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Five tetranortriterpenoids, domesticulide A-E (1-5), were isolated from seeds of Lansium domesticum Corr. together with 11 known triterpenoids (6-16). Their structures were elucidated by analysis of their spectroscopic data. Compounds 2, 3, 4, 7, 8, 10, 11, and 15 showed antimalarial activity against Plasmodium falciparum with IC(50)'s of 2.4-9.7 microg/ml.
The evolution of antimalarial drug resistance is often considered to be a single-stage process in which parasites are either fully resistant or completely sensitive to a drug. However, this does not take into account the important intermediate stage of drug tolerance. Drug-tolerant parasites are killed by the high serum concentrations of drugs that occur during direct treatment of the human host. However, these parasites can spread in the human population because many drugs persist long after treatment, and the tolerant parasites can infect people in which there are residual levels of the drugs. This intermediate stage between fully sensitive and fully resistant parasites has far-reaching implications for the evolution of drug-resistant malaria.
Artemisinin derivatives are clinically effective and safe antimalarials, but are not recommended during the first trimester of pregnancy because of the resorptions and abnormalities seen in animal reproduction studies. Understanding how, when and what toxicity occurs is crucial to any assessment of clinical relevance. Previously, DHA has been shown in the rat whole embryo culture (WEC) to primarily affect primitive red blood cells (RBCs) causing subsequent tissue damage and dysmorphogenesis. To verify the primary target of DHA in vivo and to detect consequences induced by early damage on embryo development, pregnant female rats were orally treated on gestation days (GD) 9.5 and 10.5 with 7.5 or 15 mg/kg/day DHA and caesarean sectioned on GD11.5, 12.5, 13.5, 15 and 20. A parallel in vitro WEC study evaluated the role of oxidative damage and examined blood islands and primitive RBCs. In accordance with the WEC results, primitive RBCs from yolk sac hematopoiesis were the target of DHA in vivo. The resulting anemia led to cell damage, which depending on its degree, was either diffuse or focal. Embryonic response to acute anemia varied from complete recovery to malformation and death, depending on the extent of cell death. Malformations occurred only in litters with embryonic deaths. DHA induced low glutathione levels in RBCs, indicating that oxidative stress may be involved in artemisinin toxicity; effects were extremely rapid, with altered RBCs seen as early as GD10. In establishing the relevance of these findings to humans, one should consider differences in the development of rodents and humans. While yolk sac hematopoiesis occurs similarly in the two species, early placentation and extent of exposure differ. In particular, early hematopoiesis takes only 7 days in rats (during which RBCs expand in a clonal fashion) compared with 6 weeks in humans; thus the susceptible period in relation to the duration of exposure to an artemisinin-based treatment may be substantially different.
This collaborative cross-border study was performed to determine the therapeutic efficacy of antimalarial drugs used by the National Programmes for falciparum malaria along the eastern Indo-Nepal border where there is unregulated population movement across the border. The study was conducted at sites in Jhapa District, Nepal and Darjeeling District, India. The study was conducted from August 2003 to February 2004, following the WHO 28 day treatment protocol. The efficacy of chloroquine was tested in India among 91 subjects and of sulfadoxine-pyrimethamine in Nepal among 107 subjects with laboratory-confirmed Plasmodium falciparum malaria. Of the 102 subjects who completed the study in Nepal, there were 21 (20.6%) treatment failures comprising 7 (6.9%) early treatment failures (ETF) and 14 (14.7%) late treatment failures (LTF) (5 late clinical failures [LCF] and 9 late parasitological failures [LPF]). Of the 89 subjects who completed the study in India, there were 46 (51.7%) treatment failures comprising 7 (7.9%) ETFs and 39 (43.8%) LTFs (13 LCFs and 26 LPFs). Based on WHO guidelines both countries need to review their drug policy urgently and make appropriate changes, taking into account aspects of cross-border collaboration in the control of drug-resistant malaria.
We studied three antimalarial treatments in Caala and Kuito, Angola, in 2002 and 2003. We tested chloroquine (CQ), amodiaquine (AQ) and sulfadoxine-pyrimethamine (SP) in Caala, and AQ, SP and the combinations AQ+artesunate (AQ+AS) and SP+artesunate (SP+AS) in Kuito. A total of 619 children (240 in Caala, 379 in Kuito) with uncomplicated Plasmodium falciparum malaria were followed-up for 28 days, with PCR genotyping to distinguish recrudescence from reinfection. PCR-corrected failure proportions at day 28 were very high in the CQ group (83.5%, 95% CI 74.1-90.5), high in the SP groups (Caala: 25.3%, 95% CI 16.7-35.8; Kuito: 38.8%, 95% CI 28.4-50.0), around 20% in the AQ groups (Caala: 17.3%, 95% CI 10.0-27.2; Kuito: 21.6%, 95% CI 14.3-30.6) and very low in the artemisinin-based combination groups (1.2%, 95% CI 0.0-6.4 for each combination AQ+AS and SP+AS). These results show that CQ and SP are no longer efficacious in Caala and Kuito and that the moderate efficacy of AQ is likely to be compromised in the short term if used as monotherapy. We recommend the use of AQ with AS, though this combination might not have a long useful therapeutic life because of AQ resistance.
We performed in vitro drug susceptibility assays of ten Plasmodium falciparum isolates collected from Vict Nam in 1995. All isolates were found to be highly sensitive to artesunate, dihydro-artemisinin and artemisinin. They were also sensitive to quinine. All of them were resistant to chloroquine and mefloquine in vitro. This study provides the baseline estimates of in vitro susceptibility levels of the Vietnamese isolates to artermisinin and their derivatives because the data were collected soon after these drugs were introduced countrywide. It also describes some quantitative profiles of the in vitro response of other standard antimalarial drugs in Viet Nam, which is presently limited.
The ex vivo antimalarial activity of plasma samples obtained from 20 healthy Caucasian volunteers following daily proguanil (200 mg) plus dapsone (8 mg) for malaria chemoprophylaxis inhibited five cycloguanil-resistant Thai isolates of Plasmodium falciparum. All volunteers were phenotyped as extensive metabolisers (EMs) of proguanil. Three of the five isolates were obtained from Thai soldiers who had failed malaria prophylaxis on daily proguanil (200 mg) plus dapsone (4.0 or 12.5 mg). The Thai soldiers were also classified as EMs, but had relatively lower plasma cycloguanil concentrations compared to values reported in the literature for Caucasians and black Kenyans. Although the high level of parasite resistance to cycloguanil was the most likely explanation for the Thai soldiers failing prophylaxis on proguanil plus dapsone, their low cycloguanil concentrations may have also contributed to their lack of protection. However, in areas where parasites are more susceptible to cycloguanil, such as in certain regions of Africa, proguanil plus dapsone may still be an effective chemoprophylactic drug combination.
Several reports suggest that the antimalarial mode of action of quinoline drugs may differ in their mechanistic details. The malaria parasite Plasmodium falciparum was treated in culture with chloroquine, amodiaquine, quinine and mefloquine in a dose- and time-dependent fashion. After removal of the drug, the viability of the parasites and their hemoglobin content were determined. Whereas in the presence of chloroquine and amodiaquine, there was a correlation between parasite killing and accumulation of hemoglobin, with quinine and mefloquine parasite killing was not associated with the accumulation of hemoglobin. Mefloquine inhibited the chloroquine-dependent accumulation of hemoglobin. It is suggested that whereas chloroquine and amodiaquine inhibit the digestion of hemoglobin, mefloquine and possibly quinine inhibit the ingestion of host cell hemoglobin by interfering with the ingestion process. These results may explain the demonstrable antagonism between chloroquine and mefloquine and their antipodal sensitivity to these drugs.
The antimalarial artemisinin derivatives artesunate (ART), arteether (ARE), and artemether (ARM) reveal remarkable antineoplastic activity. In the present investigation, we identified mRNA expression profiles associated with the response of tumor cells to ART, ARE, and ARM. We performed correlation and hierarchical cluster analyses of inhibition concentration 50% (IC(50)) values and basal mRNA expression levels of 464 genes deposited in the database of the National Cancer Institute, USA. Correlating IC(50) values of ART, ARE, and ARM and of 16 established antineoplastic drugs revealed that the artemisinin derivatives could not be assigned with a known class of drugs with defined mode(s) of action. The basal mRNA expression of 208 out of 464 genes (45%) correlated significantly with IC(50) values of at least one artemisinin derivative. These genes were from different classes (drug resistance genes, DNA damage and repair genes, apoptosis-regulating genes, proliferation-associated genes, oncogenes, tumor suppressor genes and cytokines). We identified two different gene clusters by hierarchical cluster analysis. One cluster contained predominately genes significantly correlated to all three artemisinin derivatives. This overlapping set of genes points to common molecular mechanisms of tumor inhibition by all three drugs in which genes affecting cellular proliferation may play an important role. The second cluster contained genes differentially associated with the response of artemisinin derivatives to cancer cells. The number of correlating drug resistance genes in this cluster increased in the order ART<ARE<ARM and was paralleled by increasing IC(50) values of the three drugs in the same order. The higher activity of ART in comparison to ARE and ARM may, thus, be explained by a lower number of drug resistance genes affecting ARTs action. The present analysis is a starting point for the generation of hypotheses on candidate genes and for a more detailed dissection of the functional role of individual genes for the activity of artemisinin derivatives in tumor cells.
Various biguanide derivatives are used as antihyperglycemic and antimalarial drugs (e.g., 1,1-dimethyl biguanide (metformin), phenylethyl biguanide (phenformin), N-(4-chlorophenyl)-N'-(isopropyl)-imidodicarbonimidic diamide (proguanil)); however, no common mechanism has been suggested in these controversial therapeutic actions. Biguanides bind endogenous metals that inhibit cysteine proteases independently, e.g., Zn(2+), Cu(2+), Fe(3+). Here, various biguanide derivatives are reported to be metal-interactive inhibitors of cathepsin B from mammals and falcipain-2 from Plasmodium falciparum. Structural homologies were identified among the Phe-Arg protease substrate motif and the metal complexes of phenformin and proguanil. Molecular modeling revealed that the position of the scissile amide substrate bond corresponds to the biguanide-complexed inhibitory metal when the phenyl groups are homologously aligned. Binding of the phenformin-metal complex within the active site of human cathepsin B was modeled with computational docking. A major binding mode involved binding of the drug phenyl group at the protease S2 subsite, and the complexed inhibitory metal shared between the drug and the protease Cys29-His199 catalytic pair. Cysteine protease inhibition was assayed with carbobenzyloxy-PHE-ARG-7-aminomethylcoumarin substrate. In the absence of metal ions, phenformin was a weakly competitive protease inhibitor (apparent K(i) several microM); however, metformin was noninhibitory. In contrast, the metal complexes of both metformin and phenformin were protease inhibitors with potency at therapeutic concentrations. Biguanide-metal complexes were more potent cysteine protease inhibitors than either the biguanide or metal ions alone, i.e., synergistic. Similar to chloroquine, therapeutic extracellular concentrations of metformin, phenformin, and proguanil caused metal-interactive inhibition of lysosomal protein degradation as bioassayed in primary tissue using perfused myocardium. The biguanide moiety is identified as a past and future structural scaffold for synthesis of many protease inhibitors. Results are discussed in relation to Zn(2+)-interactive inhibition of insulin degradation in hormone target tissues, and Fe(3+)-interactive inhibition of hemoglobin degradation in parasite food vacuoles. Previous studies on insulin hypercatabolism and insulin resistance are speculatively reviewed in light of present findings.
2-Amino-4-(3,5-diacetylphenyl)amino-1,6-dimethylpyrimidinium chloride (CNI-H0294) is a novel arylene bis(methylketone) compound that displays antimalarial activity against chloroquine- and pyrimethamine-resistant Plasmodium falciparum clones. The compound has been found to be concentrated into infected erythrocytes, with 80-179 microM accumulated when parasites were cultured in the presence of 1.0 microM CNI-H0294. Uninfected erythrocytes, in contrast, only accumulated 2.5-3.4 microM CNI-H0294 under identical conditions. Using postmitochondrial supernatants from a number of parasite clones, the compound was found to inhibit dihydrofolate reductase (EC 1.5.1.3) activity with an IC50 of 243-483 microM. Thus, while CNI-H0294 is not a powerful inhibitor of plasmodial dihydrofolate reductase, the accumulation of the compound into infected erythrocytes, when correlated to the external ED50 concentration against parasite growth in vitro, reaches concentrations sufficient to inhibit the malarial enzyme.
Two classes of drugs interact with DNA topoisomerase (topo) I, namely topoI poisons such as the camptothecins, which create DNA single-strand breaks and the catalytic inhibitors, which do not. Here, we demonstrate that the antimalarial agent chloroquine is a catalytic inhibitor of eukaryote topoI, as the drug inhibited topoI-mediated DNA relaxation. Chloroquine is known to be a topoII catalytic inhibitor and as such is able to inhibit the activity of a topoII poison, i.e. etoposide. We now show that chloroquine also inhibits the topol poison camptothecin as camptothecin-stimulated nicking of plasmid DNA was inhibited by chloroquine. These observations also apply to endogenous topoI in whole cells. Accordingly, camptothecin-induced single-strand breaks as well as cytotoxicity were antagonised by chloroquine. Further, in a band depletion assay in whole cells, chloroquine prevented camptothecin-mediated topoI trapping, indicating that chloroquine inhibits topoI by interfering with the DNA binding step of the enzyme. In contrast to camptothecin, chloroquine is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. This leads to the possibility of directing cytotoxicity to solid tumors with low extracellular pH by combining a neutral anticancer agent, i.e. camptothecin with a weak base antagonist, i.e. chloroquine. To test the feasibility of this principle, we investigated the drug combination at varying extracellular pH. We found that the antagonising effect of chloroquine on camptothecin-mediated trapping of topoI and DNA single-strand break formation was abolished at acidic extracellular pH. In a clonogenic assay, camptothecin in combination with chloroquine selectively killed cells at low pH (6.2), while camptothecin cytotoxicity was antagonised by chloroquine at normal pH (7.2). In conclusion, we show that the topoI catalytic inhibitor chloroquine inhibits camptothecin and that chloroquine can target the cytotoxic effect of camptothecin to tumor cells in acidic environments.
We propose here a new and detailed model for the antimalarial action of chloroquine (CQ), based on the its ability to inhibit degradation of heme by glutathione. Heme, which is toxic to the malaria parasite, is formed when the intraerythrocytic malaria parasite ingests and digests inside its food vacuole its host cell cytosol, which consists mainly of hemoglobin. The parasite protects itself against the toxicity of heme by polymerizing some of it to insoluble hemozoin (HZ). We show here that in Plasmodium falciparum at the trophozoite stage only ca. 30% of the heme is converted into hemozoin. We suggest that nonpolymerized heme exits the food vacuole and is subsequently degraded by glutathione, as has been shown before for uninfected erythrocytes. Marginal amounts of free heme could be detected in the membrane fraction of infected cells but nowhere else. It is well established that CQ and amodiaquine (AQ) accumulate in the parasite's food vacuole and inhibit heme polymerization, thereby increasing its efflux out of the food vacuole. We found that these drugs competitively inhibit the degradation of heme by glutathione, thus allowing heme to accumulate in membranes. Incubation of intact infected cells with CQ and AQ results in a marked increase in membrane-associated heme in a dose- and time-dependent manner, and a relationship exists between membrane heme levels and the extent of parasite killing. Heme has been shown to disrupt the barrier properties of membranes and to upset ion homeostasis in CQ-treated malaria-infected cells. In agreement with the predictions of our model, increasing the cellular levels of glutathione leads to increased resistance to CQ, whereas decreasing them results in enhanced sensitivity to the drug. These results insinuate a novel mechanism of drug resistance.
We have shown previously that chloroquine and amodiaquine inhibit the glutathione-dependent degradation of ferriprotoporphyrin IX (FP). We have also demonstrated that treatment of human erythrocytes infected with Plasmodium falciparum with chloroquine or amodiaquine results in a dose- and time-dependent accumulation of FP in the membrane fraction of these cells in correlation with parasite killing. High levels of membrane FP are known to perturb the barrier properties of cellular membranes, and could thereby irreversibly disturb the ion homeostasis of the parasite and cause parasite death. We here report on the effect of various 4-aminoquinolines, as well as pyronaridine, halofantrine and some bis-quinolines, on glutathione-mediated destruction of FP in aqueous solution, when FP was bound non-specifically to a protein, and when it was dissolved in human erythrocyte ghost membranes. We showed that all drugs were capable of inhibiting FP degradation in solution. The inhibitory efficacy of some drugs declined when FP was bound non-specifically to protein. Quinine and mefloquine were unable to inhibit the degradation of membrane-associated FP, in line with their inability to increase membrane-associated FP levels in malaria-infected cells following drug treatment. The discrepancy between chloroquine and amodiaquine on the one hand, and quinine and mefloquine on the other, is discussed in terms of the particular location of drugs and FP in the phospholipid membrane, and may suggest differences in the mechanistic details of the antimalarial action of these drugs.
Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.
In vitro, the heme cofactor of human iron(II) hemoglobin was efficiently and quickly alkylated at meso positions by the peroxide-based antimalarial drug artemisinin, leading to heme-artemisinin-derived covalent adducts. This reaction occurred in the absence of any added protease or in the presence of an excess of an extra non-heme protein, or even when artemisinin was added to hemolysed human blood. This activation of artemisinin by the heme moiety of non-digested hemoglobin clearly indicates the high affinity of this drug for heme, and its efficient alkylating ability under very mild conditions.
Plasmodium falciparum glutathione reductase (PfGR) has emerged as a drug target against tropical malaria. Here we report the expression of PfGR in Escherichia coli SG5(DE3) and isolation procedures for this protein. Recombinant PfGR does not differ from the authentic enzyme in its enzymic properties, the turnover number being 9900 min(-1). The dimeric flavoenzyme exhibits redox-dependent absorption spectra; the single tryptophan residue (per 57.2 kDa subunit) is strongly fluorescent. PfGR can be inhibited by the antimalarial drug methylene blue at therapeutic concentrations; the Ki for non-competitive inhibition is 6.4 microM. The sensitivity to methylene blue is observed also at high ionic strength so that, by analogy to human GR, analysis of crystalline enzyme-drug complexes can be envisaged.
Mechanism-based rational design and gram-scale chemical synthesis have produced some new trioxane and endoperoxide antimalarial drug candidates that are efficacious and safe. This review summarises recent achievements in this area of peroxide drug development for malaria chemotherapy.