Biomedical subjects
Publications and source records attributed to S R Meshnick.
The mechanism of action of artemisinin appears to involve two steps. In the first step, activation, intra-parasitic iron catalyses the cleavage of the endoperoxide bridge and the generation of free radicals. In the second step, alkylation, the artemisinin-derived free radical forms covalent bonds with parasite proteins.
Malarial hemozoin may play an important role as a target for antimalarial drugs and in disease pathogenesis. A new assay for hemozoin was developed in which the hemozoin was separated from cells by filtration. Trophozoites have substantially more hemozoin than rings, but there are relatively small differences between chloroquine-sensitive and chloroquine-resistant strains. The effects of hemozoin content of chloroquine and artemisinin, two antimalarial drugs, and E64 and Pepstatin A, two protease inhibitors, were measured. At concentrations at which hypoxanthine incorporation was unaffected, the hemozoin content of rings was decreased by E64, but not by the other three compounds. Artemisinin and Pepstatin A also had little effect on the hemozoin content of trophozoites. Chloroquine and E64 inhibited trophozoite hemozoin formation, but inhibited hypoxanthine uptake to a similar or greater extent. When either rings or trophozoites were exposed to several higher concentrations of chloroquine, hemozoin content was diminished, but significantly less than hypoxanthine uptake. Various concentrations of E64, in contrast, inhibited hemozoin production by both rings and trophozoites significantly more than hypoxanthine incorporation, suggesting that hemozoin production may be directly affected by E64.
[14C]Artemisinin was taken up by Plasmodium falciparum in culture and concentrated in hemozoin. In vitro, hemin and artemisinin were found to undergo a chemical reaction forming two major products which were isolated by high-performance liquid chromatography (HPLC). The m/z values of the two products were 856 and 871. Thin-layer chromatography (TLC) and HPLC of hemozoin isolated from [14C]artemisinin-treated parasites showed that the majority of the hemozoin-associated radioactivity comigrated with the synthetic adducts. When [14C]artemisinin was incubated with isolated hemozoin, [14C]artemisinin disappeared from the solution in a time-dependent manner. Some of the radioactivity present in the treated hemozoin also comigrated with the adducts on TLC. Thus, artemisinin appears to react covalently with heme in malaria hemozoin both in vitro and in situ.
The endoperoxides are a new class of antimalarial agents, of which artemisinin (qinghaosu) is the prototype. We have previously shown that artemisinin is capable of alkylating proteins in model reactions. In the present study, we showed that when Plasmodium falciparum-infected erythrocytes are treated with a radiolabeled antimalarial endoperoxide, either arteether, dihydroartemisinin, or Ro 42-1611 (arteflene), the radioactivity is largely coverted into a form which can be extracted with sodium dodecyl sulfate (SDS). Autoradiograms of SDS-polyacrylamide gels showed that six malarial proteins are radioactively labeled by the three endoperoxides. This labeling occurs at physiological concentrations of drug and is not stage nor strain specific. The labeled proteins were not the most abundant proteins seen on Coomassie-stained gels. No proteins were labeled when uninfected erythrocytes were treated with these drugs, nor when infected erythrocytes were treated with the inactive analog deoxyarteether. Thus, the antimalarial endoperoxides appear to react with specific malarial proteins.
Artemisinin (qinghaosu) and its derivatives are endoperoxide-containing compounds that are an important new class of antimalarial drugs. Tritiated dihydroartemisinin is taken up and concentrated by isolated red cell membranes but not by intact erythrocytes. More than half of the membrane-associated drug can be released by treatment with phospholipase A2 followed by extraction with ethyl acetate. The remaining drug appears to be bound to the major red cell membrane proteins. There is no association of the drug with either the membrane or cytoplasm of intact red cells. Thus dihydroartemisinin appears to be taken up by isolated membranes, where it associates with proteins but not via intact red cells.
A number of quinones have been shown to be effective antimalarials. In addition some have been shown to have oxidant effects on glucose-6-phosphate dehydrogenase-deficient red cells. To clarify the mechanism of this oxidant effect and to determine whether it is a general property of antimalarial quinones, the effects of five compounds on red cells were studied. Two, 5-hydroxy-6-demethyl pentaquine (5H6DP) and menadione, caused marked methemoglobin production and the insertion of heme into the red-cell membrane. The other three, atovaquone, daphnetin, and menoctone, did not. The insertion of heme into membranes caused diminished deformability, and this might account for the hemolytic effects. Redox potentials of all five compounds were measured. The two quinones that caused heme release, menadione and 5H6DP, had redox potentials of -141 +/- 12 and -97 +/- 25 mV, respectively, which are similar to the reported redox potentials for hemoglobin. The other three quinones had redox potentials that were either significantly higher or lower. Thus only quinones of the appropriate redox potentials are likely to be toxic to red cells.
The interaction between artemisinin and human serum was studied in vitro using [3H]dihydroartemisinin and [14C]artemisinin. Approximately 20% of added drug was covalently bound to albumin in 24 hr. The results of electrospray ionization mass spectra showed that albumin had an M(r) value of 66,745 +/- 35 and the drug-bound albumin had an M(r) of 67,223 +/- 34. The binding was blocked 15 and 58% by iodoacetamide (IA) and N-ethylmaleimide, respectively, and 80% by the combination of IA and succinic anhydride. Hemin and Fe2+ increased the binding by 40 and 10%, respectively, whereas deferoxamine inhibited the binding by 10%. Therefore, we conclude that the binding between artemisinin and albumin probably involves thiol and amino groups via both iron-dependent and -independent reactions.
Artemisinin is an important new antimalarial agent containing a bridged endoperoxide. The in vitro antimalarial activity of an artemisinin derivative, arteether, is antagonized by two iron chelators, pyridoxal benzoylhydrazone and 1,2-dimethyl-3-hydroxypyrid-4-one. Similarly, the acute toxicity of artemisinin in mice is antagonized by another chelator, deferoxamine-hydroxyethylstarch. A combination of artemisinin and hemin oxidizes erythrocyte membrane thiols in vitro, and this oxidation is also inhibited by an iron chelator. Thus, iron plays a role in the mechanisms of action and toxicity of artemisinin. The combination of artemisinin and hemin also decreases erythrocyte deformability. Iron probably catalyzes the generation of free radicals from artemisinin since alpha-tocopherol antagonizes the thiol-oxidizing activity of artemisinin and since a spin-trapped free radical signal can be seen by electron paramagnetic resonance only when artemisinin is incubated in the presence of iron.
Ultrastructural changes induced in Plasmodium falciparum by artemisinin were studied in vitro. Electron microscopic autoradiography was performed on infected erythrocytes that were exposed in vitro to 3H-dihydroartemisinin and 14C-artemisinin. These drugs consistently were located in food vacuoles and mitochondria. Two hours after administration, changes were observed in parasite mitochondria, rough endoplasmic reticulum, and nuclear envelope. At four hours, in addition to the earlier changes, nuclear membranes and, to a lesser extent, some plasma membranes formed myelin figures. In addition, there was a disappearance of ribosomes, and a destruction of food vacuole membranes. These changes may lead to the total disorganization of the parasites. Approximately 30% of the parasites manifested these alterations.
In this study, 638 patients with either Plasmodium falciparum or P. vivax malaria were treated with artemisinin (qinghaosu) that was isolated and formulated into tablets and capsules in Vietnam. In all cases, artemisinin treatment resulted in a rapid clearance of parasitemia and fever. Recrudescence rates were highest in those groups receiving treatment for five or less days (50%), but were between 10% and 23% for those groups receiving the drug for 5-10 days. A low recrudescent rate (9.5%) was also found when patients were treated with a combination of artemisinin for three days and tetracycline for five days. Thus, artemisinin represents a useful and economically feasible component of the malaria control program in Vietnam.
Artemisinin (qinghaosu) and its derivatives represent an important new class of antimalarial drugs. Previous work suggests that the antimalarial activity of artemisinin may be mediated by a reaction with intraparasitic hemin. Using cyclic voltammetry, artemisinin and dihydroartemisinin were irreversibly reduced at approximately -1 V. In the presence of concentrations of hemin as low as 50 nM, the reduction took place at much lower potentials (-0.435 to -0.460 V). Both reductions took place after adsorption onto the electrode surface. The shift of the reduction potential to more positive values is indicative of a catalytic process similar to that seen with hydrogen peroxide. The catalytic decomposition of artemisinin may play a role in the antimalarial activity of artemisinin.
De novo folate biosynthesis is required for the growth of malarial parasites and is inhibited by several important antimalarial agents. We show here that exogenous p-aminobenzoic acid (pABA) can be utilized by malaria parasites to synthesize folates. The transport of pABA into parasite infected red cells was therefore characterized. Normal red cells transport pABA in a saturable and energy-dependent manner, with a dissociation constant of 83 nM. pABA transport in parasite-infected red cells may use the same mechanism, as demonstrated by similarities in time course, concentration-response, and dissociation constant (111 nM). The transport capacity of red cells is temperature-, energy- and pH-dependent. It is inhibited by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), but not by the sodium ionophores nigericin and monensin. p-Aminosalicylic acid (PAS) inhibits pABA transport competitively, with a inhibition constant of 378 nM. Phloritin, flufanamic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DITS), which are inhibitors of the anion transporter (band 3), and oxalic acid, a substrate of this transporter, partially inhibit pABA transport into both normal and infected red cells. Interestingly, in both normal and infected red cells, the inhibitory effects of PAS and the anion transport inhibitors are additive, suggesting the involvement of 2 independent mechanisms.
Primaquine is an important antimalarial drug which causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency, probably due to oxidant generation by its metabolites. One of primaquine's metabolites, 5,6-dihydroxy-8-aminoquinoline (AQD), was found to cause chemiluminescence (CL) in vitro when incubated in the presence of luminol. This CL is inhibited by catalase and deferoxamine, unaffected by mannitol, and stimulated by superoxide dismutase (SOD), suggesting that it is mediated by H2O2. Three antioxidants (daphnetin, ferulate, and maltol), derived from Chinese herbal remedies, inhibited AQD- and H2O2-mediated CL, whereas a fourth, anisodamine, had no effect. Daphnetin also potently inhibited H2O2-mediated lipid peroxidation as measured by the production of thibarbituric acid reacting substances (TBARS). Thus, the possibility is raised that an antioxidant might be able to mitigate the oxidant hemolytic effects of primaquine.
Daphnetin is a dihydroxycoumarin that is being used in China for the treatment of coagulation disorders. It is also a chelator and an antioxidant. In vitro, daphnetin causes a 50% inhibition (IC50) of 3H-hypoxanthine incorporation by Plasmodium falciparum at concentrations between 25 and 40 microM. Several related compounds, such as scopoletin, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid show no inhibitory activity. The antimalarial activity of daphnetin is inhibited by the addition of iron. Daphnetin does not appear to be an oxidant drug, since it does not spontaneously generate superoxide in vitro. However, it does alkylate bovine serum albumin when incubated in the presence of iron. In vivo, daphnetin significantly prolongs survival of P. yoelli-infected mice.
Explore the source record for details and available documents.
Artemisinin (qinghaosu), is a promising new antimalarial drug derived from an ancient Chinese herbal remedy. When [13-14C]artemisinin is added to cultures of Plasmodium falciparum, it is converted into a product with different solubility and chromatographic properties than the parent drug. Artemisinin reacts with hemin in aqueous solution to form an adduct with an apparent molecular weight of 914 which has identical chromatographic, solubility, and electrophoretic behavior to the parasite-derived product. The reaction between artemisinin and hemin, when carried out in the presence of red cell membranes, leads to the oxidation of protein thiols. Malarial parasites are rich in hemin; artemisinin's reactivity toward hemin may explain its selective toxicity to malarial parasites.
The Michaelis-Menten inhibitory constants (Kis) and the concentrations required for 50% inhibition of the Plasmodium falciparum dihydropteroate synthetase were determined for six sulfa drugs. These drugs inhibited the in vitro growth of P. falciparum (50% lethal concentration) at concentrations of 30 to 500 nM; these concentrations were 100 to 1,000 times lower than the concentrations required for 50% inhibition and Kis (6 to 500 microM). The uptake of p-aminobenzoic acid was not inhibited by the sulfa drugs. However, infected erythrocytes took up more labeled sulfamethoxazole than did uninfected erythrocytes. Thus, the concentration of sulfa drugs by malaria parasites may explain how sulfa drugs inhibit in vitro growth of parasites through the inhibition of dihydropteroate synthetase.
The susceptibility of Pneumocystis carinii to artemisinin (qinghaosu) was determined in short-term primary culture. In untreated cultures, trophozoites increased an average of fivefold over 4 days. Inhibition of parasite growth in cultures treated with artemisinin at concentrations as low as 0.5 microM was seen. In contrast, artemisinin concentrations up to 100 microM had no effect on feeder layer cells.