Characterisation of synthetic beta-haematin and effects of the antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine on its formation.
Infrared spectroscopy, elemental analysis and X-ray powder diffraction show that the product of 30 min of reaction of haematin in 4.5 M acetate, pH 4.5 at 60 degrees C is identical to beta-haematin prepared in 4.5 M acetic acid at 70 degrees C overnight (pH 2.6). There is no evidence for formation of haem-acetate complex, which could not be isolated, even from 11.4 M acetate solution. The antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine were found to inhibit formation of beta-haematin, while 5-, 6- and 8-aminoquinoline and quinoline were found to have no effect. Quinidine was shown to form a complex with ferriprotoporphyrin IX in 40% DMSO with log K = 5.02 +/- 0.03. Log K values for halofantrine and desbutylhalofantrine are 5.29 +/- 0.02 and 5.15 +/- 0.02 respectively (solutions containing 30% acetonitrile in addition to DMSO to solubilise these drugs), which are both stronger than chloroquine under the same conditions (log K = 4.56 +/- 0.02).
Effect of Inducers, Incubation Time and Heme Concentration on IC50 Value Variation in Anti-heme Crystallization Assay
Heme detoxification through crystallization into hemozoin has been suggested as a good target for the development of screening assays for new antimalarials. However, comparisons among the data obtained from different experiments are difficult, and the IC50 values (the concentrations of drug that are required to inhibit 50% of hemozoin formation) for the same drug vary widely. We studied the effects of changes in heme concentration (precursor of β-hematin), incubation time and three inducers (SDS, Tween 20 and linoleic acid) on the IC50 of some antimalarials (chloroquine, quinine, amodiaquine, and clotrimazole). The results showed that increasing both inducer concentration and incubation time raised the IC50 of selected antimalarials. Any change in those factors caused the IC50 value to vary. Standardization of assay conditions is, therefore, necessary to increase reproducibility and reduce discrepancies in assay performance. Considering all of the variables, the best choice of inducers is in the order of SDS > Tween 20 > linoleic acid.
Potentiation of an antimalarial oxidant drug
In a previous report we described the synergistic antimalarial interaction between two structurally similar compounds, rufigallol and exifone. To explain this phenomenon, we proposed that exifone is transformed inside the parasitized erythrocyte into a xanthone with potent antimalarial properties. We speculated that the transformation process was induced by the prooxidant activity of rufigallol. On the basis of this model we hypothesized that exifone would act synergistically with other oxidant drugs. In the present study we have found a similar synergistic interaction between exifone and ascorbic acid (vitamin C) against both chloroquine-susceptible and multidrug-resistant strains of Plasmodium falciparum. The prooxidant activity of ascorbic acid against Plasmodium-infected erythrocytes is believed to result from an intraerythrocytic Fenton reaction occurring in the acidic food vacuole of the parasite. The hydroxyl radicals produced during this process are believed to attack exifone, which undergoes cyclodehydration to become 2,3,4,5,6-pentahydroxyxanthone (X5). Evidence presented to support this "xanthone hypothesis" includes the demonstration that the exifone ==> X5 transformation occurs readily in vitro under mildly acidic conditions in the presence of iron, ascorbic acid, and oxygen.