RESUMEN
The hydrophilic desferrioxamine (DFO) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (SIH) are iron chelators which inhibit in vitro proliferation of Plasmodium falciparum with similar potency (IC50 approximately 20 microM in 24- to 48-h tests). The in vivo assessment of these drugs was performed on Swiss mice infected with Plasmodium vinckei petteri with novel modes of drug administration and release. The drugs were delivered postpatently either by multiple i.p. injections or by a single i.p. or s.c. insertion of a drug-containing polymeric device which released most of the drug within 7 days at apparently first-order rates. A regimen of three daily i.p injections of 5 mg DFO for 3 consecutive days or a 70-mg dose of the drug given as an i.p. or s.c. polymer implant evoked similar delay and reduction in peak parasitemias and reduced mortality with no apparent signs of toxicity. Relatively faster, but otherwise similar results were obtained with the less hydrophilic SIH. In combination, the two drugs apparently potentiated each other. The polymeric devices were particularly useful for treating Plasmodium berghei K173-infected C57Bl mice, a suggested model of cerebral malaria, in which classical methods of DFO delivery were ineffective. The insertion of a 140-mg DFO-containing device on day 6 postinfection (parasitemia approximately 1%) led to a marked reduction in parasite proliferation, appearance of neurological sequelae and mortality of mice. Our studies indicate that polymeric devices for slow drug release might be highly advantageous for both hydrophilic and lipophilic drugs whose antimalarial efficacy might depend on the maintenance of sustained blood levels. The results obtained with slow-release devices have implications for malaria chemotherapy as well as for iron chelation therapy in iron overload conditions.
Asunto(s)
Sistemas de Liberación de Medicamentos , Quelantes del Hierro/farmacología , Malaria/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Reversed siderophores (RSFs) are artificial hydroxamate-based iron chelators designed after the natural siderophore ferrichrome. The modular molecular design of RSF derivatives allowed the synthesis of various congeners with controlled iron-binding capacities and partition coefficients. These two physicochemical properties were assessed by a novel fluorescent method and were found to be the major determinants of RSF permeation across erythrocyte membranes and scavenging of compartmentalized iron. The partition coefficient apparently conferred upon RSFs two major features: (i) the ability to rapidly access iron pools of in vitro-grown Plasmodium falciparum at all developmental stages and to mobilize intracellular iron and transfer it to the medium and (ii) the ability to suppress parasite growth at all developmental stages. These features of RSFs were assessed by quantitative determination of the structure-activity relationships of the biological activities and partition coefficients spanning a wide range of values. The most effective RSF containing the aromatic group of phenylalanine (RSFm2phe) showed 50% inhibitory concentration of 0.60 +/- 0.03 nmol/ml in a 48-h test and a 2-h onset of inhibition of ring development at 5 nmol/ml. The lipophilic compound RSFm2phe and the lipophilic and esterase-cleavable compound RSFm2pee inhibited parasite growth at all developmental stages whether inhibition was assessed in a continuous mode or after discontinuing drug administration. The antimalarial effects of RSFm2phe and cleavable RSFm2pee were potentiated in the presence of desferrioxamine (DFO) at concentrations at which DFO alone had no effect on parasite growth. These studies provide experimental evidence indicating that the effective and persistent antimalarial actions of RSFs are associated with drug access to infected cells and scavenging of iron from intracellular parasites. Moreover, the optimal antimalarial actions of RSFs are apparently also determined by improved accessibility to critical iron pools or by specific interactions with critical parasite targets.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Sideróforos/química , Sideróforos/farmacología , Animales , Antimaláricos/síntesis química , Fenómenos Químicos , Química Física , Deferoxamina/síntesis química , Deferoxamina/química , Deferoxamina/farmacología , Diseño de Fármacos , Membrana Eritrocítica/química , Membrana Eritrocítica/efectos de los fármacos , Humanos , Técnicas In Vitro , Hierro/química , Sideróforos/síntesis química , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
The antimalarial action of iron chelators is limited by factors related to drug permeation and parasite susceptibility to metal deprivation. In this study we applied iron-chelating isonicotinoyl and benzoyl hydrazones on Plasmodium falciparum cultures and assessed their antimalarial properties. The agents w ere used both individually and in combination with deferoxamine (DFO), a clinically approved iron chelator, and with hydroxyethyl-starch-DFO, a macromolecular carrier of DFO. Salicylaldehyde isonicotinoyl hydrazone (SIH) and 2-hydroxy-1-naphthylaldehyde m-fluorobenzoyl hydrazone (HNFBH) were found to be highly efficient in suppressing parasite growth at all developmental stages (IC50 24 +/- 6 micromol/L and 0.21 +/- 0.04 micromol/L, respectively, in a 36-to-42 hour test). In combination with impermeant DFO, SIH and HNFBH actions on ring forms were significantly potentiated in terms of speed of drug action and extent of inhibition. The combined effect of the hydrazones with DFO was greater than additive. Based on the capacity of SIH to extract iron from infected cells and to transfer the metal to extracellular DFOs, we propose a mechanism for a synergistic action of permeant hydrazones and impermeant (DFO) iron chelators. The application of a combination of iron chelators as antimalarials might be of therapeutic value.
Asunto(s)
Antimaláricos/farmacología , Deferoxamina/farmacología , Hidrazonas/farmacología , Quelantes del Hierro/farmacología , Isoniazida/análogos & derivados , Naftoles/farmacología , Plasmodium falciparum/efectos de los fármacos , Animales , Sinergismo Farmacológico , Compuestos Férricos/metabolismo , Humanos , Isoniazida/farmacología , Cinética , Espectrometría de FluorescenciaRESUMEN
Malaria parasites growing inside human erythrocytes differ from mammalian cells in their mode of acquisition of bioavailable iron and in their susceptibility to the antiproliferative action of iron chelators. We have assessed here three major properties associated with these phenomena: (a) the stage-dependent nature of parasite iron mobilization from the host and its integration into parasite proteins; (b) the differential permeability of the plasma membrane to iron chelators, and (c) the in situ generation of toxic chelator-metal complexes in the intracellular milieu of infected cells. We have used a combination of synthetic and natural iron chelators with similar iron-binding properties but markedly different capacities to permeate membranes. The profiles of action of these agents on the in vitro growth of Plasmodium falciparum were assessed in terms of inhibitory concentrations, speed of action, stage dependence and reversibility of effects. These profiles provided the basis for a working model of chelator action on parasitized cells. The model allowed us to predict major improvements in the antimalarial performance of iron chelators when used in appropriate combinations of slow-and fast-permeating substances. The synergistic actions found in vitro for various combinations of iron chelators are in accordance with the model and have implications for the design of therapeutic schemes.
Asunto(s)
Antimaláricos/farmacología , Quelantes del Hierro/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium/efectos de los fármacos , Animales , Antimaláricos/metabolismo , División Celular/efectos de los fármacos , Sinergismo Farmacológico , Eritrocitos/parasitología , Humanos , Hierro/metabolismo , Quelantes del Hierro/metabolismo , Plasmodium/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Hydroxamate-based iron(III) chelators exhibit potent antimalarial effects on the asexual stages of Plasmodium falciparum grown in vitro. Antimalarial activity varies with the parasite growth stage and the drug permeation properties. The hydrophilic drug desferrioxamine (DFO) is ineffective on early stages (ring forms) of the parasite due to its poor permeability but irreversibly blocks the growth of advanced stages of parasites. On the other hand, hydrophobic reversed siderophores (RSFs) are more membrane permeable and affect all parasite developmental stages; they affect ring forms irreversibly and trophozoite/schizont forms reversibly and at relatively faster rates, compared with DFO. These observations have provided the basis for postulating a possible overadditive action of the two, distinctly acting, iron chelator types for enhanced antimalarial activity. This was assessed in this study by using novel fast-acting chelators such as RSF derivatives (RSFleum2 and RSFm2) in combination with the relatively slow-acting DFO. Parasite growth was assessed in terms of nucleic acid synthesis and parasitemia. The results indicate that, at any molar ratios of the two types of drugs, the combined inhibitory effect was faster and more potent than the sum of individual effects. The combined drug action showed neither additive nor independent but overadditive properties, as well as sustained inhibition even after drug removal. The potentiating action of RSFs on the long-lasting effects of DFO on parasite growth conformed with the postulated mechanistic model of iron chelator action and iron handling by parasites. Iron chelator combinations might be of therapeutic value.
Asunto(s)
Antimaláricos/farmacología , Ácidos Hidroxámicos/farmacología , Quelantes del Hierro/farmacología , Plasmodium falciparum/efectos de los fármacos , Animales , Células CHO , División Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Sinergismo Farmacológico , Humanos , Cinética , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Hydroxamate-based chelators of iron are potent inhibitors of in vitro growth of Plasmodium falciparum. Two types of such chelators, the natural desferrioxamine and the synthetic reversed siderophore RSFileum2, are prototypes of antimalarial agents whose action spectra differ in the speed of action, stage dependence, and degree of reversibility of effects. This work explores the possibility of improving the antimalarial efficacy of these agents by using them in various combinations on in vitro cultures of P. falciparum. Growth assessment was based both on total nucleic acid synthesis and on parasitemia. The results indicate that the synthetic reversed siderophore more than complements the antimalarial action of desferrioxamine when applied during either ring, trophozoite, or mixed stages. The combined drug effects were significantly higher than the additive effect of the individual drugs. Qualitatively similar results were obtained for both reversible effects and irreversible (i.e., sustained) effects. Following an 8-h window of exposure the combined drug treatment caused parasite growth arrest and prevented its recovery, even 3 days after the treatment. The fact that such a combination of iron chelators displays a wider action spectrum than either drug alone has implications for the design of chemotherapy regimens.
Asunto(s)
Antimaláricos/farmacología , Deferoxamina/farmacología , Ácidos Hidroxámicos , Quelantes del Hierro/farmacología , Plasmodium falciparum/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1%) the incorporation on the second day in culture was only 61% of the control cultures. An effect could be observed already after two hours of incubation (30% reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage. Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress. Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.