ANTIPLASMODIAL INTERACTIONS BETWEEN ARTEMISININ AND TRICLOSAN OR KETOCONAZOLE COMBINATIONS AGAINST BLOOD STAGES OF PLASMODIUM FALCIPARUM IN VITRO LOKESH C. MISHRA, AMIT BHATTACHARYA, AND VIRENDRA K. BHASIN* Department of Zoology, University of Delhi, North Campus Delhi University, Delhi 110007, India Abstract. Emergence of drug-resistant Plasmodium falciparum strains to conventional first-line antimalarial drugs has compelled many countries to reorient their drug policies to adopt artemisinin-based combination therapies (ACTs) for treatment of uncomplicated malaria. This has increased the demand of artemisinin, already a scarce commodity. Synthesis of artemisinin is not yet commercially viable. Extensive use of available ACTs will invariably lead to emer- gence of resistance to these combinations. Thus, there is need to search for new artemisinin-based synthetic, inexpensive, synergistic combinations to reduce dependence on artemisinin. In vitro cultures of P. falciparum provide an appropriate system for identification of such new combinations. We evaluated interactions of artemisinin with triclosan or ketoco- nazole against blood stages of P. falciparum by a fixed-ratio isobologram method. Artemisinin shows mild synergistic interaction with triclosan and slight to marked antagonism with ketoconazole in vitro. These antiplasmodial interactions, however, require confirmation using in vivo model systems. INTRODUCTION Hundreds of millions of people are at risk of drug-resistant falciparum malaria infection. The worsening problems of drug resistance in many parts of the world and the limited number of antimalarial drugs available have led to increasing difficulties for adequate disease management. Artemisinin and its derivatives are among the most effective antimalarial drugs known today. They rapidly cure even drug-resistant falciparum infections. Artemisinin-based combination thera- pies (ACTs) are being used for treatment, instead of mono- therapies, to delay the emergence of resistant strains of Plas- modium falciparum to this vital class of drugs. ACTs are in- creasingly being adopted as first-line treatments in malaria- endemic regions of the world that are afflicted with P. falciparum strains resistant to conventional antimalarial drugs. The history of drug resistance is replete with instances showing the uncanny ability of parasites to rapidly acquire resistance to any chemotherapeutic assault deployed en masse to treat uncomplicated malaria cases. Clinically arte- misinin-resistant strains of P. falciparum have not yet been encountered. Thus, the demand for ACTs is increasing. Ar- temisinin is obtained from aerial parts of an herb, and this plant is globally scarce. Synthesis of artemisinin is not a com- mercially viable proposition at present. This makes ACT ex- pensive for poor people in malaria-endemic regions. There is also reason to believe that, sooner or later, resistance to ex- isting ACTs will emerge. Thus, there is need to search for synthetic synergistic drug partners of artemisinin and its de- rivatives to reduce dependence on this scarce natural product in ACTs. Use of a P. falciparum in vitro system is very potent in identifying novel lead compounds and combinations. We have evaluated the in vitro antiplasmodial activity of the syn- thetic compounds triclosan and ketoconazole as partner drugs in combination with artemisinin against erythrocytic stages of P. falciparum using a modified fixed-ratio isobologram method to study drug–drug interactions. 1 Both of these com- bination partner drugs are FDA approved for human use and demonstrated antiplasmodial activity. 2–6 In-depth studies of interactions between drugs may also provide clues to their mechanisms of action. 7 MATERIALS AND METHODS Parasite culture. Stock culture of malaria parasite P. falci- parum 3D7 strain was continuously maintained in vitro using the candle-jar method of Trager and Jensen. 8 The parasites were maintained on B + human red blood cells suspended in a complete culture medium. Each 960 mL of aqueous culture medium consisted of 10.4 g of powdered RPMI-1640 (with glutamine but without bicarbonate), 5.94 g of HEPES buffer, and 40 mg of gentamicin. Complete medium was constituted just before use by adding sterile 5% sodium bicarbonate at the rate of 4 mL per 96 mL and supplemented with 10% pooled B + serum. The stock culture parasitemia was mostly kept between 1% and 5% with subculturing every fourth day. The starting hematocrit was 5%. Drug solutions. Artemisinin, triclosan, and ketoconazole were obtained from Sigma-Aldrich (St. Louis, MO), Vivimed Laboratories Ltd. (Hyderabad, India), and Yanshu Chemicals Ltd. (Mumbai, India), respectively. Artemisinin and triclosan were dissolved separately in DMSO, and ketoconazole was dissolved in redistilled water to obtain stock solutions of 0.2, 1, and 1 mg/mL concentrations, respectively. The stock solu- tions were diluted on the day of experiment to obtain the desired concentrations for each drug. The amount of DMSO in the diluted concentrations used had no effect on parasite growth. Inhibitory concentrations assay. The inhibitory concentra- tion of each individual drug needed to prevent the growth and multiplication of P. falciparum was determined in vitro using a dose–response assay in 24-well tissue culture plates in trip- licates. Synchronous parasites were prepared 9 and challenged with a graded concentration of a drug solution for 48 hours at 37°C by the candle-jar method. 8 The medium was changed in each well after 24 hours with or without drug. The percentage inhibition of parasitemia in relation to control was calculated by examining thin-smear Giemsa-stained slides. Assay results were computed to determine the IC 50 value of each drug. * Address correspondence to Virendra K. Bhasin, Department of Zoology, University of Delhi, North Campus Delhi University, Delhi 110007, India. E-mail: virendrabhasin@hotmail.com Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 497–501 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene 497