Synergy of mefloquine activity with atorvastatin, but not chloroquine and monodesethylamodiaquine, and association with the pfmdr1 gene

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1 J Antimicrob Chemother 2010; 65: doi: /jac/dkq173 Advance Access publication 25 May 2010 Synergy of mefloquine activity with atorvastatin, but not chloroquine and monodesethylamodiaquine, and association with the pfmdr1 gene Nathalie Wurtz 1,Sébastien Briolant 2, Marine Gil 2,Véronique Parquet 2, Maud Henry 2, Eric Baret 2,Rémy Amalvict 2, Lionel Almeras 2, Christophe Rogier 2 and Bruno Pradines 2 * 1 Unité de Recherche en Physiologie et Pharmacocinétique Parasitaires UMR-MD3 Relations Hôte-Parasites Pharmacologie et Thérapeutique, Institut de Recherche Biomédicale des Armées, antenne de Marseille, Marseille, France; 2 Unité de Recherche en Biologie et Epidémiologie Parasitaires Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes UMR 6236, Institut de Recherche Biomédicale des Armées, antenne de Marseille, Marseille, France *Corresponding author. Tel: ; Fax: ; bruno.pradines@free.fr Received 19 November 2009; returned 5 March 2010; revised 11 April 2010; accepted 27 April 2010 Objectives: The aim of the study was to assess the in vitro potentiating effects of atorvastatin, a 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with mefloquine, chloroquine or monodesethylamodiaquine against Plasmodium falciparum and to evaluate whether the effects of atorvastatin could be associated with mutations or gene copy number in multidrug resistance (MDR)-like protein genes. Methods: The susceptibilities of 21 parasite strains to combinations of atorvastatin with mefloquine, chloroquine or monodesethylamodiaquine were assessed using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfmdr1, pfmdr2 and pfmrp genes. Results: Atorvastatin demonstrated synergistic effects in combination with mefloquine. The mefloquine IC 50 (50% inhibitory concentration) was reduced by 7%, 24% and 37% in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 mm, respectively. The synergistic effect of atorvastatin on the response to mefloquine was significantly associated with pfmdr1 copy number. The concentration of atorvastatin that could reduce the IC 50 of mefloquine by 50% was mm for the 12 strains that contained one copy of pfmdr1 and mm for the 9 strains that contained two copies or more. The synergistic effect of atorvastatin in combination with mefloquine was found to be significantly unrelated to mutations in pfmdr1, pfmdr2 or pfmrp genes. Conclusions: The synergy of the effect of mefloquine at concentrations relevant to its achievable plasma concentrations in patients taking 80 mg of atorvastatin daily suggests that atorvastatin will be a good candidate in combination with mefloquine for malaria treatment. Keywords: Plasmodium, malaria, resistance, drugs Introduction During the past 20 years, many strains of Plasmodium falciparum have become resistant to chloroquine and other antimalarial drugs. 1 This has prompted a search for an effective alternative antimalarial drug with minimal side effects. The emergence and spread of parasites resistant to antimalarial drugs have caused an urgent need for novel compounds to be discovered and developed. Statins, i.e. 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) reductase inhibitors, are one of the most widely prescribed classes of drugs in the developed world, with potent cholesterollowering activity and an excellent safety record. Apart from the cholesterol-lowering activity of statins, their immunomodulation and pleiotropic effects may significantly impact infection-related survival. 2,3 However, the use of a statin alone in patients with, or at risk of, severe infections is contentious. 4 Pre-operative statin use was not associated with a reduction of the rate of postoperative infection among patients who underwent cardiac surgery. 4 One of these statins, atorvastatin, showed in vitro antimalarial activity even though the presence of an HMG-CoA homologue was not revealed by BLASTX comparison of the P. falciparum genome with other protozoal HMG-CoA protein sequences. 5 Atorvastatin is 10-fold more active against P. falciparum strains than other statins. 5 Atorvastatin showed no in vitro cross-resistance # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1387

2 Wurtz et al. with chloroquine, monodesethylamodiaquine, mefloquine, quinine, lumefantrine, dihydroartemisinin, atovaquone or doxycycline, and IC 50 (50% inhibitory concentration) values for atorvastatin were found to be unrelated to mutations occurring in transport protein genes involved in quinoline antimalarial drug resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe-1. 6 Atorvastatin, used alone at 20 mg/kg of body weight, failed to prevent death from cerebral malaria or to affect parasitaemia in infected mice. 7,8 One possibility is that atorvastatin could act as an adjuvant therapy. Atorvastatin is an inhibitor of phosphoglycoprotein (Pgp), an efflux protein in cancer cells Multidrug resistance (MDR)-like proteins are involved in mefloquine resistance in P. falciparum. 12,13 The resistance to mefloquine was linked both to mutations in the pfmdr1 gene (P. falciparum multidrug resistance 1) encoding Pgh1 14,15 and to increases in copy number of the wild-type gene. 16,17 The objectives of this study were: (i) to assess the in vitro potentiating effects of atorvastatin in combination with mefloquine, chloroquine or monodesethylamodiaquine against 21 strains of P. falciparum from a wide panel of countries and with different susceptibility profiles; (ii) to evaluate whether the effects of atorvastatin could be associated with mutations in the pfmdr1 gene or with the gene copy number; and (iii) to evaluate whether the effects of atorvastatin could be associated with mutations or with copy number of other ABC transporter or MDR-like protein genes, such as pfmdr2 (P. falciparum multidrug resistance 2) (encoding PfMDR2) 18,19 or pfmrp (P. falciparum multidrug resistance-associated protein) encoding PfMRP1. 20,21 Materials and methods P. falciparum cultures In total, 21 parasite strains (familiar laboratory strains or strains obtained from isolates after growth in culture for an extended period of time) from a wide range of countries (Brazil, Cambodia, Cameroon, Djibouti, French Guyana, the Gambia, Honduras, Indochina, Niger, Republic of Comoros, Republic of the Congo, Senegal, Sierra Leone, Sudan and Uganda) were maintained in culture in RPMI 1640 (Invitrogen, Paisley, UK), supplemented with 10% human serum (Abcys S.A. Paris, France), and buffered with 25 mm HEPES and 25 mm NaHCO 3. Parasites were grown in blood type A+ human blood under controlled atmospheric conditions that consisted of 10% O 2,5%CO 2 and 85% N 2 at 378C with a humidity of 95%. All strains were synchronized twice with sorbitol before use. 22 Clonality was verified using PCR genotyping of polymorphic genetic markers, msp1, msp2 and microsatellite loci. 23,24 The potentiation evaluation for each strain was performed in three independent experiments. Drugs Atorvastatin calcium salt was purchased from Molekula (UK). Mefloquine was obtained from Roche (France, Paris). Chloroquine diphosphate was purchased from Sigma (St Louis, MO, USA). Monodesethylamodiaquine was obtained from the WHO (Geneva, Switzerland). Atorvastatin was dissolved in DMSO 1% (v/v) in RPMI. Two-fold serial dilutions, with final concentrations ranging from 0.01 to 200 mm, were prepared in 1% DMSO in RPMI and distributed into Falcon 96-well plates just before use. Mefloquine was first dissolved in methanol and then diluted in water to obtain concentrations ranging from to 400 nm. Chloroquine was resuspended and diluted in water at concentrations ranging from 5 to 3200 nm. Monodesethylamodiaquine was first dissolved in methanol and then diluted in water to obtain final concentrations ranging from to 1000 nm. In vitro assay To assess the synergy of atorvastatin with quinoline drugs, 25 ml/well of atorvastatin and 200 ml/well of the suspension of synchronous parasitized red blood cells (final parasitaemia, 0.5%; final haematocrit, 1.5%) were distributed in 96-well plates pre-dosed with mefloquine, chloroquine or monodesethylamodiaquine. Parasite growth was assessed by adding 1 mci of tritiated hypoxanthine with a specific activity of 14.1 Ci/mmol (Perkin-Elmer, Courtaboeuf, France) to each well at time zero. The plates were then incubated for 48 h in controlled atmospheric conditions. Immediately after incubation, plates were frozen and then thawed to lyse the erythrocytes. The contents of each well were collected on standard filter microplates (Unifilter GF/B; Perkin-Elmer) and washed using a cell harvester (Filter-Mate Cell Harvester; Perkin-Elmer). Filter microplates were dried and 25 ml of scintillation cocktail (Microscint O; Perkin-Elmer) was placed in each well. Radioactivity incorporated into nucleotides by the parasites was measured with a scintillation counter (Top Count; Perkin-Elmer). The IC 50 was assessed by determining the drug concentration corresponding to incorporation of 50% of the tritiated hypoxanthine by the parasite in the drug-free control wells. The IC 50 value was determined by non-linear regression analysis of log-based dose response curves (Riasmart TM, Packard, Meriden, USA). To evaluate atorvastatin modulation of quinoline resistance, isobolograms were constructed by plotting a pair of fractional IC 50 s for each combination of atorvastatin and mefloquine, chloroquine or monodesethylamodiaquine and for both parasite strains. The atorvastatin fractional IC 50 was calculated by dividing the fixed concentration by the IC 50 of the tested drug alone, and then plotted on the horizontal axis. The corresponding mefloquine, chloroquine or monodesethylamodiaquine fractional IC 50 was calculated by dividing the mefloquine, chloroquine or monodesethylamodiaquine IC 50 combined with fixed concentrations of atorvastatin, and then plotted on the vertical axis. Points lying above the straight diagonal line (corresponding to the points where there is no interaction between the drugs) are antagonistic, and points below the straight diagonal line are considered to be synergistic. In addition, three concentrations of atorvastatin (0.1, 0.5 and 1 mm), which were relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily, 25 were reanalysed separately. In addition, we calculated the concentration of atorvastatin that could reduce the IC 50 of mefloquine by 50% when used alone, [atorvastatin] mefloquine. Nucleic acid extraction Total genomic DNA of each strain was isolated using the E.Z.N.A. w Blood DNA kit (Omega Bio-Tek, GA, USA) extraction method. pfmdr1 single nucleotide polymorphisms (SNPs) pfmdr1 (PFE1150w) was amplified by PCR using the following primer pairs: 5 -AGA GAA AAA AGA TGG TAA CCT CAG-3 and 5 -ACC ACA AAC ATA AAT TAA CGG-3 to amplify codons 86 and 184; and 5 -CAG GAA GCA TTT TAT AAT ATG CAT-3 and 5 -CGT TTA ACA TCT TCC AAT GTT GCA-3 to amplify codons 1034, 1042 and The reaction mixture consisted of 200 ng of genomic DNA, 0.5 mm of forward and reverse primers, buffer (50 mm KCl/10 mm Tris, ph 8.3), 2.5 mm MgCl 2, 200 mm deoxynucleotide triphosphate (dntp) and 0.3 U of Taq DNA polymerase (Eurogentec) in a final volume of 50 ml. The thermal cycler (T3 Biometra w ) was programmed as follows: an initial 948C for 2 min; followed by 1388

3 In vitro synergy of atorvastatin and mefloquine JAC 40 cycles of 948C for 30 s, 528C for 30 s and 728C for 1 min; and a final 15 min extension step at 728C. The amplified fragments were sequenced as previously described. Sequences were analysed with the software BioEdit Sequence Alignment Editor pfmdr2 SNPs pfmdr2 (PF14_0455) was amplified by PCR using the following primer pairs: 5 -ATG GAT GTA TCA AAT TAC GAG TAT TT-3 and 5 -CTA CCA TAT CTT TTA TTT AAA AAT GAT TC-3 ; 5 -CTT CTG ATG TTC AGG AGA AAA ATA-3 and 5 -GCT ATA TCA TGA TAT ACA TTA TCC ATC TC-3 ;5 -TGT ATA TTA TTC CGG CAA CAA TAG-3 and 5 -TAT AAC TGA GCC GAT TTA ACA GC-3 ; and 5 -CTC TTG TAG GTC ATA CAG GTT CTG-3 and 5 -CTA TTT TTT TTT GTT ATT CAT ATT ATC A-3. The reaction mixture consisted of 200 ng of genomic DNA, 0.5 mm of forward and reverse primers, buffer (50 mm KCl/10 mm Tris, ph 8.3), 2.5 mm MgCl 2, 200 mm dntp and 0.3 U of Taq DNA polymerase (Eurogentec) in a final volume of 50 ml. The thermal cycler (T3 Biometra w ) was programmed as follows: 948C for 2 min for the first cycle and 30 s for subsequent cycles; 528C for 1 min for the first cycle and 1 min for subsequent cycles; and 728C for 1 min and 30 s for all cycles, for a total of 40 cycles, followed by a 15 min extension step at 728C. The amplified fragments were sequenced as previously described. Sequence analyses were performed with the software BioEdit Sequence Alignment Editor pfmrp SNPs PCR amplification followed by sequencing was used to detect SNPs in pfmrp (PFA0590w) at positions 191 and 437. The primers used for amplification and sequencing were: pfmrp-501f, 5 -TTT CAA AGT ATT CAG TGG GT-3 ; and pfmrp-1409r, 5 -GGC ATA ATA ATT GAT GTA AA-3. Copy number of pfmdr1 pfmdr1 copy number was estimated by TaqMan real-time PCR (7900HT Fast Real-Time PCR system, Applied Biosytems, Courtaboeuf, France) relative to the single copy gene, b-tubulin (PF10_0084). The following oligonucleotide primers and probes as previously reported were used with slight modifications: TGC ATC TAT AAA ACG ATC AGA CAA A-3, 5 -TCG TGT GTT CCA TGT GAC TGT-3 and 5 -VIC-TTT AAT AAC CCT GAT CGA AAT GGA ACC TTT G-TAMRA-3 for Pfmdr1; and 5 -TGA TGT GCG CAA GTG ATC C-3, 5 -TCC TTT GTG GAC ATT CTT CCT C-3 and 5 -FAM-TAG CAC ATG CCG TTA AAT ATC TTC CAT GTC T-TAMRA-3 for b-tubulin (Eurogentec, Angers, France). Individual PCRs were carried out using 1 TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nm forward primer, 900 nm reverse primer, 250 nm Taqman probe and 5 ml of template DNA in a final volume of 25 ml. The reaction mixtures were prepared at 48C in a 96-well optical reaction plate (Applied Biosystems) covered with optical adhesive covers (Applied Biosystems). The thermal cycling conditions were 508C for 2 min, 958C for 10 min and 50 cycles of 958C for 15 s and 608C for 1 min. Each sample was assayed in triplicate, and analysed with the SDS software (Applied Biosystems). PCR efficiencies of all primer pairs were evaluated on a dilution series of P. falciparum 3D7 genomic DNA and found to be sufficiently close to obviate the need for any correction factor. Therefore, the 2 2DDCt method of relative quantification was used and adapted to estimate copy numbers of the pfmdr1 gene where DDCt¼ (Ct pfmdr1 2Ct b-tubulin ) sample 2(Ct pfmdr1 2Ct b-tubulin ) calibrator. 26,27 Genomic DNA extracted from the P. falciparum 3D7 strain, which has a single copy of each gene, was used as a calibrator, whereas b-tubulin served as the control housekeeping gene in all experiments. Copy number of pfmdr2 pfmdr2 copy number was estimated as previously described for pfmdr1. The following primer pair and probe, designed by using Primer Express software v2.0 (Applied Biosystems), were used to amplify pfmdr2: 5 -AAT TAC CCA ACA CAA CCA TTA CAT ACA-3 and 5 -TCT CTC CTT TTG AAT CAT AGA ATC GA-3 ; and 5 -VIC-ACA TAA AAC CAG GTA CAA CAT GTG CTC TTG TAG GTC-TAMRA-3. Statistical analysis The differences in mefloquine IC 50 between atorvastatin concentration groups have first been tested using analysis of variance (ANOVA) for repeated measures to take into account the fact that observations made within each strain were not independent. Using the most conservative correction for interdependence between observations (i.e. Box s conservative epsilon), the differences in mefloquine IC 50 were tested for concentrations relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily (0.1, 0.5 and 1 mm). Using a random effect linear regression approach, the regression coefficients for the log-transformed mefloquine IC 50 indicated the significance of the mean fold change in mefloquine IC 50 when adding atorvastatin concentrations of 0.1, 0.5 and 1.0 mm. The Kruskal Wallis test or the Mann Whitney U-test were used when appropriate to compare equality of populations for each mutation. To account for multiple comparisons, the results of these tests were considered to be statistically significant only when P,0.005 (0.05 divided by the number of tests¼10), applying the Bonferroni correction. Results While atorvastatin demonstrated antagonistic effects in combination with chloroquine and monodesethylamodiaquine against the 21 P. falciparum strains (Figure 1), atorvastatin demonstrated synergistic effects in combination with mefloquine (Figure 2). The differences in mefloquine IC 50 between atorvastatin concentrations groups have first been tested using ANOVA for repeated measures to take into account the fact that observations made within each strain were not independent. Using the most conservative correction for interdependence between observations (i.e. Box s conservative epsilon), the differences in mefloquine IC 50 were highly significant (P, 0.001) when adding atorvastatin at 0.5 and 1.0 mm, concentrations relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily (Table 1). Using a random effect linear regression approach, the regression coefficients for the log-transformed mefloquine IC 50 indicated that the mean fold change in mefloquine IC 50 when adding atorvastatin concentrations of 0.5 and 1.0 mm (0.76 and 0.63, respectively) were also highly significant (P, 0.001). The mefloquine IC 50 was reduced by 7% [0% to 15%; 95% confidence interval (CI) 0% 14%], 24% (4% to 39%; 95% CI 17% 30%) and 37% (12% to 60%; 95% CI 31% 42%) in the presence of atorvastatin at concentrations of 0.1, 0.5 and 1.0 mm, respectively. These reductions were significant (P, 0.001) for 0.5 and 1.0 mm atorvastatin. Concentrations of atorvastatin that reduced the IC 50 of mefloquine by 50% when used alone, [atorvastatin] mefloquine, are presented in Table 2. The decrease in the mefloquine IC 50 in the presence of atorvastatin was not significantly correlated with the mefloquine IC 50 (r¼0.26, P¼0.6772) or atorvastatin IC 50 (r¼0.20, P¼0.2330). 1389

4 Wurtz et al CQ Fractional Inhibitory Concentration AVA Fractional Inhibitory Concentration 1.4 MQ Fractional Inhibitory Concentration AVA Fractional Inhibitory Concentration MDAQ Fractional Inhibitory Concentration AVA Fractional Inhibitory Concentration Figure 1. In vitro antagonism of atorvastatin (AVA) with chloroquine (CQ) or monodesethylamodiaquine (MDAQ) against the 21 strains of P. falciparum. The mean IC 50 for atorvastatin used alone was determined for each strain, and the copy number and mutations identified in the pfmdr1, pfmdr2 and pfmrp genes are presented in Table 2. The following mutations were identified for at least one strain: pfmdr1 N86Y, Y184F, S1034C, N1042D and D1246Y; pfmdr2 S208N and F423Y; and pfmrp H191Y and S437A. The copy number of pfmdr1 ranged from one to three. Only one copy of pfmdr2 was found in all of the 21 P. falciparum strains. The synergistic effect of atorvastatin on the mefloquine response was significantly associated (P¼0.0022) with the copy number of the pfmdr1 gene (Table 3). The mean IC 50 for Figure 2. In vitro synergy of atorvastatin (AVA) with mefloquine (MQ) against the 21 strains of P. falciparum. [atorvastatin] mefloquine was mm for the 12 strains with one copy of pfmdr1, mm for the 8 strains with two copies and 7.8 mm for the only strain with three copies ( mm for the 9 strains with two copies and more of pfmdr1). Discussion Mefloquine is currently one of the recommended chemoprophylactic regimens for travellers visiting malaria-endemic areas of south-east Asia, Africa and South America. Mefloquine is recommended by the French Consensus conference for chemoprophylaxis in countries with a high prevalence of resistance to chloroquine or multiresistance (group 3 countries). 28 In addition, the combination of artesunate and mefloquine has been evaluated and used mainly in south-east Asia and South America for malaria treatment. 29 Despite its efficacy against chloroquine-resistant strains, the emergence of mefloquine resistance has been documented during the last two decades in south-east Asia. Mefloquine artesunate combination therapy is beginning to fail in southern Cambodia and Thailand Furthermore, failures of antimalarial prophylaxis based on mefloquine were observed in Africa. 33 A large amount of scientific effort is directed toward elucidation of the mechanisms underlying resistance to antimalarial drugs with the hope of restoring/improving the efficacy of existing drugs and of developing new drugs that can bypass resistance mechanisms. One of the strategies for reducing the prevalence of malaria is the combinatorial use of drugs. The use of combinations protects each drug from the development of resistance and reduces the overall transmission of malaria

5 In vitro synergy of atorvastatin and mefloquine JAC Table 1. In vitro susceptibility of P. falciparum strains to atorvastatin alone, mefloquine alone and the combination of mefloquine+atorvastatin at concentrations of 0.1, 0.5 and 1.0 mm IC 50 AVA alone IC 50 MQ alone IC 50 MQ+0.1 mm AVA IC 50 MQ+0.5 mm AVA IC 50 MQ+1.0 mm AVA IC 50 geometric mean 7.24 mm 33.5 nm 31.2 nm 25.5 nm 21.2 nm IC 50 mean 95% CI Average IC 50 diminution 7% 24% 37% Average IC 50 diminution 95% CI 0% 14% 17% 30% 31% 42% P-value (versus MQ alone) 0.096,0.001,0.001 AVA, atorvastatin; MQ, mefloquine. Table 2. In vitro susceptibility to atorvastatin, the concentration of atorvastatin that decreased the mefloquine IC 50 by half ([AVA] MQ ), pfmdr1, pfmdr2 and pfmrp polymorphisms and the copy number of pfmdr1 and pfmdr2 genes in 21 P. falciparum strains Amino acid encoded by the pfmdr1 codon Amino acid encoded by the pfmdr2 codon pfmrp codon Strains MQ IC 50 (nm) AVA IC 50 (mm) [AVA] MQ (mm) copy no copy no IMT Y F S N Y 1 N Y 1 H S W Y Y S N D 2 N Y 1 Y A IMT Y Y S N D 1 N Y 1 H S D N Y S N D 1 N Y 1 H S IMT Bres Y Y S N D 2 N Y 1 Y A IMT N Y S N D 1 S Y 1 H S FCM Y Y S N D 1 N Y 1 Y A IMT K N Y C D D 1 N Y 1 Y A IMT N Y S N D 1 S F 1 H S IMT N Y S N D 1 S F 1 H S IMT K N F C D Y 2 N Y 1 Y A IMT Guy N F S D Y 1 N F 1 Y A IMT N Y S N D 1 N Y 1 H S IMT Vol Y Y S N D 2 N Y 1 Y A PA Y Y S N D 3 N Y 1 Y A 106/ Y Y S N D 2 N Y 1 Y A IMT L Y Y S N D 2 N Y 1 Y A FCR Y Y S N D 2 N Y 1 Y A IMT K N F C D D 2 N Y 1 Y A 3D N Y S N D 1 S F 1 H S HB N F S N D 1 N Y 1 H S AVA, atorvastatin; MQ, mefloquine. Values are means of mean IC 50 of three experiments for each strain. Bold font indicates point mutations. Atorvastatin, a HMG-CoA reductase inhibitor, reduced in vitro growth of P. falciparum in the micromolar range. A generally agreed upon level of efficacy would be in the low or middle nanomolar range. However, if the mechanisms of action of such a compound are sufficiently new and different from those of the commonly used antimalarial drugs, this compound could warrant further investigation. The absence of in vitro crossresistance between atorvastatin and mefloquine suggests that atorvastatin could be a good potential partner for mefloquine. 6 These different considerations and the capacity of atorvastatin to inhibit human Pgh 11 led us to evaluate atorvastatin in combination with mefloquine. Atorvastatin improved the in vitro activity of mefloquine at concentrations relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily. 25 Furthermore, the doses of atorvastatin administered to humans could be increased to 120 mg daily with only limited additional side effects. 35 A dose of 120 mg of atorvastatin increased the C max after oral administration by levels from 4- to 10-fold. Cerebral malaria shares common pathophysiological features with 1391

6 Wurtz et al. Table 3. Association of the in vitro atorvastatin (AVA) response (IC 50 ), mefloquine (MQ), AVA+MQ and polymorphism in the pfmdr1, pfmdr2 and pfmrp genes and the copy number of the pfmdr1 gene in 21 strains of Plasmodium falciparum AVA MQ AVA +MQ Genotype P-value significance a P-value significance a P-value significance a Mutation in codon 86 of pfmdr1 gene NS NS NS Mutation in codon 184 of pfmdr1 gene NS NS NS Mutation in codon 1034 of pfmdr1 gene NS NS NS Mutation in codon 1042 of pfmdr1 gene NS NS NS Mutation in codon 1246 of pfmdr1 gene NS NS NS pfmdr1 copy number (1, 2 and 3) NS NS NS pfmdr1 copy number (1 and.1) NS NS S Mutation in codon 208 of pfmdr2 gene NS NS NS Mutation in codon 423 of pfmdr2 gene NS NS NS Mutation in codon 191 of pfmrp gene NS NS NS Mutation in codon 437 of pfmrp gene NS NS NS a Mann Whitney U-test or Kruskal Wallis test; significance cut-off (0.05/10, 10 tests, Bonferroni correction). S, significant; NS, not significant. sepsis, especially with regard to the pathology of the endothelium, 36 and critically ill patients with sepsis had a significantly higher C max when compared with healthy volunteers (110.5 versus 5.9 ng/ml). 37 The synergistic effect of atorvastatin in combination with mefloquine was found to be unrelated to mutations in the MDR-like protein genes. However, the synergy of the mefloquine response with atorvastatin was associated with pfmdr1 gene copy number. The atorvastatin concentration required to reduce the mefloquine response by half was higher in strains with more than two copies. These data are promising since mefloquine artesunate combination therapy is beginning to fail in southern Cambodia and Thailand The resistance of P. falciparum to mefloquine in vitro and the clinical failures of this treatment have previously been associated with an increase in pfmdr1 copy number in Asia 16,30,38 but not in Africa. pfmdr1 amplification is detected comparatively rarely in African isolates Nevertheless, in the current study, the mefloquine IC 50 s of the isolates from Asia were reduced from 14% to 60% in combination with 1.0 mm atorvastatin and from 12% to 44% for the isolates with more than two pfmdr1 copies. The combination of atorvastatin with mefloquine and artesunate could be an alternative to maximize the efficacy and longevity of the treatment. Data on synergy of atorvastatin with artemisinin derivatives are contradictory. Wrong and Davis 42 showed that atorvastatin did not potentiate in vitro response to dihydroartemisinin on two P. falciparum strains, while we showed that atorvastatin had a synergistic effect on dihydroartemisinin activity at concentrations relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily. 43 Atorvastatin was not significantly associated with other ATPbinding cassette (ABC) transporters such as PfMDR2 or PfMRP. Nevertheless, the significance levels of the associations between the synergistic effect of atorvastatin in combination with mefloquine (P¼0.0155) or mefloquine activity (P¼0.0126) and polymorphism in the pfmrp gene (codons 191 and 437) were above the Bonferroni-corrected P-value threshold (0.005), but below However, this is a preliminary report. Twenty-one strains may not be sufficient to reach definite conclusions. The validity of the conclusion should be further investigated by analysing more strains or isolates, particularly from Asia. Atorvastatin did not increase the in vitro activity of chloroquine and monodesethylamodiaquine. The data on combination with chloroquine were in concordance with those of Wrong and Davis. 42 As predicted by the inhibition of Pgh in human cells by atorvastatin, 9 11 the association between pfmdr1 copy numbers and synergy of mefloquine effects by atorvastatin was a valid hypothesis. The transporter Pgh1 was not involved in chloroquine or monodesethylamodiaquine resistance. 44,45 PfCRT (P. falciparum chloroquine resistance transporter), which belongs to the drug and metabolite transporter (DMT) superfamily, was the transport protein involved in chloroquine 46 and monodesethylamodiaquine resistance. 47 The mode of action of atorvastatin is as yet unknown. The absence of cross-resistance with chloroquine, quinine, monodesethylamodiaquine, mefloquine, lumefantrine, dihydroartemisinin, atovaquone or doxycycline suggested different features in drug uptake and/or mode of action of atorvastatin and the other compounds. 6 The hypothesis of inhibition of Pgh1 is compelling. Atorvastatin is an HMG-CoA reductase inhibitor. Nevertheless, the presence of an HMG-CoA homologue was not revealed by BLASTX comparison of the P. falciparum genome with other protozoal HMG-CoA protein sequences. Parasites treated with mevastatin show depressed biosynthesis of dolichol and isoprenoid pyrophosphate. 48 In addition, mevastatin decreases the viability of cells by inhibiting proteasome activity. One of our future objectives is to identify the modification of the P. falciparum proteome in parasites in contact with atorvastatin. The in vitro data on mefloquine support a prospective evaluation in animal models. The different features in drug uptake and/or mode of action of atorvastatin compared with other antimalarial drugs and the synergy of the effect of mefloquine at concentrations relevant to atorvastatin plasma concentrations achievable in patients taking 80 mg of atorvastatin daily suggest that atorvastatin will be a good candidate in combination with mefloquine for 1392

7 In vitro synergy of atorvastatin and mefloquine JAC malaria treatment. All of these observations support calls for both an in vivo evaluation with a pharmacokinetic component and clinical trials of atorvastatin as an antimalarial therapy. Funding This work was supported by the Direction Centrale du Service de Santé des Armées (grant number 2007 RC 32). Transparency declarations The authors have no conflicts of interest concerning the work reported in this paper. The authors do not own stocks or shares in a company that might be financially affected by the conclusions of this article. The conclusions of this article were in no way financially influenced. References 1 Le Bras J, Musset L, Clain J. Antimalarial drug resistance. Med Mal Infect 2006; 36: Terblanche M, Almog Y, Rosenson RS et al. Statins: panacea for sepsis? Lancet Infect Dis 2006; 6: Greenwood J, Mason JC. Statins and the vascular endothelial inflammatory response. 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