Antimalarial Activity Of Chloroquine Research Articles

A Partial Convergence in Action of Methylene Blue and Artemisinins: Antagonism with Chloroquine, a Reversal with Verapamil, and an Insight into the Antimalarial Activity of Chloroquine

Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH₂ of riboflavin (RF), and FADH₂ of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH₂, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin-mediated oxidation of RFH₂ generated from N-benzyl-1,4-dihydronicotinamide (BNAH)-RF, or FADH₂ generated from NADPH or NADPH-Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor-acceptor or π complexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ-MB and CQ-artemisinins in vitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP-CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ-resistance transporter (PfCRT).

A verapamil-sensitive chloroquine-associated H+ leak from the digestive vacuole in chloroquine-resistant malaria parasites

Chloroquine resistance in the malaria parasite Plasmodium falciparum has made malaria increasingly difficult to control. Chloroquine-resistant parasites accumulate less chloroquine than their chloroquine-sensitive counterparts; however, the mechanism underlying this remains unclear. The primary site of accumulation and antimalarial action of chloroquine is the internal acidic digestive vacuole of the parasite, the acidity of which is maintained by inwardly-directed H+ pumps, working against the (outward) leak of H+. In this study we have investigated the leak of H+ from the digestive vacuole of the parasite by monitoring the alkalinisation of the vacuole following inhibition of the H+-pumping V-type ATPase by concanamycin A. The rates of alkalinisation observed in three chloroquine-resistant strains were two- to fourfold higher than those measured in three chloroquine-sensitive strains. On addition of chloroquine there was a dramatic increase in the rate of alkalinisation in the chloroquine-resistant strains, whereas chloroquine caused the rate of alkalinisation to decrease in the chloroquine-sensitive strains. The chloroquine-associated increase in the rate of alkalinisation seen in chloroquine-resistant parasites was inhibited by the chloroquine-resistance reversal agent verapamil. The data are consistent with the hypothesis that in chloroquine-resistant parasites chloroquine effluxes from the digestive vacuole, in association with H+, via a verapamil-sensitive pathway.

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.

Response to Petri: Can a proteomics strategy be used to identify the anti-malarial activity of chloroquine?

Response to Petri: Can a proteomics strategy be used to identify the anti-malarial activity of chloroquine?

Can a proteomics strategy be used to identify the anti-malarial activity of chloroquine?

Interest in the mechanism of action of chloroquine is intense partly because of the emergence of drug-resistant parasites. Chloroquine resistance has been genetically linked to mutations in a parasite protein (PfCRT) that might confer resistance by inhibiting chloroquine accumulation in infected erythrocytes. Now chloroquine-binding proteins in malaria-infected erythrocytes, surprisingly, have been identified as human aldehyde dehydrogenase 1 and quinone reductase 2, raising the interesting possibility that the target of the anti-malarial activity of chloroquine might be a host enzyme.

Does oxidative stress have a role in the antimalarial activity of chloroquine?

Does oxidative stress have a role in the antimalarial activity of chloroquine?

Access to hematin: the basis of chloroquine resistance.

The saturable uptake of chloroquine by parasites of Plasmodium falciparum has been attributed to specific carrier-mediated transport of chloroquine. It is suggested that chloroquine is transported in exchange for protons by the parasite membrane Na+/H+ exchanger [J Biol Chem 272:2652-2658 (1997)]. Once inside the parasite, it is proposed that chloroquine inhibits the polymerization of hematin, allowing this toxic hemoglobin metabolite to accumulate and kill the cell [Pharmacol Ther 57:203-235 (1993)]. To date, the contribution of these proposed mechanisms to the uptake and antimalarial activity of chloroquine has not been assessed. Using sodium-free medium, we demonstrate that chloroquine is not directly exchanged for protons by the plasmodial Na+/H+ exchanger. Furthermore, we show that saturable chloroquine uptake at equilibrium is due solely to the binding of chloroquine to hematin rather than active uptake: using Ro 40-4388, a potent and specific inhibitor of hemoglobin digestion and, by implication, hematin release, we demonstrate a concentration-dependent reduction in the number of chloroquine binding sites. An equal number of chloroquine binding sites are found in both resistant and susceptible clones, but the apparent affinity of chloroquine binding is found to correlate with drug activity (r2 = 0.93, p < 0.0001). This completely accounts for both the reduced drug accumulation and activity observed in resistant clones and the "reversal" of resistance produced by verapamil. The data presented here reconcile most of the available biochemical data from studies of the mode of action of chloroquine and the mechanism of chloroquine resistance. We show that the activity of chloroquine and amodiaquine is directly dependent on the saturable binding of the drugs to hematin and that the inhibition of hematin polymerization may be secondary to this binding. The chloroquine-resistance mechanism regulates the access of chloroquine to hematin. Our model is consistent with a resistance mechanism that acts specifically at the food vacuole to alter the binding of chloroquine to hematin rather than changing the active transport of chloroquine across the parasite plasma membrane.

Poly ICLC enhances the antimalarial activity of chloroquine against multidrug-resistant Plasmodium yoelii nigeriensis in mice.

Swiss mice infected with multidrug-resistant Plasmodium yoelii nigeriensis were treated with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethyl cellulose (Poly ICLC), a potent interferon (IFN) inducer and immune enhancer, in combination with chloroquine (CQ), which completely eliminated the malaria parasite from these animals. The enhancement of the antimalarial activity of poly ICLC was found to be completely reversed by the cytochrome P-450 inducer, phenobarbitone. No effect of Nw nitro-L-arginine (NLA), an inhibitor of nitric oxide, was seen on the enhancement of the antimalarial activity of CQ by Poly ICLC. These results suggest the possible involvement of cytochrome P-450 enzyme-mediated mechanism in the enhancement of the antimalarial activity of CQ by Poly ICLC.

Antimalarial properties of soy-bean fat emulsions

Intralipid ® and Ivelip ® are commercial preparations of soy-bean lipid extracts used for intravenous supplementation of lipids in various clinical conditions. They were found to inhibit the growth of Plasmodium falciparum in culture with an IC 50 of 8.07 ± 2.13 and 13.32 ± 2.05 mg.ml −1, respectively. Intralipid ® rapidly and efficiently inhibited nucleic acid synthesis in cultured P. falciparum, exhibiting full inhibitory activity in less than 2 h. Ivelip ® injected intraperitoneally, was found by the 4-day suppressive test to be active in vivo against P. vinckei petteri within the normal recommended regimen for dietary lipid supply (0.5–4 g.kg −1), but it was impossible to obtain a radical cure even with very high doses (6.4 g.kg −1). Ivelip ® was less effective against P. berghei and P. yoelii nigeriensis. As Ivelip ® showed no interference with the antimalarial activity of chloroquine, it could be considered for use in the treatment of severe human malaria in association with 4-aminoquinolines to expedite the clearance of parasites.

Inhibitory effect of chloroquine on the peroxidase activity of ferriprotoporphyrin IX

The effect of the antimalarial chloroquine {7-chloro-4-[4-(diethylamino)-1-methylbutylamino]quinoline} on the oxidation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)(abts) promoted by ferriprotoporphyrin IX [(3,7,12,17-tetramethyl-8,13-divinylporphyrin-2,18-dipropanoato)iron(III)] and H2O2 was studied. Under the conditions [abts][H2O2][catalyst], the kinetic results showed a first-order dependence on both the peroxide (up to 8 × 10–3 mol dm–3) and the catalyst concentration (up to 4 × 10–6 mol dm–3), although a saturation effect was observed at higher concentrations. The values of the apparent second-order rate constants in the absence and presence of chlorcquine were (1.71 ± 0.05)× 105 and (1.23 ± 0.04)× 105 dm6 mol–2 s–1 at pH 6.86, 303.1 ± 0.2 K, and I= 0.10 mol dm–3 NaCl. Therefore, the antimalarial inhibits the peroxidase activity by about 28% due the formation of a catalytically inactive complex with the haem. A first-order dependence on the concentration of abts was also verified, followed by a zero-order dependence at concentrations higher than 5 × 10–3 mol dm–3. Some EPR spin-trapping experiments, and comparative studies in the presence of bromide ions, provided evidence for the participation of analogues of the horseradish peroxidase compounds I and II rather than hydroxyl radicals, in the peroxidase activity of the haem. The results are discussed in the context of the antimalarial activity of chloroquine.

A Novel Pyrrolidonoalkaneamine (WR268954) That Modulates Chloroquine Resistance of Plasmodium falciparum In Vitro

With the recent observations of efflux of chloroquine from Plasmodium falciparum and modulation of chloroquine resistance by calcium channel blockers, such as verapamil, a great deal of attention has been focused on the development of new modulators that can potentiate the efficacy of chloroquine. We report a new compound, WR268954, that has weak intrinsic antimalarial activity compared to chloroquine. In vitro, it increased the susceptibilities of chloroquine-resistant P. falciparum strains to chloroquine and quinine, but did not affect the chloroquine-susceptible strains. In the presence of 2,000 nM of WR268954, the 50% inhibitory concentration of chloroquine for drug-resistant P. falciparum decreased 90-fold in comparison with the control (chloroquine only). The same concentration of WR268954 increased the potentiation of chloroquine in resistant strains to a level approximately equivalent to that observed for the sensitive strain. This compound also potentiates the efficacy of quinine in drug-resistant parasites. However, WR268954 did not enhance the efficacy of mefloquine in the mefloquine-resistant parasites. In this report, the data show the synergistic effect of WR268954 on the antimalarial activity of chloroquine in drug-resistant strains of P. falciparum, but only an additive effect on drug-sensitive strains of parasites. Compound WR268954 belongs to a pyrrolidino alkane amine class whose in vitro chloroquine resistance modulator activity supports the basis for the synthesis of this class of compounds.

Cloning of a fresh isolate of Plasmodium falciparum and drug sensitivity of the clones

A freshly isolated strain of Plasmodium falciparum was cloned by limited dilution using a co-culture of infected erythrocytes on monolayers of functionally active rodent hepatocytes. 15 clones were isolated, and the anti-malarial activity of chloroquine, quinine, mefloquine and halofantrine against the clones, the original isolate, and a culture-adapted isolate was determined using a 48 h radioisotope microdilution method. The multiplication rates of all clones and the culture-adapted isolate were estimated by counting the number of parasitized cells on Giemsa-stained thin smears. Variations found in drug sensitivity and multiplication rate of different clones provided strong evidence of heterogeneity of a single strain parasite population. No morphological variation was detected by light microscopy. The use of a hepatocyte feeder layer improved the adaptation of cloned parasites to continuous culture conditions and thus enabled us to clone directly a fresh isolate, without losing clones during the culture-adaptation process.

Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones

Interaction between the flavones casticin and artemetin and the antimalarial activity of chloroquine and qinghaosu (QHS) was examined using an in vitro growth assay based on [ 3H]hypoxanthine incorporation in synchronized cultures of a cloned line of Plasmodium falciparum. Casticin, and to a lesser extent artemetin, selectively enhanced the inhibition of growth by QHS, but had little effect on the activity of chloroquine. The findings suggest that flavones indigenous to Artemisia annua, from which QHS is isolated, might significantly alter the clinical potential of this novel antimalarial drug in the treatment of chloroquine-resistant malaria.

The antimalarial action of chloroquine and mechanisms of resistance.

Specificite de stade de l'action de la chloroquine. Processus d'alimentation des parasites et action de la chloroquine-chloroquine et ferriprotoporphyrine X: chloroquine en tant qu'agent lysosomotrope. Resistance de P. falciparum a la chloroquine

Susceptibility of human malaria parasites to chloroquine is pH dependent.

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the latter--either by changing the external pH or by adding the acidotropic agent NH4Cl--led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 X 10(-7) M, 1.8 X 10(-7) M, and 3.3 X 10(-7) M, respectively. Marked resistance to CQ (IC50 = 9.8 X 10(-7) M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.

Study of the mechanisms of the antimalarial action of chloroquine and dabequine: Formation of complexes with hemin

Study of the mechanisms of the antimalarial action of chloroquine and dabequine: Formation of complexes with hemin

Chloroquine accumulation in isolated rat liver lysosomes

Indeed, qualitative observations on lysosomes in vivo [2-61 indicate that the intralysosomal pH is 3-6. Further- more, studies with isolated lysosomes have shown that the pH within the organelles is lower than that of the suspending medium when the pH of the latter is > 5, and that the pH difference across the lysosomal membrane becomes greater as the pH of the medium is increased [7-91. In the in vitro studies the intra- lysosomal pH was estimated by measuring the dis- tribution of the lipid-soluble weak base methylamine between the lysosomes and the medium [7-91. Weak bases are accumulated within the lysosomes in the intact cell [lo]. Homewood et al. [ 1 l] have suggested that the antimalarial action of chloroquine is due to its being accumulated in the lysosomes of the malarial parasite. This leads to an increase in the intralysosomal pH, to an inhibition of the lysosomal hydrolysis of haemoglobin, and, consequently, to arrested growth of the parasite [l l] . According to Homewood et al. [ 1 l] the extent of chloroquine accumulation is governed by the pH difference between the intra- and extralysosomal spaces and the buffering capacity of the lysosomes. De Duve et al. [lo] , however, consider that the amount of chloroquine accumulated in lysosomes, as estimated for instance by Wibo and Poole [ 121 in cultured fibroblasts, is too great to be accounted for solely by this mechanism, and suggest that an active

Effect of PABA on chloroquine resistance in Plasmodium berghei yoelii.

IN mice1 paraaminobenzoic acid(PABA) in the diet of the host is necessary for the growth of blood infections of Plasmodium berghei. The area of the parasite metabolism in which PABA is involved is believed to be closely associated with the point of action of certain antimalarial drugs including pyrimethamine, proguanil and the sulphonamides. Several drugs of this group are antagonized by PABA2. The effect of PABA on the antimalarial action of chloroquine has received little attention as chloroquine does not seem to act directly on the pathways of PABA metabolism. Kirakosyan3, however, has shown that chloroquine is more effective against a sensitive strain of P. berghei berghei in mice when PABA is absent from the diet of the host.

Lysosomes, pH and the anti-malarial action of chloroquine.

MALARIA parasites1 or mammalian liver2 suitably exposed to chloroquine rapidly form autophagic vacuoles; in the case of the parasites, these are visible under the light microscope because they contain clumps of pigment, the remains of haemoglobin digestion (Fig. 1). These striking morphological changes are well known but not correspondingly well understood.

COMPLEX FORMATION BETWEEN CHLOROQUINE AND FERRIHAEMIC ACID IN VITRO, AND ITS EFFECT ON THE ANTIMALARIAL ACTION OF CHLOROQUINE.

RESISTANCE of malarial parasites to Chloroquine and its analogues was first reported in 19611, and during the past several years has caused increasing concern. Among the possible mechanisms to be considered in the development of drug resistance is production by the parasite of a metabolite which can interact with Chloroquine and thereby interfere with the action of the drug. This communication presents spectrophotometric evidence that ferrihaemic acid, a product of haemoglobin breakdown by the malarial parasite, can bind strongly to chloroquine in vitro. The anti-malarial action of chloroquine against P. berghei infection in mice is markedly inhibited by such complex formation.