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Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 497–501 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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

* Address correspondence to Virendra K. Bhasin, Department of Zoology, University of Delhi, North Campus Delhi University, Delhi 110007, India. E-mail: virendrabhasin@hotmail.com

demonstrated antiplasmodial activity. 26 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.

497

498

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Drug combination preparations. For each combination as- say, drug strength was made so that IC 50 of the individual drugs fell between third and fourth 2-fold serial dilutions, while the other four drug combinations were made in fixed ratios (Tables 2 and 3). Six combinations prepared in nM ratios of artemisinin/triclosan were 32:0, 25.6:1780, 19.2:3560, 12.8:5340, 6.4:7120, and 0:8900, respectively; and six concen- trations of artemisinin (nM)/ketoconazole ( M) combina- tions were 32:0, 25.6:3.36, 19.2:6.72, 12.8:10.08, 6.4:13.44, and 0:16.8, respectively. Combination plate preparation. Drug dilutions of combina- tions were made in sterile, flat-bottom, 96-well tissue culture plates as described elsewhere. 1 Each microtiter plate contains wells in eight rows (AH) and 12 columns. Wells in row H, columns 3 to 11, contained a 2× stock concentration, and serial dilutions were made from solutions in these wells up- ward to wells in row B. Each well contained a total volume of 200 L of drug, presynchronized infected RBCs (0.8% para- sitemia at 2.5% hematocrit), and complete culture medium. Simultaneously, parasite control wells without drug were pre- pared in row A, columns 3 to 11, and rows A to H, columns 2 and 12, although only column 2 was taken for IC 50 calcula- tions. The wells in column 1, rows A to H, were kept with a normal RBC suspension in complete medium, without drug. Six combinations (in triplicate) were tested in two plates. Ex- cept for row H, columns 3 to 11, in all wells 100 L of com- plete medium was added. Serial dilution was done from row H to row B, with transfer of 100 L each time after mixing, and 100 L from the wells in row B was discarded finally. The plates were kept in a candle jar and candle-lighted to increase CO 2 concentration. 8 The plates were incubated at 37°C for 48 hours with a brief interruption after 24 hours, and re-gassed by burning the candle. Slide preparation and staining. The plates were removed from candle jar, and the material from each test well was transferred into the corresponding well numbered 1.5-mL mi- crocentrifuge tube. After a fast spin, the supernatant was re- moved and the pellet was mixed thoroughly to prepare thin blood smear slide for each test well. The slides were air-dried, methanol-fixed, and stained in Giemsa stain for 40 minutes. After they were stained, slides were removed from the cou- pling jar, washed in running tap water, and air-dried. The Giemsa-stained slides were examined to count the number of parasites in random adjacent microscopic fields, equivalent to 3000 erythrocytes at 1000× magnification. Percent parasit- emia was calculated. Construction of isobologram. For the combination assay, IC 50 was calculated from two sets of doseresponse graphs (Figures 1 and 2), each containing a drug-alonecurve and four drug-combination curves. Fractional inhibitory concen- tration (FIC) values were calculated separately for each drug concentration present in the combination by the following formula:

FIC =

Fraction of drug concentration required to produce IC 50 when used in combination

Fraction of drug concentration required to produce IC 50 when used alone

Drug interaction assessment. The sum of the fractional in- hibitory concentrations (FICs) of both the drugs for a par- ticular combination shows the interaction pattern 10 between the two drugs.

498 MISHRA AND OTHERS Drug combination preparations. For each combination as- say, drug strength was made

FIGURE 1. Doseresponse curves of artemisinin (A) and triclosan (B) are presented for six different combination solutions with their serial dilutions. IC 50 values were calculated for both of the drugs present in combinations 1 to 6 separately. In these examples, two sets of curves of the first replicate are presented. Six sets of such curves were obtained.

Sum FIC FIC =

IC 50 of A in mixture

 
 

IC 50 of A alone

IC 50 of B in mixture

+

IC 50 of B alone

RESULTS

Sensitivity of P. falciparum. The sensitivity of malaria parasite, strain 3D7, to artemisinin, triclosan, and ketocona- zole was assessed in vitro against erythrocytic stages. The computed IC 50 values given in Table 1 were used for combi- nation assays.

498 MISHRA AND OTHERS Drug combination preparations. For each combination as- say, drug strength was made

FIGURE 2. Doseresponse curves of artemisinin (A) and ketoco- nazole (B) are presented for six different combination solutions with their serial dilutions. In these examples, two sets of curves of the first replicate are presented. Six sets of such curves were obtained.

ARTEMISININ AND TRICLOSAN ANTIPLASMODIAL INTERACTIONS

499

TABLE 1

Baseline sensitivity of P. falciparum (strain 3D7) to artemisinin, tri- closan, and ketoconazole in terms of IC 50

Drug

Mean IC 50 ± SE*

Artemisinin

Triclosan

Ketoconazole

3.97 ± 0.08 nM 1.105 ± 0.059 M 2.101 ± 0.0253 M

* Standard error (n 3).

Sensitivity to drug combinations. From combination ex- periments, the observed IC 50 values were analyzed in relation to data obtained with the single compounds by using the method of Berenbaum, 11 which yielded FICs for the drug combination. Mean FIC values were calculated separately for both the drugs present in combinations 1 to 6. In these six preparations, combinations 1 and 6 contain drug A and drug B alone, respectively, so their IC 50 corresponded to the IC 50 obtained by their individual doseresponse assay. From each triplicate, six sets of doseresponse curves were obtained. The mean FICs of interaction between artemisinin/triclosan (Fig- ure 3A) and artemisinin/ketoconazole (Figure 3B) were plotted in an isobologram for combination preparations 1 through 6. Interaction between artemisinin and triclosan. The FIC values of artemisinin and triclosan fixed-ratio combinations showed a synergistic interaction with combination 2 and trends toward mild synergism with combinations 3, 4, and 5 (Table 2). Interaction between artemisinin and ketoconazole. Interac- tion of artemisinin with ketoconazole showed an additive pat- tern with combination 2, moderate antagonism with combi- nation 3 and 4, and marked antagonism with combination 5 (Table 3).

DISCUSSION

Artemisinin and its four derivatives (dihydroartemisinin, artesunate, artemether, and arteether) are rapidly acting an-

ARTEMISININ AND TRICLOSAN ANTIPLASMODIAL INTERACTIONS 499 T ABLE 1 Baseline sensitivity of P. falciparum (strain 3D7)

FIGURE 3. (A) Isobologram showing the interaction between ar- temisinin and triclosan. (B) Isobologram showing the interaction be- tween artemisinin and ketoconazole. Mean FICs of drugs A and B were taken on the y and x axes, respectively, along with X and Y error bars. The straight line joining both axes shows additive interaction.

timalarial drugs effective against even chloroquine-resistant malaria infections. To improve their efficacy and delay the onset of resistance to this vital class of antimalarial drugs, they are used in combination with other effective antimalarial drugs. At present, they play an essential role in malaria man- agement and control. This, however, depends on ensuring that they are affordable, readily available, and of acceptable quality. Unfortunately, the quality and effectiveness of these antimalarials have been declining, and the supply is often inefficient and unreliable. Fake preparations of the frequently used ACTartemether and lumefantrine combinationhave hit some of the Southeast Asian markets. 12 The global scar- city of artemisinin, leading to its high cost, and the lack of quality control in resource-poor countries where malaria is rampant are some of the factors for these maladies. The wide- spread misuse of ACTs suggests that they will succumb to resistance sooner rather than later. The current grim situation demands reduced dependence on artemisinins and a search for synthetic, inexpensive partner drugs to augment the anti- plasmodial activity of new combinations. In vitro cultures of P. falciparum blood stages form a good first screen for identifying potential partner drugs for ACTs to be developed as anti-malaria therapies. Combining arte- misinin with another in vitro-effective schizonticidal drug may lead to altered pharmacokinetics, decreases in efficacy, and modified toxicity levels of combined drugs. Many of these aspects cannot be predicted using this system. However, an in vitro drugdrug interactions interpretation for antiplasmodial activity by an isobologram is obtainable and can provide in- formation regarding synergistic, additive (indifferent), or an- tagonistic outcomes in inhibiting parasite growth and multi- plication. Isobolograms are prepared either by the fixed-ratio method, 1 where concentration ratios of both drugs differ by a fixed ratio, or by the checkerboard method, 13 where one drug concentration is kept constant while the other is varied. Both of these methods have been used in drug-combination studies for antitumor, antibacterial, and antifungal agents. 14 16 The fixed-ratio method using known synergistic antimalarial drugssulfadoxine and pyrimethaminein combination as- say was validated (data not presented) and found to be accu- rate, simple, and straightforward. In-depth study of antima- larial drug interactions is of great significance to both devel- opment of combination therapies and understanding the mode of action of these drugs. 7 The candidate partner drug in ACT should have an inde- pendent mode of action to delay the selection of resistance. Artemisinins are sesquiterpene lactones containing an endo- peroxide bridge. There are several views regarding the mode of action of artemisinin on P. falciparum. The mechanism of action is considered to be at least two-step processes, involv- ing an activation step and alkylation. In the activation step, intraparasitic iron catalyzes cleavage of the endoperoxide bridge and generates a highly reactive free radical, which in the second step forms a covalent bond with parasite proteins and incapacitates them, 17,18 eventually killing the parasite. It seems to act on many targets within a nanodomain vicinity of the parasite. Perhaps this is one reason why resistance to this vital class of drug has not emerged despite its long historic use for fever resolution in China. A yeast model showed artemisi- nin to interact with the electron transport chain, generating a local reactive oxygen species and causing depolarization of the mitochondrial membrane. 19 It has also been shown that

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TABLE 2

Interaction between artemisinin and triclosan against P. falciparum (strain 3D7) at different combination solutions

Combination

Ratio of drugs (in 100 L)

Mean FIC 50 ± SE*

solution

Drug A

Drug B

Drug A (artemisinin)

Drug B (triclosan)

FICs, interaction

  • 1 5

0

1.0 ± 0.029

0.0

1.0, ADD

  • 2 4

1

0.416 ± 0.0078

0.1093 ± 0.0027

0.525, SYN

  • 3 3

2

0.568 ± 0.0087

0.386 ± 0.0079

0.954, SLT-SYN

  • 4 2

3

0.3233 ± 0.0047

0.494 ± 0.006

0.817, SLT-SYN

  • 5 1

4

0.1483 ± 0.00145

0.666 ± 0.0245

0.824, SLT-SYN

  • 6 0

5

0.0

1.0103 ± 0.0399

1.0103, ADD

* Standard error (n 3). SYN, synergistic; SLT-SYN, slightly synergistic; ADD, additive.

artemisinins exert their antimalarial action by disrupting cal- cium homeostasis; they promote discharge of calcium ions from intracellular stores of malaria parasite by specifically inhibiting a metabolic enzyme: the malarial, calcium- dependent, endoplasmic reticulum ATPase. 20 Inhibition of this enzyme causes uncontrolled release of calcium stored in the endoplasmic reticulum into cytoplasm, causing parasite death. Triclosan is synthetic 2-hydroxydiphenyl ether that has been evaluated as a drug partner with artemisinin. Triclosan has a favorable safety profile and is well tolerated by hu- mans. 2 It is neither an acute oral toxicant nor does it act as a carcinogen, mutagen, or teratogen. 2 It is found in many com- mercial products, such as mouthwashes and deodorants. Its use as a systemic antimicrobial has not been established, but antimalarial activity was demonstrated when it was adminis- tered subcutaneously 6 in an in vivo system. Triclosan also inhibits the growth of the blood stages of P. falciparum in vitro and is effective against chloroquine-resistant and chlo- roquine-sensitive strains. 6 Triclosan inhibits the fatty acid bio- synthesis II (FAS II) pathway in P. falciparum through inhi- bition of Fab enzyme, which is necessary for the conversion of crotonyl-ACP (acyl carrier protein) in butyryl-ACP. 6,21 The FAS II pathway is completely lacking in humans and is, therefore, potentially an attractive target for antimalarial therapeutics. 22 On the basis of these facts, triclosan was con- sidered to be a suitable partner drug for evaluation with ar- temisinin. A modified fixed-ratio isobologram method 1 was used to determine the effect of triclosan in combination with artemisinin in strain 3D7 of P. falciparum. The experiments conducted in combination showed a mild synergistic interac- tion in each combination ratio (Table 2), with synergism at combination 2. But even a weak synergistic interaction may be of importance for its mechanistic implications in the design and development of new agents. 15 Both artemisinin and tri-

closan also showed differential stage-specific inhibitions with high mortality of schizont and trophozoite blood stages, re- spectively (data not shown). This observation substantiates the fact that they have different modes of action on the para- site. The pharmacokinetic characteristics of the partner drug are important considerations in determining the suitability of the drug for practical use. Both partners in drug combinations should have similar pharmacokinetic characteristics, so as not to leave any drug alone in circulation unprotected by the other drug for extended period of time; otherwise, it will de- feat the purpose of combination therapy. It is known that artemisinin has a short half-life of a few hours. 23 Similar stud- ies with triclosan in humans are lacking. Intravenous admin- istration of radiolabeled triclosan to rats showed that the plasma elimination half-time was 9 hours. 24 It will be of interest to evaluate this combination in vivo malaria model systemas well as triclosan in combination with different ar- temisinin derivativesto establish the synergistic effects of this new antimalarial agent. Ketoconazole was also evaluated for drug interactions with artemisinin. This synthetic compound is an imidazole and has wide-spectrum antimicrobial activity, including P. falci- parum. 5 Ketoconazole inhibits the enzyme 14- -demethylase, thereby interrupting the synthesis of ergosterol, an important component of plasma membrane of apicomplexan parasites like P. falciparum. 25 This enzyme is also necessary for con- version of lanosterol into zymosterol, which is converted to ergosterol. 25 It also inhibits growth of falcipain (FP) crystals by a surface-binding mechanism and interferes with other heme crystal-sensitive pathways in P. falciparum, leading to death of the parasite. 3 Falcipain-2 (FP-2) plays an important role in degradation of erythrocyte proteins like hemoglobin, thus rendering an important target in the design of novel antimalarial drugs. 26 These considerations led to selection of this compound in the present studies. Our experiments

TABLE 3

Interaction between artemisinin and ketoconazole against P. falciparum (strain 3D7) at different combination solutions

Combination

Ratio of drugs (in 100 L)

 

Mean FIC 50 ± SE*

solution

Drug A

Drug B

Drug A (artemisinin)

Drug B (ketoconazole)

FICs, interaction

  • 1 5

0

1.019 ± 0.01266

0.0

1.019, ADD

  • 2 4

1

1.217 ± 0.03037

0.301 ± 0.005686

1.518, ADD

  • 3 3

2

1.233 ± 0.03851

0.809 ± 0.02771

2.042, SLT-ANT

  • 4 2

3

1.208 ± 0.05028

1.793 ± 0.06917

3.001, SLT-ANT

  • 5 1

4

0.780 ± 0.01472

3.110 ± 0.03166

3.890, MKD-ANT

  • 6 0

5

0.0

1.001 ± 0.004096

1.001, ADD

* Standard error (n 3). ADD, additive; SLT-ANT, slightly antagonistic; MKD-ANT, markedly antagonistic.

ARTEMISININ AND TRICLOSAN ANTIPLASMODIAL INTERACTIONS

501

showed a combination of artemisinin and ketoconazole to be largely antagonistic in antiplasmodial activity in vitro (Table 3). The antimalarial activity of ketoconazole is enhanced with an increase in oxygen concentration. 27 Older P. falciparum- infected erythrocytes are more susceptible to ketoconazole than younger infected RBCs, as older RBCs are more sus- ceptible to oxidative stress. 28 Because we used the candle-jar method, where the concentration of oxygen in the cultures could not be controlled, it would be premature to conclude that interactions between artemisinin and ketoconazole were antagonistic without supporting evidence from in vivo or in vitro model system wherein oxygen levels could be con- trolled. Although the concentrations of gentamicin used in the experiments do not affect the parasite growth in vitro, 29 even small amounts may affect drug interactions.

Received September 26, 2006. Accepted for publication November 7,

2006.

Financial support: This work was supported in part by the Depart- ment of Biotechnology, Ministry of Science and Technology, Gov- ernment of India. L.C.M. and A.B. received fellowships from the Council of Scientific and Industrial Research, New Delhi. The Ameri- can Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.

Authorsaddress: Lokesh C. Mishra, Amit Bhattacharya, and Viren- dra K. Bhasin, Department of Zoology, University of Delhi, Delhi 110007, India, Telephone: +91-11-27667989, E-mail: virendrabhasin@ hotmail.com.

REFERENCES

  • 1. Fivelman QL, Adagu IS, Warhurt DC, 2004. Modified fixed ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother 48: 40974102.

  • 2. Bhargava HN, Leonard PA, 1996. Triclosan: applications and safety. Am J Infect Control 6: 209218.

  • 3. Chong CR, Sullivan DJ Jr, 2003. Inhibition of heme crystal growth by antimalarials and other compounds: implications for drug discovery. Biochem Pharmacol 66: 22012212.

  • 4. Pfaller MA, Krogstad DJ, 1981. Imidazole and polyene activity against chloroquine-resistant Plasmodium falciparum. J Infect Dis 144: 372375.

  • 5. Pfaller MA, Segal JJ, Krogstad DJ, 1982. Activity of ketocona- zole and its deacyl derivative against Plasmodium falciparum and Candida isolates. Antimicrob Agents Chemother 22: 917

919.

  • 6. Surolia N, Surolia A, 2001. Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP of plasmo- dium falciparum. Nat Med 7: 167173.

  • 7. Bell A, 2005. Antimalarial drug synergism and antagonism:

mechanistic and clinical significance. FEMS Microbiol Lett 253: 171184.

  • 8. Trager W, Jensen JB, 1976. Human malaria parasites in continu- ous culture. Science 193: 673675.

  • 9. Lambros C, Vanderberg JP, 1979. Synchronization of Plasmo- dium falciparum erythrocytic stages in culture. J Parasitol 65:

418420.

  • 10. Gupta S, Thapar MM, Wernsdorfer WH, Bjorkman A, 2002. In vitro interaction of artemisinin with atovaquone, quinine, and mefloquine against Plasmodium falciparum. Antimicrob Agents Chemother 46: 15101515.

  • 11. Berenbaum MC, 1978. A method for testing synergy with any number of agents. J Infect Dis 137: 122130.

  • 12. Newton PN, McGready R, Fernandez F, Green MD, Sunjio M, Bruneton C, Phanouvong S, Millet P, Whitty CJM, Talisuna AO, Proux S, Christophel EM, Malenga G, Singhasivanon P,

Bojang K, Kaur H, Palmer K, Day NPJ, Greenwood BM, Nos- ten F, White NJ, 2006. Manslaughter by fake artesunate in Asiawill Africa be next? PLoS Med 3: e197.

  • 13. Martinez-Irujo JJ, Villahermosa ML, Alberdi E, Santiago E, 1996. A checkerboard method to evaluate interactions be- tween drugs. Biochem Pharmacol 51: 635644.

  • 14. Ryungsa K, Tanabe K, Inoue H, Toge T, 2002. Mechanism(s) of antitumor action in protracted infusion of low dose 5-fluoro- uracil and cisplatin in gastric carcinoma. Int J Oncol 20: 549

555.

  • 15. Hall MJ, Middleton RF, Westmacott D, 1983. The fractional in- hibitory concentration (FIC) index as a measure of synergy. J Antimicrob Chemother 11: 427433.

  • 16. Meletiadis J, Mouton JW, Meis JFGM, Verweij PE, 2003. In vitro drug interaction modeling of combinations of azoles with ter- binafine against clinical Scedosporium prolificans isolates. An- timicrob Agents Chemother 47: 106117.

  • 17. Meshnick SR, 1994. The mode of action of antimalarial endo- peroxides. Trans R Soc Trop Med Hyg 88: 3132.

  • 18. Ridley RG, 2003. Malaria: to kill a parasite. Nature 424: 887889.

  • 19. Li W, Mo W, Shen D, Sun L, Wang J, 2005. Yeast model uncovers dual roles of mitochondria in the action of artemisinin. PLoS Genet 1: e36.

  • 20. Ludwig UE, Webb RJ, van Goethem IDA, East JM, Lee AG, Kimura M, ONeill PM, Bray PG, Ward SA, Krishna S, 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424: 957961.

  • 21. Beeson JG, Winstanley PA, McFadden GI, Brown GV, 2001. New agents to combat malaria. Nat Med 7: 149150.

  • 22. Rao SP, Surolia A, Surolia N, 2003. Triclosan: a shot in the arm for antimalarial chemotherapy. Mol Cell Biochem 253: 5563.

  • 23. White NJ, 1997. Assessment of the pharmacodynamic property of antimalarial drugs in vivo. Antimicrob Agents Chemother 41:

14131422.

  • 24. Siddiqui WH, Buttar HS, 1979. Pharmacokinetics of triclosan in rat after intravenous and intravaginal administration. J Envi- ron Pathol Toxicol 2: 861871.

  • 25. Docampo R, Moreno SNJ, 2001. Bisphosphonates as chemo- therapeutic agents against trypanosomatid and apicomplexan parasites. Curr Drug Targets Infect Disord 1: 5161.

  • 26. Hogg T, Nagarajan K, Herzberg S, Chen L, Shen X, Jiang H, Wecke M, Blohmke CJ, Hilgenfeld R, Schmidt CL, 2006. Structural and functional characterization of falcipain-2, a he- moglobinase from the malarial parasite Plasmodium falci- parum. J. Boil. Chem. 281: 2542525437.

  • 27. Pfaller MA, Krogstad DJ, 1983. Oxygen enhances the antima- larial activity of the imidazoles. Am J Trop Med Hyg 32: 660

665.

  • 28. Tiffert TH, Ginsburg H, Krugliak M, Elford BC, Lew VL, 2000. Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum. Proc Natl Acad Sci USA 97: 331

336.

  • 29. McClom AA, McHardy N, 1984. Evaluation of a range of anti-

microbial agents against the parasitic protozoa, Plasmodium falciparum, Babesia rodhaini and Theileria parva in vitro. Ann Trop Med Parasitol 78: 345354.