Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

Abstract

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC50), assessed by parasite incorporation of [ 3 H]hypoxanthine, were between 0.2 and 1.1 M. CLT concentrations of 2 M and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (Ϸ48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 M CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria. In the course of an investigation on the homeostasis of human red cells infected in vitro with the human malaria parasite Plasmodium falciparum, we found that clotrimazole (CLT), an imidazole derivative used as an antifungal agent (1), had a powerful growth-inhibiting effect on the parasite. The vast clinical experience, proven tolerance, and unique lack of acquired fungal resistance to CLT prompted a detailed investigation of its antimalarial effects in cultures of P. falciparum to assess its clinical potential. In this study, we report the in vitro effects of CLT on P. falciparum cultures by assessing the timeand concentration-dependent changes in parasite growth, parasite morphology, stage-specific development, and parasite replication. Materials and MethodsCultures. Five different laboratory strains of P. falciparum [A4 (2), W2, NF54, HB3, and FCR] were cultured in human erythrocytes by standard methods (3) under a low oxygen atmosphere. The culture medium was RPMI 1640, supplemented with 40 mM Hepes, 25 mg͞liter gentamicin sulfate, 10 mM D-glucose, 2 mM glutamine, and either 8.5% (vol͞vol) pooled human serum (A4 clone), or 10% heat-inactivated plasma (strains W2, NF54, HB3, and FCR). Culture media, with or without CLT, were changed daily, unless otherwise indicated. Parasites were synchronized at the ring stage with D-sorbitol (4) for all experiments. Initial parasitemias varied between 2% and 7% for the different e...

Clotrimazole Binds to Heme and Enhances Heme-dependent Hemolysis

“…Previous reports (3,4) indicate that CLT inhibits the growth of CQ-resistant strains of malaria. Furthermore, the low IC 50 value of CLT against malaria makes it a practical antimalarial drug because a plasma CLT concentration of 2 M is achievable with an oral dose of 1 g. Therefore, CLT is promising as a new antimalarial drug.…”

“…The antifungal agent clotrimazole (CLT) (Fig. 1) inhibits the growth of chloroquine-resistant P. falciparum strains in vitro (3,4).…”

The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum

“…Five different strains of P falciparum (A4, 15 W2, and FCR3 in study I; FCR3K ϩ and ITgC32p21 in study II) were cultured in human erythrocytes by standard methods 16,17 under a low-oxygen atmosphere. The culture medium was RPMI 1640, supplemented with 40 mM HEPES, 25 mg/L gentamicin sulfate, 10 mM D-glucose, 2 mM glutamine, and either 8.5% (vol/vol) serum (strains FCR3K ϩ , ITgC32p21, and A4 clone) or 8% heat-inactivated plasma (strains W2 and FCR3).…”

Clotrimazole Scaffold as an Innovative Pharmacophore Towards Potent Antimalarial Agents: Design, Synthesis, and Biological and Structure–Activity Relationship Studies

“…6 CLT is a well-known antimycotic drug which exhibits a weak in vitro antimalarial activity against different Pf strains and importantly, irrespective of their CQ sensitivity. 7,8 Biological activities of CLT are mediated by its ability to interact with ferri-protoporphyrin IX (Fe(III)-FP), which is present as the prosthetic group in several enzymes, such as (i) 14R-lanosteroldemethylase (14-LD), the fungal cytochrome inhibited by CLT, (ii) human P450 cytochromes, which are inhibited by CLT causing altered metabolism of both xenobiotics and endogenous presence of sodium hydride, to afford nitriles 25a and 25b, respectively, which were subsequently converted into the corresponding ketone derivatives 26a,b. 19 Following the synthetic procedure already described, Grignard reaction, followed by Table 1.…”