast, rumen protozoa and human parasitic protozoa. Conclusion These data highlight the need to re-evaluate the carbon and redox balance of this important human pathogen, ultimately leading to a better understanding of how the parasite is able to adapt to the variable environments encountered during parasite development and disease progression.

The function of transporters in peripheral blood mononuclear cells (PBMC) has been characterized, but less is known about cytochrome P450 (CYP) enzyme function in these cells. Given that cytokines are dysregulated in many diseases, the purpose of this work was to assess the impact of cytokines on the expression of CYPs, transporters and chemokine receptors in PBMC. Human PBMC were incubated with cytokines for 48 h. ATP-binding cassette (ABC)B1, ABCC1, ABCC2, CYP2B6, CYP3A4, CXCR4 and CCR5 expression were measured by quantitative polymerase chain reaction and flow cytometry at 0, 4, 8, 24 and 48 h. Enzyme activity was assessed using fluorescent probes. We show here functional activity of CYP3A4 and CYP2B6 in PBMC. Furthermore, cytokines had a significant impact on the mRNA and protein expression of all proteins. For example, interleukin-2 (IL-2) had a marked impact on ABCB1 mRNA (% control 4745 +/- 11961) and protein (% control 200 +/- 57). Increases in drug efflux transporter expression, in response to cytokines, resulted in reduced cellular accumulation of digoxin [decrease of 17% and 26% for IL-2 and interferon-gamma (IFN gamma) respectively] and saquinavir (decrease of 28% and 30% for IL-2 and IFN gamma respectively). The degree to which drug transporter and chemokine receptor expression changed in response to cytokines was positively correlated (e.g. ABCB1 and CXCR4, r(2) = 0.545). These data have important implications for diseases in which cytokines are dysregulated and for which pharmacological intervention targets immune cells.

It has been estimated that in 2013 there were approximately 198 million cases of malaria (with an uncertainty range of 124–283 million) and an estimated 584,000 deaths (with an uncertainty range of 367,000–755,000), with the majority of deaths amongst African children under 5 years of age [1]. As a result of global efforts, including in transmission control (e.g. removal of breeding sites using insecticides and prevention of human contact through screens and bed nets), improved antimalarial chemotherapy and early effective case management, malaria mortality rates have fallen by 47 % globally and by 54 % in Africa since 2000 [1].

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pf dhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanil-dapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolate-artemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2003 and 2005.

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.

Malaria is a disease in desperate need of new chemotherapeutic approaches. Certain microtubule inhibitors, including vinblastine and taxol, have highly potent activity against malarial parasites and disrupt the normal microtubular structures of intra-erythrocytic parasites at relevant concentrations. While these inhibitors are useful tools, their potential as anti-malarial drugs is limited by their high toxicity to mammalian cells. In contrast, two classes of antimitotic herbicide, namely dinitroanilines (e.g. trifluralin and oryzalin) and phosphorothioamidates (e.g. amiprophosmethyl), exhibit moderate activity against the major human malarial parasite Plasmodium falciparum in culture but very low mammalian cytotoxicity. We examined the dynamics and kinetics of uptake and subcellular compartmentation of [14C]trifluralin in comparison with [3H]vinblastine. We wished to determine whether the relatively modest activity of trifluralin was the consequence of poor uptake into parasite cells. Trifluralin accumulated in parasite-infected erythrocytes to approximately 300 times the external concentration and vinblastine at up to approximately 110 times. Accumulation into uninfected erythrocytes was much lower. Uptake of trifluralin was rapid, non-saturable and readily reversed. It appears that the hydrophobic nature of trifluralin leads to accumulation largely in the membranes of the parasite, reducing the levels in the soluble fraction and limiting access to its microtubular target. By contrast, vinblastine accumulated predominantly in the soluble fraction and uptake was saturable and mostly irreversible, consistent with binding predominantly to tubulin. The results indicate that synthesis of more polar trifluralin derivatives may be a promising approach to designing microtubule inhibitors with more potent antimalarial activity.

The selective antimalarial activity of chloroquine and related compounds stems from the extensive saturable uptake of these drugs into malaria parasites. Chloroquine resistant strains of Plasmodium falciparum have evolved a mechanism to reduce the saturable uptake. The molecular mechanism of saturable chloroquine uptake is controversial and attention is currently focused on mutually exclusive models of active chloroquine uptake and intracellular chloroquine binding. We sum up recent evidence which conclusively proves that the saturable accumulation of chloroquine is due to intracellular binding to ferriprotoporphyrin IX rather than active transport into the parasite via the sodium/hydrogen exchanger. We discuss recent findings that the affinity of chloroquine binding to ferriprotoporphyrin IX is reduced in resistant parasites. The mechanism responsible for reduced binding affinity can be overcome by verapamil and various lysosomotropic agents, and is thought to be the basis of chloroquine resistance.

Unsymmetrical dispiro- and spirotetraoxanes have been designed and synthesized via acid-catalyzed cyclocondensation of bis(hydroperoxides) with ketones. Incorporation of water-soluble and polar functionalities, via reductive amination and amide bond formation, produces several analogues with low nanomolar in vitro antimalarial activity. Several analogues display an unprecedented level of oral antimalarial activity for this class of endoperoxide drug.

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b(3), K1CB(2), PR70CB(1), PR71CB(2) and TM(4)CB8-2.2.3). Fifty percent inhibitory concentration (IC(50)) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30mug), NADPH (1nM) and phosphate buffer pH 7.4, carried out at 37 degrees C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994(b3), PR(70)CB(1), PR(71)CB(2)) and sensitive (TM(4)CB8-2.2.3, K1CB(2)) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [(14)C]MQ as a tracer. The time courses of [(14)C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [(14)C]MQ was observed in the resistant compared with sensitive clones.

Plasmodium falciparum is the most important parasitic pathogen in humans, causing hundreds of millions of malaria infections and millions of deaths each year. At present there is no effective malaria vaccine and malaria therapy is totally reliant on the use of drugs. New drugs are urgently needed because of the rapid evolution and spread of parasite resistance to the current therapies. Drug resistance is one of the major factors contributing to the resurgence of malaria, especially resistance to the most affordable drugs such as chloroquine. We need to fully understand the antimalarial mode of action of the existing drugs and the way that the parasite becomes resistant to them in order to design and develop the new therapies that are so urgently needed. In respect of the quinolines and artemisinins, great progress has been made recently in studying the mechanisms of drug action and drug resistance in malaria parasites. Here we summarize from a historical, biological and chemical, perspective the exciting new advances that have been made in the study of these important antimalarial drugs.

The aim of this study was to synthesise pure enantiomers of potent antimalarial 1,2,4-trioxanes, which are related to the natural antimalarial artemisinin, and then to assay each against a panel of Plasmodium falciparum strains. The working hypothesis was that if the artemisinin derivatives interact with a specific protein-target site, then there should be stereoselective differences in their activity. In five different P. falciparum isolates, however, the trioxane enantiomers (+)-7 a, (-)-7 a and (+)-7 b, (-)-7 b, showed the same level of in vitro antiparasitic activity.