Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

Quinoline resistance in malaria is frequently compared withP-glycoprotein-mediated multidrug resistance (mdr) in mammaliancells. We have previously reported that nonylphenolethoxylates,such as NP30, are potential Plasmodium falciparum P-glycoproteinsubstrates and drug efflux inhibitors. We used in vitro assaysto compare the ability of verapamil and NP30 to sensitize twoparasite isolates to four quinolines: chloroquine (CQ), mefloquine(MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize(reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesserextent, CQ. The presence of 2 µM verapamil had no effecton mefloquine resistance; however, the presence of verapamilmodulated the activities of QN and QD in a manner parallel tothat observed for CQ. Genetic analysis of putative quinolineresistance genes did not suggest an association between knownpoint mutations in pfcrt and pfmdr1 and NP30 sensitization activity.We conclude that the sensitization action of NP30 is distinctboth phenotypically and genotypically from that of verapamil.

INTRODUCTION

Top

Introduction

The incidence of malaria is increasing, with an estimated 300million to 500 million cases of malaria and 3 million deathsfrom malaria annually (30). Despite optimistic predictions ofthe eventual control and eradication of malaria made in thepast, the spread of drug-resistant parasites, particularly thoseresistant to chloroquine (CQ), has led to a resurgence of thedisease.

New antimalarials have been introduced in recent years (11,22); however, they are significantly more expensive than CQand pyrimethamine-sulfadoxine (Fansidar), the treatment optionsmost commonly used in developing nations. The appearance ofmalaria resistant to CQ was first reported in the late 1950sin South America (33) and Southeast Asia (15) and has sincespread to nearly all regions where malaria is endemic (18).The emergence of CQ-resistant falciparum malaria, particularlyin Africa, has been a global health disaster (18) and is directlyresponsible for the increased number of deaths from Plasmodiumfalciparum infections (30).

The mechanism by which quinoline-based antimalarials inhibitthe growth of blood-stage parasites remains controversial; however,they appear to interfere with the production of the malariapigment hemazoin (for a review, see reference 28). Quinolineuptake into the food vacuole, where polymerization occurs, maybe a passive process (27); and drug-resistant parasites appearto have developed or adapted mechanisms to remove quinolinesfrom this environment (32). Quinoline resistance in P. falciparumis frequently compared to multidrug resistance in mammaliancells, a comparison that is supported by the observation thatCQ resistance in P. falciparum can be reversed by known P-glycoproteinsubstrates such as verapamil (19), chlorpromazine (3), promethazine(21), chlorpheniramine (4), and citalopram (12). P. falciparumcontains several multidrug resistance-like proteins, includingPgh1, which is encoded by pfmdr1 (14). Site-directed mutagenesisof Pgh1 has demonstrated that Pgh1 is associated with resistanceto mefloquine (MF), quinine (QN), quinidine (QD), and halofantrine(26). On the other hand, mutations in Pgh1 were not directlyresponsible for CQ resistance. A gene on chromosome 7, pfcrt(9), which encodes a vacuolar transport protein, has been linkedto CQ resistance. Parasite transfection studies have demonstratedthat mutations in pfcrt are sufficient to confer CQ resistancein vitro (13). However, in clinical studies pfcrt mutationsappear to be necessary, but not sufficient, to predict CQ treatmentoutcomes in vivo (10, 24). Other host and, possibly, parasitefactors contribute to parasite clearance following CQ therapy.These include host immune response (premunition) and a possiblerole for pfmdr1 (26) and other gene products in mediating higherlevels of CQ resistance. Furthermore, the observation that thereversal of CQ resistance by verapamil is not uniformly effective(31) is consistent with the hypothesis that multiple gene productsmodulate the responsiveness of malaria parasites to CQ.

NP30, an uncharged polyethoxylated nonylphenol surfactant containingan average of 30 ethoxylate units (Fig. 1), appears to belongto a novel class of resistance reversal agents since its structuredoes not contain a nitrogen atom and the surfactant has no ionizablegroups (7, 8). We therefore wished to determine if NP30 hadquinoline sensitization properties that were similar to or differentfrom those of verapamil, the agent most frequently used to examineCQ resistance in vitro.

View larger version (7K):
[in this window]
[in a new window]
FIG. 1. NP30. NP30 consists of a nonylphenol group onto which an average of 30 ethoxylate residues have been polymerized.

MATERIALS AND METHODS

Top

Materials and Methods

P. falciparum cultures. Human malaria parasites were grown in type A-positive bloodobtained by venipuncture of volunteers. Cultures were maintainedby the method of Trager and Jensen (29) by using RPMI 1640 supplementedwith 10% human serum (either type A or AB [Wisent Inc., St.Bruno, Quebec, Canada]) and 50 µM hypoxanthine (Gibco).Patient isolates were obtained from pretreatment blood samplesfrom patients enrolled in ethically approved studies at theTropical Disease Unit, University of Toronto (16, 34). The resistancegenotypes (point mutations in pfcrt and pfmdr1) of these isolateswere determined as described previously (1, 2, 6, 23, 24).

In vitro drug susceptibility testing. In vitro drug susceptibility testing was performed by usinga lactate dehydrogenase assay modified to be specific for thepresence of P. falciparum lactate dehydrogenase (5, 17, 25).The concentration of drug that resulted in a 50% inhibitionof viability of the parasite cultures (IC₅₀) was determinedby a nonlinear regression analysis using the computer programSigma Plot 2000 (Jandel Scientific). The IC₅₀s represent themeans of four determinations.

NP30. Samples of NP30 were the kind gift of Union Carbide and weremade up as 1% (wt/vol) stock solutions in water after extensivedrying by lyophilization to obtain accurate weight measurements.Checkerboard assays (two-dimensional arrays of the surfactantconcentration versus the quinoline concentration) were initiallyundertaken to determine the IC₅₀s of NP30 and the quinolinesalone and in combination. A single concentration of NP30 (0.003%[wt/vol]; 21 µM) was used in assays for comparison ofthe activity of NP30 with that of verapamil on the basis ofthe fact that this concentration of NP30 offered a favorabledegree of sensitization with minimal direct antiparasitic effect.

RESULTS

Top

Results

We have previously determined that NP30 both has anti-P. falciparumeffects and can reverse CQ resistance in some isolates (8).To test the hypothesis that NP30 and verapamil interacted witha common site, we determined whether the presence of 2 µMverapamil enhanced the anti-P. falciparum effects of NP30. Thepresence of verapamil did not modify the anti-P. falciparumactivity of NP30 (Fig. 2), suggesting that verapamil and NP30have distinct sites of action. Examination of the capacity ofverapamil to sensitize two P. falciparum isolates, designatedisolate 1 (which originated in West Africa) and isolate 2 (whichoriginated in the Indian subcontinent), to CQ indicated thata high degree of sensitization could be obtained in isolate1 but that only a limited degree of sensitization could be obtainedin isolate 2 (Fig. 3). When 21 µM NP30 was present, theIC₅₀ of CQ for isolate 1 was nearly identical to that for theuntreated sample; however, the IC₅₀ of CQ for isolate 2 wasreduced to a value less than that for the verapamil-treatedsample. This finding suggests that fully verapamil-sensitiveCQ resistance is independent of the site of action of NP30.

View larger version (18K):
[in this window]
[in a new window]
FIG. 2. Effect of verapamil on the anti-Plasmodium action of NP30. The IC₅₀ of NP30 for Plasmodium cultures was determined in the absence (closed bars) and presence (open bars) of 2 µM verapamil. The values represent the means ± standard errors of the means of three independent assays.

View larger version (22K):
[in this window]
[in a new window]
FIG. 3. Effect of verapamil or NP30 on the IC₅₀s of four quinolines in two P. falciparum patient isolates. The IC₅₀s of CQ, MF, QN, and QD were determined in the presence of drug alone, 2 µM verapamil, or 21 µM NP30 for two isolates. The values represent the means ± standard errors of the means of four independent assays. Asterisks indicate that the values were significantly different (P < 0.05) from the control values.

Previous work has indicated that some forms of quinoline resistancemay be unique to CQ and has suggested that resistance to MF,QN, and QD may result from mutations in a common efflux protein,such as Pgh1 (26). We therefore determined what effect the presenceof verapamil and NP30 had on the IC₅₀s of MF, QN, and QD inthe two isolates. The IC₅₀s of MF for isolates 1 and 2 decreasedsignificantly in the presence of NP30; however, verapamil hadno effect (Fig. 3). NP30 resulted in a marked reversal of QNand QD resistance; however, the effect of verapamil in the presenceof QN and QD was variable and not as dramatic as that of NP30(Fig. 3). We therefore conclude that (i) NP30 and verapamilhave dissimilar sensitization properties, (ii) MF resistanceresults from a verapamil-insensitive but NP30-sensitive mechanism,and (iii) QN and QD resistance can be reversed by either verapamilor NP30.

The use of two isolates with different CQ resistance phenotypessuggested that isolate 1 and isolate 2 might not share commonpoint mutations in putative quinoline resistance genes. We thereforedetermined if previously characterized point mutations in thepfcrt and pfmdr genes were present (Table 1). The point mutationpatterns seen in the pfcrt and pfmdr genes were not able todifferentiate between the two distinct CQ resistance phenotypes(NP30 sensitive versus NP30 insensitive) observed.

View this table:
[in this window]
[in a new window]
TABLE 1. Genotype sequences of putative drug resistance genes in P. falciparum patient isolates^a

DISCUSSION

Top

Discussion

Quinoline resistance in the erythrocytic stages of P. falciparumis frequently compared to multidrug resistance in mammaliancells, in part because of the initial observation that chloroquineresistance could be reversed by verapamil. NP30 is also ableto reverse some forms of quinoline resistance in P. falciparumcultures; however, the results obtained in this study suggestthat its resistance reversal properties are different from thoseof verapamil. This is seen most clearly in the case of isolate1, in which CQ resistance could be reversed only by verapamil,while MF resistance could be reversed only by NP30. While thissuggests that MF and CQ resistance is due to the expressionof independent NP30-inhibitable and verapamil-inhibitable mechanisms,the results observed for QN and QD do not support this hypothesis.The QN and QD IC₅₀s for isolate 1 can be dramatically decreasedby the presence of either NP30 or verapamil. This suggests thatboth the verapamil-sensitive and the NP30-sensitive resistanceelements are a necessary part of the QN and QD resistance process,since disruption of either makes the parasites sensitive tothe drug's presence. If the two resistance mechanisms ran ina parallel fashion, we would expect that the presence of eitheragent alone would have little effect because of redundancy.Both agents are effective; therefore, it appears that they havea serial arrangement (e.g., they may facilitate transport betweendifferent compartments within the parasite). The same relationshipbetween QN and QD resistance and the MF and CQ resistance patternswas also seen in isolate 2, in which the degree of reversalseen for QN and QD in isolate 2 supports the hypothesis thatQD and QN efflux requires both the CQ and MF efflux mechanismsto be functioning simultaneously.

While the results obtained with CQ and MF for isolate 1 stronglysuggest that the actions of verapamil and NP30 can be independentof each other, the results obtained with CQ for isolate 2 suggestthat there can also be some overlap in their actions. The presenceof either verapamil or NP30 results in a moderate, but significant,drop in the CQ IC₅₀ for isolate 2. When both verapamil and NP30were added to the same assay, their effects did not appear tobe additive (data not shown), suggesting that this result isnot due to the presence of a mixed population of verapamil-and NP30-sensitive parasites. CQ resistance can be either fullyor partially sensitive to the presence of verapamil, and recentreports suggest that this behavior may be associated with thepresence of specific sequences in the pfcrt gene (20). It wouldbe of interest to know if these pfcrt polymorphisms were relatedto or were independent of the NP30 CQ sensitization potential.

It would appear that in our original report of the sensitizationproperties of NP30 (8) we used a group of parasite isolatesthat were sensitized by the presence of NP30. Our present findingsindicate that there is a subset of P. falciparum isolates thatdisplay CQ resistance that is insensitive to NP30. The differencebetween the NP30 sensitivities of these isolates cannot be explainedby previously reported point mutations in pfcrt and pfmdr1 associatedwith quinoline resistance, since these mutations were presentin the two isolates used in this study. Further work is requiredto characterize the interaction of both verapamil and NP30 withresistance elements in P. falciparum.

NP30 has both anti-P. falciparum and sensitization abilities.The concentration of NP30 chosen for the evaluation whose resultsare presented in Fig. 2 (21 µM) was chosen because itpermitted us to observe a high degree of sensitization, despitethe presence of the direct anti-P. falciparum action of theagent. Higher levels of NP30 in the assays may result in a higherdegree of CQ resistance reversal in isolate 2 than that shownin Fig. 2; however, it is of interest that QN and QD resistancereversal was nearly complete at this concentration. This impliesthat CQ resistance in this isolate is sensitive to the presenceof NP30; however, the concentration of NP30 required for CQresistance reversal may be higher than that required for QNand QD resistance reversal.

The finding that NP30 can be used to reverse MF, QN, and QDresistance in vitro may prove to be clinically useful, sincethis agent does not have the pharmacological effects on thehuman host that previously described agents, such as verapamiland promethazine, have. Furthermore, the low cost of this materialmay make its use in developing countries an economically viableproposition.

ACKNOWLEDGMENTS

MF was a gift from Hoffmann-La Roche. This work was supportedby an operating grant from CIHR (grant MT-13721) and a CareerScientist Award from the Ontario Ministry of Health (to K.C.K.).

FOOTNOTES

* Corresponding author. Mailing address: Clinical Science Division, Rm. 7316, Medical Sciences Building, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada. Phone: (416) 978-0356. Fax: (416) 978-8765. E-mail: ian.crandall{at}utoronto.ca.

REFERENCES

Top