Atorvastatin Is 10-Fold More Active In Vitro than Other Statins against Plasmodium falciparum

Abstract

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, influence a broad array of pathogenic microorganisms. Lovastatin reduces the intracellular growth of Salmonella enterica serovar Typhimurium in cultured macrophages, while atorvastatin does the same in a mouse model (2). Lovastatin additionally reduces the growth of Candida albicans by inhibiting the sterol pathway (11). Statins interfere severely with the growth of protozoan parasites of the Trypanosomatidae family such as Trypanosoma cruzi and various Leishmania species (8, 12, 13). HMG-CoA reductase has been detected in Trypanosoma and Leishmania (3, 8). The presence of an HMG-CoA homolog was not revealed by BLASTX analysis of the Plasmodium falciparum sequence with other protozoal HMG-CoA protein sequences. However, as reported previously, treatment in vitro of Plasmodium falciparum with 120 or 240 μM mevastatin inhibited parasite growth (4, 9). The susceptibilities to simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt (Calbiochem, Merck, Germany), and atorvastatin calcium salt (Molekula, United Kingdom) were assessed in vitro against chloroquine-susceptible P. falciparum strains 3D7 (Africa), D6 (Sierra Leone), and IMT031 (Gabon) and chloroquine-resistant strains W2 (Indochina), Bre1 (Brazil), and FCR3 (The Gambia). Lovastatin and mevastatin were converted to the active form by dissolving the lactone form in 100 μl of 100% ethanol, adding 200 μl of 0.2 M KOH, and then adding 0.2 M HCl for neutralization to pH 7.2 (5). Simvastatin, simvastatin sodium salt, pravastatin sodium salt, lovastatin, fluvastatin sodium salt, mevastatin, mevastatin sodium salt, and atorvastatin calcium salt were dissolved in dimethyl sulfoxide 1% (vol/vol) in RPMI. Twofold serial dilutions, with final concentrations ranging from 1.5 μM to 200 μM, were prepared in dimethyl sulfoxide 1% in RPMI and distributed into Falcon 96-well plates just before use. The isotopic microdrug susceptibility test used was described previously (10). Table ​Table11 presents the 50% inhibitory concentrations (IC50) of the different statins for P. falciparum. Simvastatin, fluvastatin, lovastatin, and atorvastatin, in the salt active forms, are more active than simvastatin, mevastatin, and lovastatin, in the lactone form. Pravastatin and mevastatin sodium or potassium salts are inactive against P. falciparum (>200 μM). The results indicate that susceptibility to the salts of simvastatin, fluvastatin, lovastatin, and mevastatin is not dependent on the status of chloroquine resistance. The results observed with the simvastatin salt were similar to those reported by other authors (5). Atorvastatin salt, in the range of 5 to 12 μM, is 10-fold more active against P. falciparum than the other salts. Atorvastatin IC90s ranged from 14.8 to 39 μM. The activity of atorvastatin is independent of the status of chloroquine resistance (4.8 to 5.8 μM against chloroquine-resistant strains versus 5.3 to 11.8 μM for the susceptible strains). TABLE 1. In vitro activities of statins against chloroquine-susceptible (3D7, D6, and IMT031) and chloroquine-resistant (W2, Bre1, and FCR3) P. falciparum strains The chemical structures of simvastatin, lovastatin, mevastatin, and pravastatin are closely related. Those of fluvastatin and atorvastatin are very different from the others. The structural differences between atorvastatin and the other statins could explain differential activity. However, we cannot rule out the action of calcium in the differential activity of atorvastatin. Multiple daily doses of 2.5 to 80 mg of atorvastatin produced steady-state maximum plasma concentrations of 1.95 to 252 μg liter−1 (in the range of 0.2 to 0.3 μM for the maximum) (1). In L6 cells (rat skeletal muscle cell line), atorvastatin at 100 μM induced death in 27% of the cells (7). Although the atorvastatin IC50 for P. falciparum exceeds these reported plasma concentrations, it may be below toxic concentrations. Parasites treated with mevastatin show depressed biosynthesis of dolichol and isoprenoid pyrophosphate (4). In addition, mevastatin decreases the viability of cells by inhibiting proteasome activity. Atorvastatin is an inhibitor for phosphoglycoprotein, an efflux protein, and may be a substrate for this transporter as well (6). A phosphoglycoprotein in P. falciparum, Pgh1, is implicated in quinoline resistance. In conclusion, the present observation suggests that atorvastatin is a good candidate for further studies on the use of statins in malaria treatment.