To kill or not to kill, that is thequestion: cytocidal antimalarial drugresistancePaul D. Roepe1,2

1 Department of Chemistry, Georgetown University, 37th and O Streets NW, Washington DC 20057, USA2 Department of Biochemistry and Cellular and Molecular Biology, Georgetown University, 37th and O Streets NW, Washington DC

20057, USA

Opinion

Elucidating mechanisms of antimalarial drug resistanceaccelerates development of improved diagnostics andthe design of new, effective malaria therapy. Recently,several studies have emphasized that chloroquine (CQ)resistance (CQR) can be quantified in two very distinctways, depending on whether sensitivity to the growthinhibitory effects or parasite-kill effects of the drug arebeing measured. It is now clear that these cytostatic andcytocidal CQR phenotypes are not equivalent, and recentgenetic, cell biological, and biophysical evidence sug-gests how the molecular mechanisms may overlap.These conclusions have important implications for elu-cidating other drug resistance phenomena and empha-size new concepts that are essential for the developmentof new drug therapy.

Altered drug transport in CQR Plasmodium falciparum:history and curiositiesAll scientists have their favorite older research articleswritten by colleagues to which they refer often over theyears. Sometimes they are particularly unforgettablebecause they perturb the scientist’s preconceived sensethat things are mostly understood for the topic at hand.One such personal favorite is a 1986 study of drug trans-port in P. falciparum infected red blood cells (iRBCs; seeGlossary) by Geary, Jensen, and Ginsburg [1], wherein thekey question, ‘how does CQ accumulation differ for CQRversus CQ sensitive (CQS) malarial parasites?’ is asked. Inthe study, iRBCs are exposed to mM levels of external CQ.At mM levels, well-known differences in CQ accumulationfor CQS versus CQR parasites that are seen at nM levels ofexternal CQ vanish, as recently confirmed [2].

Reading this paper today, any new student of antimalar-ial drug resistance phenomena would ask several pointedquestions. It is now well established that at lower concen-trations (1–10 nM), differences in CQ accumulation are

1471-4922/$ – see front matter

� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.01.004

Corresponding author: Roepe, P.D. (roepep@georgetown.edu).Keywords: Plasmodium falciparum; cytostatic; QTL analysis; drug transport;autophagy.

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easily measured for CQS versus CQR parasites, and thatthis is intricately linked to the mechanism of CQR [3,4]. Thisobservation is in fact central to an entire field that beginswith the proposal of Fitch [5] and Macomber and Spintz [6]over 45 years ago, wherein free heme in the acidified para-site digestive vacuole (DV), released upon catabolism of RBChemoglobin (Hb), is the molecular target of CQ and relatedquinoline antimalarial drugs. The implications of this pro-posal have guided malaria research for decades. Recentwork indeed shows, in atomic detail, how these drugs reactwith free heme [7,8]. We now know that heme–drug inter-actions stall the growth of DV hemozoin (Hz), the formationof which is essential to parasite physiology [9–12]. We alsoknow that the key determinant for P. falciparum CQR is anintegral membrane protein localized to the DV membrane[13]. A series of studies have shown that mutated versions ofthis protein, PfCRT (Plasmodium falciparum chloroquineresistance transporter), catalyze increased efflux of CQ outof the DV for CQR parasites, and away from heme target[14–18]. Although the thermodynamics and kinetics of thistransport are still debated [3] and additional questionsremain for how CQR and multidrug resistance (MDR) over-lap [19–21], overall this has led to a very satisfying model forCQ pharmacology and the mechanism of CQR. Namely, inCQR, increased efflux of CQ from the DV leads to decreasednet accumulation of CQ because fewer complexes are formedbetween CQ and the target, DV localized heme, relative towhat occurs in CQS parasites. In support of this, clearlyreduced DV CQ accumulation is seen for CQR parasites atnM levels of external CQ [2]. Relatedly, easily measured 10–100 nM differences in CQ sensitivity are routinely seen forCQS versus CQR parasites, and many mutant pfcrt alleles,created by different drug selection histories in variousregions, have been found around the globe [22,23]. A populartheory has been that these different alleles confer the dif-ferent levels of CQR or MDR that exist.

Or do they? The above model for CQR has been eluci-dated primarily by studying how nM chloroquine concen-trations ([CQ]) interact with iRBCs. Dozens of drugtransport studies have examined iRBC transport at nMlevels, and thousands of CQ susceptibility measurementshave calculated nM drug IC50 values for various strainsand isolates. These IC50 values yield levels of CQR that areuniversally 5–15-fold, which are expressed by calculating

Glossary

Amodiaquine (AQ): a common antimalarial drug related to CQ.

Artemether (ATM): a methyl ether derivative of artemisinin and currently a

preferred treatment for malaria, particularly in combination with lumefantrine.

Artemisinin combination therapy (ACT): two antimalarial drugs used together,

with one partner drug being artemisinin or one of its common derivatives (i.e.,

artemether).

Batty approach: refers to a method for testing efficacy of antimalarial drugs in

mouse models of malaria, developed by Kevin Batty and colleagues, wherein

drug is administered after the number of malarial parasites in the mouse has

climbed to a high level [51].

Bolus: a dose of drug given in such a way as to rapidly achieve the desired

effect, often in pulse fashion.

Chloroquine (CQ): the most widely used antimalarial drug to date, a four amino

quinoline derivative of the natural product quinine (QN).

Chloroquine resistance (CQR): decreased sensitivity to CQ in malarial

parasites.

Chloroquine sensitive (CQS): refers to the natural sensitivity to chloroquine for

wild type strains of P. falciparum.

Cmax: maximum or peak concentration of drug achieved after administration.

Cytocidal chloroquine resistance (CQRCC): CQR phenotype as quantified by the

shift in LD50 or cytocidal potency.

Cytostatic chloroquine resistance (CQRCS): CQR phenotype as quantified by

the shift in IC50 or cytostatic potency.

Digestive vacuole (DV): lysosomal-like organelle within mature RBC stages of

P. falciparum involved in digestion of red cell Hb.

Fifty percent inhibitory concentration (IC50): dose of a drug required to slow

the rate of growth of a cell population by 50%.

Fifty percent lethal dose (LD50): dose of a drug required to kill 50% of a cell

population.

HB3 (CQS) • Dd2 (CQR) genetic cross: as described in [13] and references

within, a library of clonal progeny from mating these two strains of P.

falciparum.

Hemozoin (Hz): crystalline form of heme found within the parasite DV.

Mefloquine (MQ): a quinoline methanol relative of QN, developed by the

Walter Reed Army Institute of Research as a potent antimalarial drug substitute

for CQ.

Multidrug resistance (MDR): resistance to multiple, structurally divergent

drugs.

Peter’s suppression test: refers to a commonly used method to test the efficacy

of an antimalarial drug in a mouse model of malaria, initially described by

Wallace Peters of the London School of Hygiene and Tropical Medicine [52],

wherein drug is administered alongside, or shortly after, inoculation of the

mouse with malarial parasites.

Plasmodium falciparum chloroquine resistance transporter (PfCRT): integral

membrane protein found within the DV membrane that is mutated in CQR P.

falciparum.

Plasmodium falciparum infected red blood cell (iRBC): a human RBC infected

with a P. falciparum malarial parasite; usually only one parasite is found within

the RBC.

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1): integral

membrane protein found within the DV membrane that is often, but not

always, either mutated or overexpressed in CQR P. falciparum.

Proteasome: large cylindrical protein complexes whose purpose is to degrade

damaged or misfolded proteins.

Quantitative trait loci (QTL): DNA segments linked to a quantifiable phenotypic

trait.

Quinine (QN): a natural product antimalarial drug derived from the cinchona

tree that was the preferred antimalarial treatment until the 1940s.

TMIC: aggregate time for which a drug exceeds the minimum inhibitory

concentration (MIC).

Verapamil (VPL): a calcium channel blocker used to treat hypertension and

cardiac arrhythmia that is also well known for ‘resistance reversal’ effects in

some strains of CQR P. falciparum.

Vinca alkaloids: class of anticancer drugs originally derived from Catharanthus

roseus; well known for antimitotic effects via inhibition of tubulin polymerization.

Opinion Trends in Parasitology March 2014, Vol. 30, No. 3

CQR/CQS strain IC50 ratios. The 2–10-fold differences inCQ accumulation measured for CQS versus CQR parasitesseem entirely compatible with this �10-fold degree ofresistance. Nonetheless, in re-reading Geary et al. [1]today, where differences in drug accumulation are notobserved, one might ask which drug concentrations arerelevant, or, what is the concentration of drug to whichparasites in the human body are exposed?

Occasionally forgetting the laboratory and thinking onlyabout the clinic is importantThe answer to that question is ‘initially, mM, not nM’ [24–26]. Yet, prior to 2009, only one study [1] compared CQaccumulation for CQS versus CQR parasites at levels cor-responding to the initial clinical situation. At those higherlevels of drug, differences in accumulation for CQS and CQRparasites vanish [1,2]. So what is really going on at thehigher levels of drug and how might this affect our under-standing of CQR? How resistant are CQR parasites to thesehigher concentrations? Since lowered drug accumulationdoes not appear to be involved at these concentrations, ifthey are resistant, what is the mechanism?

A straightforward explanation begins to reveal itself byrecognizing that, for many drugs used to treat infectiousdiseases, low doses have growth inhibitory or cytostaticeffects, whereas higher doses often (but not always) havecell kill or cytocidal effects. This well-established concepthas been central to elucidating multiple layers of resis-tance to anticancer and antibacterial drugs but has notbeen highlighted for antimalarial drug resistance untilrecently. Why? This additional question is discussed below.

First, returning to Geary et al. the reason those experi-ments are particularly unforgettable is because when amalaria patient enters a clinic, he/she will have up to 1011–1012 parasites in his/her body. Next, when CQ, artemether(ATM), or other potent antimalarials are administered, ifthe parasites are sensitive to the drug(s), then parasitemiadrops by several logs (to �109) within hours. Plasma [CQ]is �1 mM [24–26], with Cmax well above CQ LD50 for CQSparasites, but below CQR CQ LD50. Correspondingly,patients infected with CQS malaria show fast reductionin parasitemia upon use of CQ, but patients infected withCQR malaria do not, which is why CQ therapy fails forthese patients. The parasite intraerythrocytic life cycle is 2days long, thus it takes �2 days to observe growth inhibi-tion by the drug, as quantified by IC50. However, inpatients, parasites must be cleared within hours, not days.Clearly, an ideal antimalarial drug does not merely slow orhinder the growth of parasites but rather it kills parasitesrapidly within the initial 48 h of therapy. Rigorously, IC50

quantifies growth inhibition potency and not necessarilycell kill potency. LD50 quantifies cell kill potency. Thus, inelucidating drug potencies and drug resistance phenom-ena, have we been measuring all that is necessary toquantify clinically relevant CQR?

We probably have not been measuring all that is neces-sary. Over 10 years ago, Fidock and colleagues performed animportant experiment that asked if mutant PfCRT proteinis necessary for elevated CQ IC50 in drug-resistant P. falci-parum [27]. When mutant PfCRT was expressed in a CQSparasite background without any CQ selection, CQ IC50 forthe transfectants in fact matched CQ IC50 for highly drug-selected strains. Meaning that mutant PfCRT was suffi-cient, or very nearly sufficient, to recapitulate CQR asdefined by CQ IC50. In principle, the CQR problem seemednearly solved, and the model was satisfyingly fairly consis-tent with the Fitch, Macomber, and Spintz hypothesis [5,6].

However, PfCRT is only responsible for 10–20% ofthechangeincytocidalactivityofCQinthesameCQRstrains[28]. That is, mutant PfCRT confers nearly all CQ cytostatic

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0 h 6 h 24 h 48 hTime

Key: Growth in the presence of drug

LD50 assay

IC50 assay

Growth in the absence of drug

72 h

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Figure 1. Schematic of LD50 versus IC50 assays. In the conventional IC50 assay (top), asynchronous Plasmodium falciparum cultures at 2% hematocrit and 1% parasitemia

are continuously treated with serial dilutions of drug for 72 h at 378C under 5% CO2 atmosphere. After 72 h, Sybr Green I is added and fluorescence is measured at excitation

and emission wavelengths of 485 nm and 538 nm, respectively, to quantify relative growth. In the LD50 assay (bottom; see [32]), P. falciparum cultures at 2% hematocrit and

2% parasitemia are treated with serial dilutions of chloroquine (CQ) in bolus fashion for 6 h and then the drug is completely washed away. After 48 h of additional incubation

in the absence of drug, Sybr Green I is added and fluorescence is measured as above.

Opinion Trends in Parasitology March 2014, Vol. 30, No. 3

resistanceasdefinedbyIC50shift [13,27],butamuchsmallercomponentofCQcytocidalresistanceasdefinedbyLD50shift[28]. This is not as radical as it might appear. Some cancerchemotherapeuticsshowcytostaticpotentialviainhibitionofthecell cycle (e.g.,bybindingtotubulin),butexhibitcytocidalpotency via induction of programmed cell death (e.g., apop-tosis). Accordingly, tubulin mutations can confer resistanceto cytostatic doses of vinca alkaloids, whereas mutations inapoptosis proteins are needed to confer cytocidal resistanceto the same drug [29–31].

Another prediction from the ‘nM IC50 model’ for CQR isthat if heme is the molecular target of quinoline drugs suchas CQ, then the ability of this class of drugs to inhibit heme-to-hemozoin conversion should correlate with antiplasmo-dial potency. Indeed, for CQ and quinine (QN) analogs, itdoes, assuming the hemozoin formation assay is doneunder close-to-physiological conditions (reviewed in [7]).Nevertheless, as recently learned, this is true only whenpotency is defined via IC50 [11]. When potency is defined byLD50, the correlations vanish [11]. This evidence supportsthe notion that the mechanisms underlying the cytostaticversus cytocidal actions of quinoline drugs are distinct. Ifmechanisms of action are distinct, resistance to those twomechanisms can be distinct.

Back to the question ‘why’?Differences in cytocidal (LD50) versus cytostatic (IC50)activities of antimalarial drugs historically have not beenemphasized for a very simple reason; namely, assays foreasily quantifying LD50 have not been available. Even now,with two approaches recently published [32,33], thesesemi-high-throughput methods have limitations. The onlyunambiguous assay for cytocidal activity is a tedious ‘limit-ing dilution’ approach alluded to by Young and Rathod 20years ago [34] that has recently been developed further[35]. It remains as tedious as it was 20 years ago and

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requires at least a month of live parasite culturing to evenbegin quantifying cidality.

A much faster (2–3 days) and vastly less expensive LD50

assay was recently published [32]. This assay relies on out-growth of viable parasites after bolus dose of drug (Figure 1).However, the limitations include difficulty in unequivocallydistinguishing parasites that are dead versus quiescent.Sometimes, a high dose of a cytocidal drug does not kill amicrobebutinducesahibernatoryorquiescentstatethatalsoprevents normal rates of outgrowth. One way to control forthiswiththePaguioassayistoexaminemultipletimepoints,and to examine cell cycle kinetics [32]. With these controlmeasurements, meaningful LD50 for well-understood diffu-sible drugs such as CQ can be obtained [28].

Initial results with this assay were very striking butconsistent with the questions raised by re-reading Gearyet al. [1]. First, in contrast to the well-established notionthat all CQR is �10-fold (via ratios of CQR/CQS strain CQIC50), CQR parasites can in fact show 10–200-fold levels ofresistance by LD50 ratios [28,32]. Cytocidal resistances canbe higher and they span a wider range of values. Second,the rank order of resistance to drugs via IC50 (e.g., resis-tance to CQ > QN > MQ > AQ for a CQR strain) is notnecessarily the same rank order as defined by LD50. Third,verapamil (VPL), which is well known to re-normalize IC50

for some CQR parasites by negating altered drug accumu-lation, does not re-normalize LD50 [32], yet another cluethat cytostatic and cytocidal layers of CQ pharmacologymust be molecularly distinct (Box 1).

This assay was also recently used in a quantitative traitloci (QTL)analysisoftheinvaluableHB3(CQS)�Dd2(CQR)genetic cross [28]. As is well known, IC50-directed QTL of thiscross has elucidated the primary role of PfCRT in P. falci-parum CQR phenomena [13], as well as the importantmodulatory role of PfMDR1 (Plasmodium falciparum multi-drug resistance protein 1) [19–21]. As recently reported [28],

Box 1. Outstanding questions

� What are the molecular targets for the cytocidal activity of CQ and

other quinoline-based antimalarial drugs?

� What is the range of cytocidal CQR in the field?

� Do other antimalarial drugs have distinct cytostatic versus

cytocidal targets?

� How are LD50 versus IC50 data for a candidate drug best leveraged

in preclinical development?

The answers to these questions will have significant influence on

more rapid development of new antimalarial therapy active against

drug-resistant malaria.

Opinion Trends in Parasitology March 2014, Vol. 30, No. 3

LD50-directed QTL of the same cross reveals another layer toCQR phenomena that is particularly relevant for the cyto-cidalactivityofCQ(Figure2).Importantly, thechromosomallocus harboring pfcrt is also revealed in LD50-directed QTL,consistent with analysis of CQ LD50 for PfCRT transfectantsdescribed above. However, two new chromosomal loci, notpreviously associated with CQR phenomena, are alsorevealed. Analysis of genes within these loci (see [28]) sug-gests that vesicle traffic, proteasome, and lipid metabolismpathways are potentially relevant for cytocidal CQR. Onepathway relevant to cell death that intersects with all threeprocesses is autophagy. Interestingly then, autophagy hasrecently been implicated in cell death for several cell types,including the related apicomplexan Toxoplasma gondii [36].

2 × LD50

LD50

2 × IC50

IC50

Figure 2. Re-routing of PfATG8 positive vesicles in infected red blood cells (iRBCs) treat

treating malarial parasites with cytostatic (IC50) versus cytocidal (LD50) dosages of CQ.

diamidino-2-phenylindole (DAPI) nuclear staining (blue), and overlay of the three image

(see [28] for methods). CQS P. falciparum treated with IC50 or 2 � IC50 levels of CQ (top tw

protein ATG8 that partially overlaps with the apicoplast (not shown, see [28]), whereas p

expanded punctate PfATG8 staining, including near the periphery of the parasite [28].

with LD50 levels of CQ that has recently been linked to cytocidal chloroquine resistan

transporter (PfCRT) mutations [28]. Scale bar = 5 mm.

Cytocidal resistance (elevated LD50) is resistance to celldeath. This invokes altered control of cell signaling relevantfor regulation of a cell death pathway. Perhaps not coinci-dentally then, alterations in an autophagy-like cascade haverecently been quantified for CQR parasites [28]. Also, con-sistent with its partial effect on CQ LD50, mutant pfcrt aloneis not responsible for these alterations [28]. Much workremains to be done, but one prediction is that mutationsare likely to be found in multiple autophagy genes for CQR P.falciparum isolates that show high CQRCCbecause there aremultiple ways to regulate balance between the cell survivaland cell death consequences of this cascade [37].

A new ‘two-tiered’ model for CQR and a new paradigmfor antimalarial drug discoveryWe are left with a more complex, but more satisfyingunderstanding, which may begin to reconcile numerousobservations that were not necessarily compatible with a‘nM IC50’ view of CQR phenomena. Meaning, effects of CQseen at concentrations closer to mM peak plasma [CQ]. Forexample, CQ DNA intercalation [38], particularly potentinduction of oxidative damage [39], accumulation of oddHb-containing vesicles [40], altered multiplicity of schizog-ony [41], and changes in additional important proteins forCQR parasites (e.g., glutathione [42] and PfATG8 [28]).

TRENDS in Parasitology

ed with cytocidal dose of chloroquine (CQ). Different cellular effects are seen upon

From left to right these panels show transmittance, PfATG8 staining (green), 40,6-

s for fixed iRBCs harboring CQ sensitive (CQS) strain HB3 Plasmodium falciparum

o rows) exhibit somewhat punctate cytosolic distribution of the autophagy marker

arasites treated at higher cytocidal dose (bottom two rows) show more intense and

CQ resistant (CQR) parasites show a dampened PfATG8 response upon treatment

ce (CQRCC) and that is not due to Plasmodium falciparum chloroquine resistance

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Opinion Trends in Parasitology March 2014, Vol. 30, No. 3

These phenomena have been seen in a variety of experi-ments but are often not emphasized with respect to CQR orCQ pharmacology, presumably because they do not neces-sarily correlate with CQ IC50, DV CQ accumulation, orPfCRT status. Nevertheless, perhaps they are more rele-vant for CQ cytocidal potency, which is not correlated withCQ IC50, DV CQ accumulation, or PfCRT status either.PfCRT changes are necessary but not sufficient for highlevels of CQRCC, and additional facets to CQ pharmacologyare necessary for parasite kill. A common thread connect-ing these facets could be generation of, or response to,oxidative damage. Relatedly, to explain the only partial(10–20%) effect of PfCRT on raising CQ LD50, perhapssome CQ–heme complexes formed at higher cytocidal[CQ] promote cell death via oxidative damage as proposed[42], whereas CQ–heme interactions at lower cytostatic[CQ] are merely growth inhibitory because they stall hemo-zoin production but do not produce sufficiently toxic levels ofheme or heme–drug conjugate. This might explain yetanother puzzling observation, wherein multiple CQ–hemecomplexes with different solubility characteristics and oxi-dative potential are now known to be formed at different[CQ] and heme concentrations [43,44]. Like many transpor-ters, PfCRT is probably better able to kinetically competewith passive diffusion of its substrate at lower concentra-tions (e.g., IC50 levels of CQ) but not at much higher con-centrations (e.g., LD50 levels of CQ), making it more relevantfor cytostatic resistance. This broader view may also helpexplain why the CQ transport activity of different mutantPfCRT isoforms does not appear to correlate with CQRmeasured for the cognate strains of P. falciparum [45].

The broader view drives home a key principle recog-nized for some time [46], but that has been somewhatneglected in antimalarial drug discovery efforts. Namely,the cytocidal potency of candidate antimalarial drugs isjust as important, if not more important, than the cyto-static potency. This is because malaria patients may enterthe clinic with such a high parasite burden that hundredsof billions of parasites must be killed quickly. Preventingparasite growth does not do this, regardless how low thedrug IC50 is. A corollary is that understanding cytocidalresistance is just as necessary as understanding cytostaticresistance. In some cases, drug discovery gets ‘lucky’because conventional IC50 assays might reveal somedegree of cytocidal potential. This is the case for artemi-sinin and its relatives. A very good way to slow prolifera-tion of a mass population of parasites versus time is to killsome of them, which 10 nM levels of ATM can do veryquickly [33]; hence, ATM IC50 and LD50 lie close together.However, for other drugs (e.g., CQ), this is not the case atIC50 dose, because the drug can be washed away during theIC50 assay and none or very few of the parasites are foundto be dead [32,41]. In these cases, higher doses or muchlonger exposure times are required to reveal cytocidalactivity. A simple conclusion is that, notwithstanding pro-gress [32,33,35], there is a real need for even better andmore convenient cytocidal assays. Ideally, these assaysshould be able to inspect both Cmax and TMIC regimensand have the capability to examine different stages ofparasite development, because cytocidal potency can varyfor these different stages [41,47]. At a given dose, some

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drugs might even show cytostatic activity for one stage, butcytocidal for another.

Integrating these concepts into design of combinationdrug therapy is also essential. Most now agree that com-bination drug therapy will remain the standard of care formalaria for quite some time, but as more delayed clearancephenomena for patients treated with artemisinin combi-nation therapies (ACTs) are found [48,49], it is clear thatdevelopment of new drug combinations effective againstCQR malaria are desperately needed. Ideally, these shouldbe additive or synergistic at both IC50 and LD50 levels ofquantification. Importantly then, recent evidence indicatesthat synergy as quantified by IC50 potency does not neces-sarily correlate with synergy as quantified by LD50 for thesame drug pairs [50]. Experimental quantification of bothis required. Such analyses will probably result in bettercorrelation between in vitro predictions and measurementof drug activity in animals.

With regard to this last point, the above analysis yieldsyet another provocative question: if common in vitro assayshave not quantified cytocidal potency, then what is the realpredictive ability of typical animal studies? Do theyaccount for distinction between cytostatic versus cytocidalpotency? As described elsewhere [51], there are importantdistinctions between mouse models of malaria. Cytostaticdrug activity in a mouse model is revealed by the standardPeter’s suppression test, wherein drug is administeredalongside or shortly after inoculation with a rodent-infec-tious Plasmodium sp. By contrast, the Batty approach [52]allows parasitemia in the mouse to increase before admin-istration of drug, similar to what occurs in a humanpatient. Results from this format more closely reflect theinitial clinical situation, wherein 1011–1012 parasites mustbe reduced to <109.

Concluding remarksThe best antimalarial drugs show relatively fast kill of largenumbers of parasites, along with excellent growth inhibitory(cytostatic) activity. The best antimalarial drug combina-tions will work together to achieve those two goals at lowerdoses (lower toxicity) and with improved PK/PD, versusMDR strains of P. falciparum and Plasmodium vivax. CQand related quinolines have been highly successful drugsthat show excellent cytostatic and cytocidal activity, andthat remain effective partner drugs in combination therapy.PfCRT mutations that result in decreased DV concentra-tions of CQ are sufficient for most, if not all, of the shift in nMCQ IC50 observed in CQR parasites. However, cytocidalCQR is not correlated with DV accumulation of CQ orinhibition of heme-to-hemozoin conversion; correspond-ingly, PfCRT mutations are only responsible for 10–20%of the shift in mM CQ LD50 for these same strains [28].Further elucidation of cytocidal targets and cytocidal resis-tance mechanisms are key remaining pieces to the antima-larial drug resistance puzzle.

AcknowledgmentsThe author thanks Drs Michael Ferdig (Notre Dame University),Anthony Sinai (University of Kentucky), and David Sullivan (JohnsHopkins University) for numerous insightful conversations, and AmilaSiriwardana (Georgetown University) for help in constructing the figures.

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