A double-headed pro-drug that overcomes chloroquine resistance.

Abstract

Chloroquine (CQ) was developed as an antimalarial drug during World War II, being the most effective and affordable for prophylaxis and treatment of malaria until resistance started to spread throughout the world in the early 1960s. The mechanism of action of the drug is a matter of debate, let alone the means that the parasite has developed to resist its pleiotropic actions. One such mechanism suggests that CQ interferes with the detoxification of noxious ferriprotoporphyrin IX (FP) which is produced when the intraerythrocytic malaria parasite digests the hemoglobin of the host cell. The drug would do so by inhibiting the biomineralization of FP inside the digestive vacuole and by inhibiting the degradation of free FP that escapes into the cytosol by reduced glutathione (GSH). Increasing the GSH levels has been shown to confer resistance to CQ and vice versa.This mechanism provided an incentive for several scientists from France, Belgium and Germany to contemplate using this for drug development. Capitalizing on these findings, Davioud-Charvet et al. have now devised a pro-drug that is hydrolyzed intracellularly to an inhibitor of glutathione reductase (GR), the enzyme responsible for the reduction of oxidized glutathione to GSH and a 4-aminoquinoline that mimics the action of CQ. The first moiety was discovered in a chemical library of 1,4-naphtoquinones, using a high-throughput screening for GR inhibition. This has yielded several alcanoic acid derivatives which not only inhibited recombinant parasite and human GR protein in a competitive manner, but also had antimalarial activity in the micromolar range [1xA prodrug form of a Plasmodium falciparum glutathione reductase inhibitor conjugated with a 4-anilinoquinoline. Davioud-Charvet, E. et al. J. Med. Chem. 2001; 44: 4268–4276CrossRef | PubMed | Scopus (102)See all References][1]. The authors then produced esters from these alcanoic acid derivatives and the alcohol N-{3-[(7-chloro-4-quinolyl)amino]-5-(hydroxymethyl)phenyl} -2-piperidinoacetamide, which they had characterized as an antimalarial in a previous study, although not developed further because of its high toxicity [2xSynthesis and in vitro and in vivo antimalarial activity of new 4-anilinoquinolines. Delarue, S. et al. J. Med. Chem. 2001; 44: 2827–2833CrossRef | PubMed | Scopus (44)See all References][2]. The esters inhibited the GSH-dependent destruction of FP efficiently, but had no anti-GR activity. However, the ester that has been selected for further studies displayed antimalarial activity in the 20–30 nM range. Whereas the activity of the parent aminoalcohol changed in parallel to the CQ-resistance of the various Plasmodium falciparum strains tested in culture, the ester was invariably active with IC50 similar to those of CQ against the most sensitive strain. As expected, the ester caused a dramatic reduction in cellular GSH concentration, indicating that the ester has been hydrolyzed to release the GR-inhibiting naphtoquinone, thus enhancing the action of the 4-aminoquinoline in a GSH-poor cytosol. These results indicate that the GSH-related action CQ could be the major mechanism underlying drug resistance. Most importantly, the selected ester tested in vivo with the Plasmodium berghei mouse model showed antimalarial activity by reducing infection and delaying mortality from 11 days in controls to 23–25 days in animals treated for four days only with 40 mg kg−1 of the ester. The ester was neither toxic to the mice or to a human diploid embryonic lung cell line (hMRC5).Although the concepts of pro-drugs that are activated intracellularly and inhibition of an enzyme for the enhancement of drug action are not novel to pharmacology, they were used here in a systematic and critical manner. In essence, such double-headed pro-drugs could be generated with other inhibitors of the various enzymes involved in glutathione metabolism. Many details on the action of this pro-drug remain to be elucidated, but it can serve as a lead compound for further improvement of its pharmacodynamic and pharmacokinetic properties.