Can we teach an old drug new tricks?Jun-Hong Ch’ng1, Laurent Renia2, Francois Nosten3,4 and Kevin S.W. Tan1

1 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore2 Singapore Immunology Network, Agency for Science Technology and Research, Biopolis, Singapore3 Shoklo Malaria Research Unit, Mae Sot, Tak, Thailand4 Center for Clinical Vaccinology and Tropical Medicine, Oxford, UK

Opinion

Although resistance to chloroquine (CQ) has relegatedit from modern chemotherapeutic strategies to treatPlasmodium falciparum malaria, new evidence sug-gests that higher doses of the drug may exert a differ-ent killing mechanism and offers this drug a new leaseof life. Whereas the established antimalarial mecha-nisms of CQ are usually associated with nanomolarlevels of the drug, micromolar levels of CQ trigger adistinct cell death pathway involving the permeabiliza-tion of the digestive vacuole of the parasite and arelease of hydrolytic enzymes. In this paper, we pro-pose that this pathway is a promising antimalarialstrategy and suggest that revising the CQ treatmentregimen may elevate blood drug levels to trigger thispathway without increasing the incidence of adversereactions.

Antimalarial resistance as an obstacle to eradicationA widely endorsed three-pronged strategy to achieve ma-laria eradication has been recently reviewed and called foraggressive control in high-burden areas, continued shrink-age of the malaria map and the research and developmentof new tools and techniques [1]. In accordance with thethird directive, there has been a recent drive towards thedevelopment of novel antimalarials which utilize uniquemechanisms of action to surmount the problem of drug-resistant parasites. One possible mechanism that hasrecently been the subject of intense investigation is theprogrammed cell death (PCD) pathway in Plasmodiumfalciparum [2–13]. In this paper, we propose that CQ-induced PCD is a potentially useful antimalarial strategyworth investing due to the low cost of the drug, widespreadavailability, stability and tolerability.

The PCD pathway in the malaria parasiteGiven the expanding literature on PCD pathways in uni-cellular organisms [14], the existence of such a pathwaywas investigated in P. falciparum in the hope of defining anovel antimalarial pathway. A recent review has highlight-ed some of the conflicting findings from various studies anddiscussed the challenges in deciphering valid conclusionsfrom these studies using different strains, stages andinducers [15]. However, our recent work [3] has accountedfor some of these discrepancies and the growing body of

Corresponding authors: Ch’ng, J.-H. (chngjunhong@hotmail.com);Tan, K.S.W. (mictank@nus.edu.sg).Keywords: malaria; Plasmodium falciparum; chloroquine; lysosomotropic; drugresistance; programmed cell death; apoptosis; digestive vacuole; regimen; dosage;high dose; dose escalation.

220 1471-4922/$ – see front matter � 2012 Elsevier Ltd. All rights

evidence supporting Plasmodium spp. PCD [2–13] hasgenerally outnumbered studies opposing its existence[16,17].

Although nanomolar levels (3–300 nM) of CQ werefound to be ineffective, micromolar levels (3–30 mM) ofCQ were able to induce classical apoptotic hallmarks [2].A simple pathway was established where cysteine proteaseactivation resulted in mitochondrial depolarization, anamplification of cysteine protease activity and DNA frag-mentation (Figure 1). This pathway bore striking similari-ties to the mammalian apoptotic pathway, was amenableto apoptosis inhibitors and differed from unregulated ne-crosis.

As the parasite genome does not encode homologs totypical apoptosis mediators such as caspases (which areclan CD cysteine proteases), Apaf-1, Bax or other Bcl-2proteins, various clan-specific protease inhibitors wereused to demonstrate the essential involvement of clanCA cysteine proteases in facilitating this pathway [2]. Asthese proteases are almost exclusively localized to thelysosome-like digestive vacuole (DV) of the parasite, thisgave rise to the hypothesis that the DV may have becomepermeabilized by CQ treatment such that constituentproteases were released to trigger PCD.

It was further demonstrated that micromolar quantitiesof CQ resulted in the permeabilization of the DV mem-brane, with the abrupt and rapid redistribution of Ca2+ andfluorescence-tagged CQ out of the DV [3]. Interestingly,this was only induced at micromolar and not nanomolarlevels of CQ and occurred without visible rupture of the DVmembrane as viewed under electron microscopy.

Other ‘non-antimalarial’ lysosome-disrupting com-pounds were also shown to trigger DV permeabilization,and this was accompanied by PCD hallmarks [3]. Theselysosomotropic molecules were shown to be equally effec-tive against both CQ-sensitive and -resistant parasites,showing that the PCD pathway in CQ-resistant parasiteswas not compromised. This suggested that the early eventof DV permeabilization was sufficient for initiating thePCD pathway and raised the possibility of using lysosomo-tropic drugs to kill parasites via destabilizing the DV.

Notably, CQ had the highest efficacy of the lysosomo-tropic compounds tested and triggered DV permeabiliza-tion at only 3 mM. The other compounds required muchhigher concentrations (between 100 and 200 mM) and thisrestricted their therapeutic usefulness. In light of this, wenow discuss the feasibility of using CQ to induce DVpermeabilization.

reserved. doi:10.1016/j.pt.2012.02.005 Trends in Parasitology, June 2012, Vol. 28, No. 6

PfCRT

µM of CQaccumulates

in DV

Otherlysosomotropic

compounds

Ca2+

release

DV partiallypermeabilized

zVAD / E64d 4HT

Clan CA cysteineproteases leak

from DV

Mitochondriadepolarization

zVAD / E64d BAPTAActinomycin DCycloheximide

Signal amplification:Further release ofclan CA proteases

from DV?

DNA degradation(Ca2+ dependent &

transcriptionallyregulated)

VP

Approximate timingpost-CQ treatment:

0 – 3 hrs 3 – 4 hrs 4 – 6 hrs 6 – 8 hrs 8 – 10 hrs

TRENDS in Parasitology

Figure 1. Summary of the programmed cell death (PCD) pathway in Plasmodium falciparum. Within 3 h of exposure to micromolar levels of the lysosomotropic

antimalarial chloroquine (CQ), the digestive vacuole (DV) of the parasite is compromised, leading to the release of constituent clan CA proteases. These proteases then

trigger mitochondrial depolarization within 4 h, which then amplifies the cysteine protease activity after approximately 6 h. Finally, DNA fragmentation can be observed

after 8–10 h. The pathway may be inhibited at different points by clan CA protease inhibitors such as E64d or zVAD, mitochondrial depolarization inhibitor

4-hydroxytamoxifen (4HT), Ca2+-chelator BAPTA-AM and transcription/translation inhibitors actinomycin D and cycloheximide. The accumulation of CQ in the DV is reduced

in CQ-resistant parasites possessing the pfcrt mutation as this gene encodes an efflux transporter, but this transporter can be inhibited by verapamil (VP). However, the

PfCRT transporter is unable to impede the accumulation of other lysosomotropic compounds such as promethazine or chlorpromazine and the DV remains susceptible to

permeabilization. This figure is based on data from previous publications [2,3].

Opinion Trends in Parasitology June 2012, Vol. 28, No. 6

An ‘old’ drug with a new mechanism of actionOwing to the higher costs and/or unavailability of alterna-tive effective antimalarials, the use of CQ in malaria-endemic countries continues to be prevalent despite thepresence of CQ resistance [18,19]. The primary antimalar-ial mechanism of CQ lies in its ability to enter into the DVto interfere with hemazoin polymerization. Even at nano-molar levels, this diprotic weak base accumulates withinthe acidic DV, resulting in the accumulation of toxic hemewhich eventually kills the parasite. This explains thenanomolar IC50 values commonly observed in CQ-sensitiveparasites and accounts for the rapid clearance of CQ-sensitive parasites when serum levels of the drug aresustained at nanomolar levels.

However, the novel PCD pathway highlighted here isonly observed in parasites treated with micromolar levelsof CQ and this raises questions about the clinical relevanceof the findings. Can blood levels of CQ reach such micro-molar levels for the DV to be permeabilized and for PCD tobe triggered?

Although serum CQ levels do escalate into the micro-molar levels soon after drug administration, such concen-trations are not sustained and quickly decrease in patientsadhering to the World Health Organization (WHO) recom-mended treatment regimen (10 mg/kg on Days 0 and 1, and5 mg/kg on Day 2) [20] (Box 1). Whereas sustained nano-molar levels of CQ are effective against CQ-sensitivestrains, CQ-resistant strains bearing drug-efflux transpor-ters (such as PfCRT and MDR1) are able to flush CQ out ofthe DV and evade clearance.

A slight modification to the regimen that involved smal-ler repeated dosing (10 + 5 + 5 mg/kg on Day 0 and5 + 5 mg/kg on Day 1) was reported to dramatically in-crease the duration, to approximately 24 h, at which CQwas sustained at 3 mM [20]. This occurred without raisingthe peak CQ levels or increasing adverse side effects.Moreover, the rate at which parasites were cleared wassignificantly improved with 50% of parasites being cleared

in approximately one-third of the time taken with thestandard regimen. Given evidence showing that micromo-lar levels of CQ are sustained in the blood following thisrevised regimen, it is conceivable that the rapid clearanceof parasites may be a result of parasites undergoing PCD inaddition to the classical CQ antimalarial effects.

In another study, patients in Guinea–Bissau wereshown to be prescribed an even higher total dose ofCQ, consuming approximately 6.5 mg/kg dose twicedaily for 5 days without any increase in adverse reac-tions [21]. The continued effectiveness of CQ and the lowand stable proportion of CQ-resistant parasites wereattributed to this high dosing of the drug which, webelieve, is likely to have led to sustained micromolarblood concentrations. Such micromolar levels of the drugmay have triggered PCD in parasites, accounting for thecontinued effectiveness of CQ even where CQ-resistantparasites are prevalent.

In a third study performed in children diagnosed withsevere malaria, a continuous intravenous infusion of CQ(25 mg/kg, 0.83 mg/kg/h) was demonstrated to graduallyelevate and maintain CQ blood concentrations to beyond3 mM [22]. This profile was unlike those given two doses ofintravenous CQ (5 mg/kg infused over a 4 h period, at 0 hand 12 h) which saw fluctuating blood levels. Although notthe primary aim of this study, the results indicate thatblood levels of CQ can be maintained stably at high levelsto trigger CQ-induced PCD in parasites.

These studies suggest that CQ may still have its place inthe arsenal of effective antimalarial drugs provided that itis administered at an optimized regimen or formulationwhich ensures sustained micromolar levels of the drug inthe blood to trigger parasite PCD without increasing theseverity and likelihood of adverse side effects.

Proceeding with cautionBut how could an approach as simple as CQ dose escalationhave been overlooked previously? One study in Brazil

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Box 1. Summary of treatment regimens mentioned in this article, brief pharmacodynamic and pharmacokinetic information

and toxicity data of chloroquine

WHO treatment regimen

Day 0: 10 mg/kg

Day 0: 5 mg/kg (6 h later)

Day 1: 5 mg/kg

Day 2: 5 mg/kg

Pussard et al. [20]

Day 0: 10 + 5 + 5 mg/kg

Day 1: 5 + 5 mg/kg

Wildling et al. [25] (Gabon)

Day 0: 15 mg/kg

Day 1: 10 mg/kg

Day 2: 10 mg/kg

WHO treatment regimen (practical)

Day 0: 10 mg/kg

Day 1: 10 mg/kg

Day 2: 5 mg/kg

Ursing et al. [21]

Day 0: 6.5 + 6.5 mg/kg

Day 1: 6.5 + 6.5 mg/kg

Day 2: 6.5 + 6.5 mg/kg

Day 3: 6.5 + 6.5 mg/kg

Day 4: 6.5 + 6.5 mg/kg

De Andrade et al. [23] (Brazil)

Day 0: 20 mg/kg

Day 1: 20 mg/kg

Day 2: 10 mg/kg

WHO prophylaxis regimen

5 mg/kg weekly or

10 mg/kg weekly but divided into 6 daily doses

White et al. [22]

Day 0–1: 25 mg/kg continuous

intravenous drip at 0.83 mg/kg/h

Sexton et al. [24] (Rwanda)

Day 0: 10 mg/kg

Day 1: 10 mg/kg

Day 2: 10 mg/kg

Day 3: 10 mg/kg

Day 4: 10 mg/kg

Most of this data is adapted from the Roll Back Malaria Website at (http://rbm.who.int/cmc_upload/0/000/014/923/am2_1-1.htm), accessed on 2

February, 2012.

CQ has a high oral bioavailability, being absorbed rapidly in the gut. Plasma concentrations peak within 3 h but decrease rapidly due to the very

high volume of distribution. CQ accumulates readily in adipose tissue and also in melanin-containing tissues such as the skin and eye. CQ is also

concentrated in erythrocytes, but more so in parasitized erythrocytes. CQ is metabolized slowly into monodesethyl- and bisdesethylchloroquine

and is eliminated slowly with a half-life of approximately 10 days.

Adverse reactions are rare at the usual dosages, but pruritus is common among dark-skinned people. Headaches, nausea and blurred vision

may also be experienced if administered without food. Visual impairment due to long-term and high-dosage use has been observed. Biannual

retina screening is recommended for those consuming 300 mg of CQ per week for over 5 years.

The therapeutic index for CQ is low and acute poisoning is extremely dangerous. Poisoning may result after a single bolus ingestion of 1.5–2 g

of CQ (approximately 3� the daily treatment dose), with cardiotoxicity resulting in hypotension and arrhythmias, and leading to circulatory and

respiratory collapse, convulsions and death.

Opinion Trends in Parasitology June 2012, Vol. 28, No. 6

utilizing a double-dose regimen (20 mg/kg on Days 0 and 1,10 mg/kg on Day 2) showed that this regimen was moreeffective than the standard regimen, clearing parasitemiaafter Day 3 [23]. However, CQ-resistant parasites stillsurfaced after Day 6, and the high dose regimen was notrecommended as standard of care in Brazil as it merelydelayed the recurrence of parasitemia. A separate study inRwanda assayed the efficacy of a prolonged higher dose(10 mg/kg daily for 5 days) and resulted in a similaroutcome [24]. Although patients with the higher dosehad a reduced parasitemia at Day 7, the geometric meanparasite densities at Day 14 were shown to be similar inboth groups, suggesting that this regimen was unable tosuppress proliferation of drug-resistant parasites. A thirdstudy conducted in Gabon showed that a marginal increasein CQ dosage (15 mg/kg on Day 0, 10 mg/kg on Days 1 and2) was also ineffective in overcoming CQ-resistant para-sites, although this dosage reportedly reduced the inci-dence of high-grade resistance [25].

Although blood CQ levels were not monitored in thesethree studies, differences between the regimens assessedand those highlighted earlier provide an important clue tothe outcomes of CQ dose escalation: it may be the combi-nation of an increased CQ consumption and splitting intomultiple smaller doses that is required to sustain blood CQlevels in the micromolar range. It is conceivable that thethree unsuccessful trials had raised the peak levels of bloodCQ compared to the standard regimen but failed to sustainhigh CQ levels beyond the initial period. The need fordetailed studies into the pharmacokinetics of CQ bloodlevels in patients undergoing various regimens had been

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previously highlighted [26], and such studies could provideinsight into the possibility of using CQ to induce PCD andaccount for differences in the conflicting reports.

Another factor possibly accounting for the differences instudy conclusions was the distribution of CQ-resistantparasites: the studies with successful outcomes were donein areas with lower incidences of resistant parasites at thepoint the study was conducted [27–29]. Although CQ-re-sistant parasites have indeed been shown to exhibit areduced extent of PCD features by 10 h, it is not clear ifthese parasites are truly resistant to CQ-induced PCD or ifthey only show a delayed PCD response [2]. What is clear isthat these CQ-resistant parasites do have a fully functionalPCD pathway as evidenced by the effectiveness of otherlysosomotropic compounds [3].

A possible CQ revival: promises and challengesIf indeed a change in the recommended CQ treatmentregimen is all that is required to exploit this novel PCDpathway, clinical trials to optimize the treatment regimenwill be well worth considering given the low production costof the drug, widespread availability, stability and goodtolerability. Nonetheless, careful attention needs to be paidto the dangers of overdose.

The low therapeutic index of CQ and severity of associ-ated outcomes (nausea, diplopia, convulsions, respiratoryand cardiac arrest) clearly indicates that any modificationto the approved regimen needs to be carefully and thor-oughly assessed [30,31]. Although all the CQ dose escala-tion studies used in this article showed no increase in thelikelihood of adverse reactions, interindividual variability

Opinion Trends in Parasitology June 2012, Vol. 28, No. 6

in pharmacokinetics and pharmacodynamics has alreadybeen observed and may require that regimens need to becarefully assessed [32].

Closely related to the issue of overdosing is the issue ofpatient compliance and self-medication [21]. Where malar-ia is endemic and accessibility to doctors is low, great carehas to be taken to minimize the dangers of self-medicationespecially when the dose being recommended is close totoxic levels. Furthermore, poor patient compliance shouldalso be presumed, and this is especially problematic ifmultiple daily dosing is required for the therapy to beeffective and safe. One possible solution to this is thereformulation of CQ to a time-release tablet. Although thiswill require time, effort and funding to develop the chal-lenges to doing so will probably be considerably less thanthat involved in developing novel antimalarials and canhelp ensure a more successful treatment outcome.

Another important consideration is the eventuality ofPCD resistance. Although resistance to the cell deathpathway proposed seems improbable (the enzymes medi-ating PCD are the same enzymes essential for hemoglobinhydrolysis and it is unlikely that the silencing of a few ofthese proteases will compromise the PCD pathway), itwould be unwise to dismiss this possibility altogether.Research on another pathogenic human parasite, Leish-mania, showed that resistance to drug-induced cell deathfavored the acquisition of multidrug resistance [33]. Brief-ly, tolerance to certain apoptotic-inducing drugs simulta-neously resulted in cross-resistance to other drugs withdifferent cellular targets but had the same mode of killing.If a similar situation were realized in the context of ma-laria, parasites resistant to CQ-induced PCD may exhibitcross-resistance to other antimalarials utilizing similarpathways. It is therefore of utmost importance to avoidantimalarial monotherapy so as to minimize the risk ofresistance development.

As combinational chemotherapy remains the most pru-dent means of safe-guarding the precious arsenal of anti-malarial drugs, it is important to bear in mind (even fromthe onset of strategy conception) possible drugs which maybe administered in conjunction. As this revised regimen islikely to trigger a DV-mediated PCD pathway that isspecific to the later stages of the erythrocytic stage para-sites (trophozoites and schizonts), antimalarials targetingthe other pathways and stages could be useful for combi-national therapy: atovaquone (mitochondria targeting),sulfadoxin–pyrimethamine (antifolates), tetracyclins (api-coplast targeting) and artemisinin derivatives (targetingring stage parasites) represent potential candidates forcombinational therapy.

The way forwardIn the face of mounting drug resistance, the hope of reviv-ing the effective use of CQ requires a coordinated researcheffort to reliably and responsibly determine its suitability.Translational laboratory-based research may assess thesusceptibility of field isolates to undergo CQ-induced PCDor if multidrug-resistant isolates are actually PCD resis-tant. Animal studies would aid in our understanding of thephysiological and pharmacological implications of CQredosing options and to validate the effectiveness of novel

time release CQ formulations. With the emergence of CQresistance in Plasmodium vivax, studies should be encour-aged to see whether a similar approach could be proposedfor this prevalent parasite.

Field studies involving the monitoring of blood CQlevels in regions where CQ is routinely prescribed beyondthe WHO standard regimen (especially when it is con-sumed in smaller repeated doses) will be essential inreviewing the potential success or failure of this approach,whereas clinical trials to determine the tolerability andefficacy of an optimized CQ regimen/formulation will ulti-mately decide the fate of such an approach. These studiesshould also consider pharmacodynamic variations in high-risk groups such as pregnant women and young childrenwho bear the heaviest malaria burden and have highesttreatment failure rates [26].

Basic research into new parasite-specific lysosomotropiccompounds, combinations of DV-destabilizing drugs or mo-lecular linking of such compounds may yield viable alter-natives which exploit the PCD pathway. Although therehave recently been several studies demonstrating a varietyof PCD stimuli in P. falciparum [2–13], it might be expectedthat the translation of these inducers into novel antimalar-ials will likely be a time-consuming and costly endeavor.

Although the possibility of effectively redeploying CQmust not be mistaken for a magic bullet, it remains aninteresting and feasible possibility in the eradication agen-da. Accompanied by the development of other novel effec-tive antimalarials to combat drug resistance, the proposedstrategy will need to be introduced in conjunction withimprovements to accessibility to combinational therapies,surveillance and vector control. Malaria is aggressive andwe must be also – bold, but responsibly so.

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