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Experimental Parasitology 115 (2007) 387–392

Roxithromycin potentiates the effects of chloroquine and mefloquineon multidrug-resistant Plasmodium falciparum in vitro

T.H. Min a, M.F.M. Khairul a, J.H. Low a, C.H. Che Nasriyyah a, A. Noor A’shikin b,M.N. Norazmi c, M. Ravichandran b, S.S. Raju a,*

a Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysiab Department of Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

c School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Received 23 March 2006; received in revised form 5 October 2006; accepted 10 October 2006Available online 21 November 2006

Abstract

Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium

falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), aknown p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQand reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronizedP. falciparum (Dd2 strain) cultures. The IC50 values of CQ and MQ were 60.0 ± 5.0 and 16.0 ± 3.0 ng/ml; these were decreased substan-tially when combined with ROM. Isobolograms indicate that CQ–ROM combinations were relatively more synergistic (mean FICI 0.70)than MQ–ROM (mean FICI 0.85) with their synergistic effect at par with CQ–verapamil (VRP) (mean FICI 0.64) and MQ–VRP (meanFICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.

� 2006 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Plasmodium falciparum; Chloroquine resistance; Mefloquine resistance; Roxithromycin; p-glycoprotein inhibitor

1. Introduction

Malaria is one of the biggest health problems in theworld with 500 million new cases occurring worldwide eachyear, resulting in 1.5–2.7 million deaths (Kondrachine andTrigg, 1997). The situation is rapidly deteriorating with theemergence of chloroquine (CQ) and mefloquine (MQ)resistant Plasmodium falciparum (P. falciparum) in manyareas. The spread of CQ and MQ resistant P. falciparum

throughout Southeast Asia, Africa and South Americahas necessitated alternative treatment regimes for malaria(Bruce-Chwatt et al., 1986; Noronha et al., 2000; Wich-mann et al., 2003).

In order to preserve the efficacies of current and futurechemotherapeutic agents new strategies must be continu-

0014-4894/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.exppara.2006.10.004

* Corresponding author. Fax: +609 7653370.E-mail address: ssraju97@rediffmail.com (S.S. Raju).

ously developed, evaluated and implemented. Combinationtherapy is an excellent way to combat drug resistance andavoids the wait for the development of new antimalarialdrugs. Various drugs with different mechanisms of actionmay enhance efficacies and extend the therapeutic ‘lifespan’ of existing antimalarial agents.

Roxithromycin (ROM), a macrolide antibiotic, is ananalogue of azithromycin (AZM). AZM has a similar activ-ity to doxycycline against malaria in vitro (Yeo and Riek-mann, 1995) and in vivo (Anderson et al., 1995). Tayloret al. (2001) showed that combinations of CQ–doxycyclinewere effective against CQ-resistant P. falciparum. SinceAZM has a strong antimalarial activity against CQ-resis-tant P. falciparum strains (Biswas, 2001) and a reported syn-ergistic antimalarial effect with CQ (Ohrt et al., 2002), it ishoped that ROM could produce the same effect.

ROM contains the 14-lactone ring derived from erythro-mycin (Kanfer et al., 1998). Erythromycin is bactericidal

388 T.H. Min et al. / Experimental Parasitology 115 (2007) 387–392

against susceptible microorganisms, inhibiting protein syn-thesis by binding to the 50S unit of the ribosome. It is pos-sible a similar mechanism occurs in plasmodia. ROM (LikeAZM) readily penetrates cells, achieving a 20 to 30 foldhigher intracellular concentration, thus making it highlysuitable for the treatment of diseases caused by intracellu-lar pathogens such as Legionella, Chlamydia, Rickettsia

spp., Mycobacterium spp. and Brucella spp. (Pechere,2001).

ROM has an intrinsic antimalarial activity like that ofazithromycin (Pradines et al., 2001), and is a known p-gly-coprotein inhibitor (Corallo and Rogers, 1996). p-glyco-protein inhibitors are reported to have a chemosensitizingeffect on MDR cancers and on CQ-resistant P. falciparummalaria (Martin et al., 1987). Because of these propertiesand its potential to kill intercellular pathogens, we hopethat concomitant administration of ROM, individuallywith CQ and then MQ, would reverse or prevent CQ/MQ-resistance. We therefore studied these effects ofROM with CQ and MQ on Dd2 (CQ and MQ resistantstrains of P. falciparum), compared with similar combina-tions using VRP with CQ/MQ (a known synergistic combi-nation). We further studied the effects of ROM and CQ/MQ combinations on 3D7 (CQ sensitive strain) and W2(MQ sensitive strain).

2. Materials and methods

2.1. Parasite clones

Well characterized clones of Dd2 (CQ and MQ resis-tant) (MRA-156), 3D7 (CQ sensitive) and W2 (MQ sensi-tive) strains of P. falciparum were used for the drugassays. These were kindly provided by Malaria researchand reference reagent resource center (MR4), ATCC, USA.

2.2. Drug assays

The susceptibility of Dd2, 3D7 and W2 strains to theassayed drugs were evaluated using a modified semi-auto-mated microdilution technique. Cultures were synchro-nized with 5% D-sorbitol (Sigma cell culture, Louis, USA)and differential stages were checked by thin smears. Ifgreater than 80% of parasites were observed at the ringstage of infection and more than 0.5% of infected erythro-cytes were evident, the isolates were then used in drug testswith CQ and MQ. Drug tests were conducted on sterile 96-well flat bottomed culture plates in RPMI 1640 (Gibco�,Invitrogen Cooperation, Aucland, NZ) supplemented withgentamicin (1 mg/ml), (0.9%) glucose, HEPES, NaHCO3

with 5% erythrocytes and 10% human pooled serum inaccordance with the methods of Desjardins et al. (1979).The plates with cultures were incubated in an atmosphereof 5% CO2, 5% O2 and 90% N2 using a modified candlejar method (Trager and Jensen, 1976).

ROM (Sigma–Aldrich, Steinheim, Germany) and MQHCl (Mepha, Switzerland) were dissolved in 95% ethanol,

then diluted in distilled water, RPMI and CCM (after dilu-tion the concentration of ethanol was 0.12%). CQ diphos-phate (Sigma–Aldrich, Steinheim, Germany) was dissolvedin distilled water. Fifty microliter of the drug solution wasadded to 150 ll RBC-medium-mix (containing 5% RBCand a parasitaemia of around 0.5%) in each well, and thedrug assays were repeated three times.

Growth was measured using radioactive 3H-hypoxan-thine (0.5 lCi/ml/well; Amersham Life Sciences, Bucking-hamshire, UK), which was spiked at 24 h post-incubation(Desjardins et al., 1979). After 48 h the cultures were har-vested onto Unifilter GF/C 96-well glass filters (Perkin-El-mer Life Sciences, Boston, USA) using a cell harvestersystem (Inotech, Dottikon, Switzerland). The filter plateswith parasite cultures were spiked with scintillation fluidand were analyzed for radioactivity by means of an auto-mated beta counter (Plate CHAMELEON multilablecounter 425–104, Hidex Oy, Turku, Finland).

2.3. Statistical analysis

All data were analyzed by SPSS 11.0 software. The IC50

of each drug alone and in combination were evaluated bylog probit. Fractional IC50 (FIC50) and FIC index (FICI)were calculated using the formulae:

FICI ¼ FIC50Aþ FIC50B

FIC50A ¼ IC50 of drug A at fixed conc: of drug B

IC50 of drug A

FIC50B ¼ IC50 of drug B at fixed conc: of drug A

IC50 of drug B

FICI data was interpreted (<1 synergism; >1 antagonism;=1 additive) according to Ohrt et al. Growth curve datawas plotted using SPSS 11.0 software. Isobolograms wereconstructed for the combinations, MQ and ROM, andCQ and ROM, by using Microsoft Excel. FIC indices onthe FICI = 1 line are considered additive interaction, whilstthe FIC indices above the line are considered antagonisticinteraction and below, synergistic interaction.

The growth rate (% of growth) was calculated by divid-ing the total number of infected erythrocytes in the pres-ence of drug(s) (total erythrocytes minus uninfectederythrocytes), by the maximum number of infected eryth-rocytes with no drug administered, multiplied by 100.

3. Results

The IC50 of ROM against Dd2 clones of P. falciparum

was 3.8 ± 0.9 lg/ml, whilst the IC50 values of CQ andMQ were 60.0 ± 5.0 and 16.0 ± 3.0 ng/ml, respectively.By adding ROM, the concentration of MQ/CQ needed toachieve the same antiparasitic activity was markedlyreduced (Tables 1 and 2). Similarly, in the presence ofCQ/MQ the concentration of ROM needed to achievethe same antiparasitic activity was also reduced (Tables 1and 2). In comparison, the IC50 of CQ against 3D7 clones

Fig. 1. Isobologram showing the synergistic effect between ROM and CQ(average FIC of 0.70) in vitro against Dd2 clones of P. falciparum,compared to VRP and CQ combination (average FIC of 0.64). The IC50 ofthe drug combination was plotted as fractional of IC50 (FIC50).

Fig. 2. Isobologram showing the synergistic interaction between ROMand MQ (average FIC of 0.86) in vitro against Dd2 clones of P. falciparum,compared to VRP and MQ combination (average FIC of 0.60). The IC50ofthe drug combination was plotted as fractional of IC50 (FIC50).

Table 1Interaction between ROM and CQ at fixed concentrations of ROM andCQ

ROM at fixed concentration (lg/ml) CQ (ng/ml) FIC50 Index

2 10.2 0.71 12.6 0.480.5 21.6 0.490 60 1

CQ at fixed concentration (ng/ml) ROM (lg/ml) FIC50 Index

32 0.75 0.7316 1.36 0.638 2.15 0.70 3.77 1

Fractional IC50 Index (FIC50 Index) of ROM and CQ showing synergisticinteraction. CQ, chloroquine; ROM, roxithromycin FIC50 Index < 1,synergism; FIC50 Index > 1, antagonism; FIC50 Index = 1, additive.

Table 2Interaction between ROM and MQ at fixed concentrations of ROM andMQ. Fractional IC50 Index (FIC50 Index) of RQ and MQ showingsynergistic interaction. MQ, mefloquine; ROM, roxithromycin FIC50

Index < 1, synergism; FIC50 Index > 1, antagonism; FIC50 Index = 1,additive

ROM at fixed concentration (lg/ml) MQ (ng/ml) FIC50 Index

2.83 6.34 0.932 6.92 0.780 19.21 1

MQ at fixed concentration (ng/ml) ROM (lg/ml) FIC50 Index

14.67 0.57 0.889.28 1.7 0.840 4.71 1

T.H. Min et al. / Experimental Parasitology 115 (2007) 387–392 389

(CQ-sensitive P. falciparum) was 4.38 ± 0.81 ng/ml and theIC50of MQ against W2 clones (MQ-sensitive P. falciparum)was 3.8 ± 0.2 ng/ml; ROM could not reduce the IC50 val-ues of CQ and MQ.

The mean FIC indices of ROM + CQ and ROM + MQwere 0.62 and 0.85, respectively, indicating synergisticinteractions between them; these were comparable toVRP + CQ (mean FICI 0.64) and VRP + MQ (mean FICI0.60) interactions. Constructed isobolograms (Figs. 1 and2) clearly demonstrate the synergistic interaction betweenROM and CQ, and ROM and MQ; with comparabilityto VRP + CQ and VRP + MQ isobolograms, respectively.ROM and MQ showed less synergy than ROM and CQ.The isobolograms of CQ + ROM on 3D7 clones (CQ-sen-sitive P. falciparum) revealed an antagonistic interaction(Fig. 3) and the isobolograms of MQ + ROM on W2clones (MQ-sensitive P. falciparum) revealed an additive/antagonistic interaction (Fig. 4).

Growth curves were drawn by plotting percentagegrowth (% of parasitemia) of Dd2 clones against fixedMQ or CQ concentrations with fixed ROM concentrations.These showed the parasites treated with CQ/MQ alone,grew better in comparison to the parasites treated with

combination ROM + CQ/ROM + MQ. When ROM wasadded to CQ/MQ, the growth curves shifted to the left.This shift to the left of the control curve, indicates the inter-action between ROM and CQ/MQ to be either synergisticor at least additive (Figs. 5 and 6).

4. Discussion

Our results suggest ROM exhibits antimalarial activityon all three assayed strains of P. falciparum; Dd2 (CQand MQ resistant), 3D7 (CQ sensitive) and W2 (MQsensitive) corroborating the findings of Pradines et al.(2001). Our results (FIC50 indices, isobolograms andgrowth curves) clearly indicate the synergistic interaction

Fig. 3. Isobologram showing the antagonistic effect between ROM andCQ (average FIC of 2.62) in vitro against 3D7 clones (CQ-sensitive P.

falciparum). The IC50 of the drug combination was plotted as fractional ofIC50 (FIC50).

Fig. 4. Isobologram showing the additive/antagonistic interactionbetween ROM and MQ (average FIC of 1.26) in vitro against W2 clones(MQ-sensitive P. falciparum). The IC50 of the drug combination wasplotted as fractional of IC50 (FIC50).

Fig. 5. Growth curves of P. falciparum (Dd2 strain)—72 h post-incuba-tion with ROM and CQ. Shifting of growth curves to left indicatessynergistic/additive interaction between ROM and CQ.

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between the combinations of ROM and CQ/MQ againstCQ/MQ-resistant P. falciparum parasites. However, nosynergistic interaction between the combinations of ROMand CQ/MQ, against CQ/MQ-sensitive P. falciparum par-asites was observed. Similarly AZM, an analogue of ROM,exhibited antimalarial effects in vitro against two strains ofP. falciparum (Ohrt et al., 2002). Combinations of CQ andAZM showed a range of activity, from additive to synergis-tic interactions, for the eight CQ-resistant isolates testedwhereas quinine was synergistic with AZM (Ohrt et al.,2002).

Macrolides have a slow antimalarial action and shouldbe administered together with fast acting antimalarialdrugs such as CQ and MQ, to reverse drug resistanceefficiently (Yeo and Riekmann, 1995). Pradines et al.(2001) previously showed the activity of ROM improveswith a longer incubation time (IC50 is 18.6 lM at 48 h

and 4.8 lM at 96 h for W2 strain). We therefore incubatedthe microtiter plates for 72 h and the IC50 obtained forROM (3.8 ± 0.9 lg/ml) was similar to the value obtainedafter 96 h incubation by Pradines et al.

ROM, a single-base macrolide (Labro, 1997), canachieve a higher intracellular concentration in almostall the cells (Pechere, 2001). Macrolides are less activeagainst Borrelia burgdorferi at low pH in acidic endo-some and their activity was improved by alkalization ofthat compartment with CQ (Donta, 2003). CQ is a dip-rotic weak base, which can accumulate to extensive val-ues in the acidic food vacuole of the parasites bydiffusing across the vacuolar membrane where it becomesprotonated. The protonated CQ accumulates at mM lev-els in the vacuole from nM levels in the plasma (Yayonet al., 1984). CQ may therefore enhance accumulation ofROM in the acidic food vacuole of the parasites com-pared to MQ, a monobase. This is consistent with ourresults, which showed the CQ and ROM combinationto be more synergistic compared to the MQ and ROMcombination.

Pgh1, a gene product of Pfmdr1, can be inhibited by p-glycoprotein inhibitors such as ROM. Pgh1, a typicalmember of the ATPase Binding Cassette (ABC) superfam-ily and an analogue of p-glycoprotein, plays a role in CQand MQ resistance (Foote et al., 1990). Pgh1 is localizedto the membrane of the parasite food vacuole and playsa direct role in transporting drugs (Cowman et al., 1991).Verapamil, a Ca2+ channel blocker and known p-glycopro-tein inhibitor can reverse CQ resistance in P. falciparum

(Martin et al., 1987). However, there are other studiesshowing the amplification of the pfmdr1 gene and itsproduct Pgh1 result in alterations to CQ sensitivity (Priceet al., 2004). Cowman et al. (1991) indicated the increasedpfmdr1 gene copy number would reduce CQ resistance,however, our findings do not support this. According to

Fig. 6. Growth curves of P. falciparum (Dd2 strain)—72 h post-incubation with ROM and MQ. Shifting of growth curves to left indicates synergistic/additive interaction between ROM and MQ.

T.H. Min et al. / Experimental Parasitology 115 (2007) 387–392 391

their observations, concomitant administration of ROMshould have increased the MQ response more than theCQ response.

Pharmacokinetic studies of ROM indicate the meanpeak plasma concentration of ROM (at steady state, aftera dose of 150 mg twice daily) is 9.3 lg/ml, with 96% boundto plasma protein at trough concentrations; however bind-ing is saturable and only about 86% is bound at usual peakconcentrations (Lassman et al., 1988; Puri and Lassman,1987; Wise et al., 1987). Our results show that IC50 ofROX against CQ/MQ resistant P. falciparum is 3.8 ±0.9 lg/ml. From pharmacokinetic data and IC50 values ofROM it can be concluded that plasma levels of ROM (pro-tein bound free) equivalent to IC50, could not be achievedwith the usual therapeutic dose. It is possible however toachieve a higher intra-erythrocytic concentration than theIC50 value of ROM; it is known to achieve a high tissueconcentration approximately 20 times the plasma concen-tration (Bouvier d’Yvoire et al., 1998; Ishiguro et al.,1989). Moreover, our study has shown concentrations ofROM as low as 0.5–2 lg/ml, could lower IC50 values ofCQ by 3- to 6- fold. These low free drug concentrationsof ROM could be achieved by increasing its dose to200 mg twice daily, however this dose of ROM may haveto be increased further to potentiate the effect of MQ.

The synergistic interaction between ROM and CQ/MQcombinations was at par with VRP and CQ/MQ combina-tions. We conclude that ROM is a potential antimalarialagent and that it enhances the antimalarial effect of CQand MQ. Thus the combinations of ROM and CQ/MQwould give a positive resolve for reversing drug resistancein malaria. Currently we are carrying out in vivo studiesto substantiate these in vitro findings.

Acknowledgments

This study was supported by IRPA EA long-term Grant(Grant No. 305/PPSP/6112250) from the Ministry ofScience, Technology and Innovation through the Schoolof Medical Sciences, Universiti Sains Malaysia. We thankMR4 for providing us CQ and MQ resistant P. falciparum

strain Dd2 and thank Mepha Sdn Bhd, Malaysia forsupplying Mefloquine HCl.

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