Modulators of the efficacy and toxicity of drugs in malaria treatment

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Modulators of the efficacy and toxicityof drugs in malaria treatmentAlexis Nzila1,2 and Roma Chilengi1,2

1 Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research Program, PO Box 230, 80108, Kilifi, Kenya2 Nuffield Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK

Review

The burgeoning problem of drug resistance in malariarequires the urgent discovery of new drugs. Discovery ofan antimalarial agent that fulfills the requirements of amass-treatment drug is challenging. Such an antimalar-ial drug should be safe for infants and children, beefficacious during a treatment course of 3–5 days, havegood intestinal absorption, and be inexpensive. Suchconditions are difficult to meet, which explains (at leastin part) the paucity of antimalarial drugs. Developmentof antimalarial drugs has been based on two orthodoxapproaches: drugs are discovered from medicinal plantsor synthetic chemistry. However, recent observation ofhuman chemotherapy and pharmacopeia suggests thatother strategies could be used to discover new drugs orto extend the therapeutic lifetime of failing drugs. In thisreview, we present evidence that the use of modulatorsof the efficacy and toxicity of drugs will lead to thediscovery of new drugs and extend the therapeutic life-time of existing agents.

IntroductionThe emergence of Plasmodium falciparum parasites resist-ant to artemisinin derivatives is a growing concern becausecurrent strategies of antimalarial chemotherapy rest onartemisinin-based combination therapy (ACT) [1]. Forinstance, Coartem1 (a combination of lumefantrine andartemether) has been adopted as the first-line treatmentfor uncomplicated malaria in many African countries [2,3].Other combinations, such as piperaquine and dihydroarte-misinin or pyronaridine and artesunate, are undergoingclinical evaluation and have now reached phase III/IV trials[4,5]. Artemisinin derivatives are also being evaluated aspotential replacements for quinine in the treatment ofsevere malaria [6]. Thus, the spread of artemisinin resist-ance would not only compromise the efficacy of currentantimalarial combinations, but also prevent the develop-ment of an alternative to quinine in the treatment of severemalaria. Table 1 lists current antimalarials in use or inclinical development and their resistance status [1,7–16]. Toovercome this potential shortfall, non-artemesinin-basedcombinations have been proposed as an alternative, butthe paucity of available antimalarials limits this strategy.More than ever, new antimalarials are urgently needed.

The discovery of drugs against malaria is more challen-ging than it is for other diseases (Box 1). In addition tomeeting standard pharmacological properties, to becomean antimalarial for mass treatment, a drug should meet

Corresponding author: Nzila, A. ([email protected]).

0165-6147/$ – see front matter � 2010 Published by Elsevier Ltd. doi:10.1016/j.tips.2010.03.002

the following key criteria: safety in infants and children;effectiveness in a 3–5 days treatment course, with pre-ferred once-daily administration; development as a tabletor syrup (hence the drug should have a good intestinalabsorption profile); and affordability (ideally it should costless than US $1 per course).

If the same criteria had to be applied, for instance, todrugs against cancer (a disease against which drugs areconstantly being developed), the anticancer armamentar-ium would not be as vast as it is now. Specifically, thesafety and bio-availability of drugs administered by theoral route would have limitations. For instance, 5 fluoro-uridine, a drug that is central to the treatment of solidtumors (it is estimated that each year, more than US $1billion are spent on this drug), and pemetrexed (one of themost potent antifolate cancer drugs) would not have beendeveloped because of their poor oral bio-availability[17,18].

The development of new antimalarials has primarilybeen based on two orthodox approaches: a drug is discov-ered as a component of a medicinal product or plant (e.g.artemisinin and quinine) or as a synthetic molecule thatmight be completely novel or a modification of an existingone (which is how most current antimalarials have beendiscovered). To become a drug, a compound should beefficacious and have acceptable pharmacokinetic and phar-macodynamic properties. It is estimated that 95–99% ofdrug candidates fail during the development process as aresult of one of these aforementioned reasons.

Recent observation of human pharmacopeia indicatesthat many drugs with limited efficacy or poor pharmaco-kinetics and pharmacodynamics have been combined withmodulators to increase their efficacy or their pharmaco-logical properties, thereby restoring or enhancing theirutility as a drug. This approach has been used in thetreatment of infectious and non-communicable diseases.

Here, we present examples of howmodulators of efficacyand safety have been exploited in the treatment of selecteddiseases. We also provide suggestions on how this strategycan be used in malaria to extend the therapeutic life ofexisting drugs or to discover new antimalarial agents.

Modulators of efficacy: ‘chemosensitisers’Modulators of efficacy (chemosenstisers) are drugs that arenot active on their own, but increase the efficacy of otherdrugs when used in combination. This approach has beencentral to the treatment of bacterial infection and is animportant research topic in the treatment of cancer.

Available online xxxxxx 1

Table 1. Most important antimalarial agents and their resistance patterns

Antimalarial(s) Drug family Main use in humans Resistance status Ref.

Chloroquine 4-amino-quinoline No longer in use Widespread [12]

Amodiaquine (AQ) 4-amino-quinoline Treatment of uncomplicated

malaria

Reported [16]

Quinine Amino-alcohol Treatment of severe malaria

and uncomplicated malaria

Reported [7]

Mefloquine Amino-alcohol Prevention of malaria, and

treatment of uncomplicated malaria

Widespread in

South-East Asia

[8]

Single drugs

and non-ACT1

Pyrimethamine/

sulfadoxine

Antifolate Only in IPT2 and treatment of

uncomplicated malaria in pregnancy

Widespread [13]

Proguanil (PG) Triazine Prophylaxis against malaria Widespread [13]

PG/atovaquone (ATV) Triazine/naphtoquinone Prophylaxis against malaria Widespread against

PG and reported

against ATV

[11]

Artemisinin derivatives Sesquiterpene Used as monotherapy for

uncomplicated malaria

Reported in

South-East Asia

[1]

ACT1 Lumefantrine/arteme-

ther1 (coartemTM)

Aryl-amino-alcohol/

sesquiterpene

Treatment of uncomplicated malaria.

Currently, first-line treatment in

many Africans countries

Decrease LM activity;

Artemisinin resistance

reported

[1,10]

combinations

in clinic

AQ/artesunate3 4-amino-quinoline/

sesquiterpene

Treatment of uncomplicated malaria Reported for both drugs [1,16]

Mefloquine/artesunate Amino alcohol/

sesquiterpene

Treatment of uncomplicated malaria Reported for both drugs [1,8]

ACT in

development

(phase III/IV)

Piperaquine

(PQ)/dihydroartemisinin3

(Eur-artekinTM)

4-amino-quinoline/

sesquiterpene

Treatment of uncomplicated malaria Reported for both drugs [1,14]

Pyronaridine

(PRN)/artesunate3

(PyramaxTM)

Benzonaphthyridine/

ses-quiterpene

Treatment of uncomplicated malaria Reported for both drugs [1,15]

ACT: artemisinin-based combination therapy.

IPT: Intermittent preventive treatment.

These compounds are all derivatives of artemisinin and the emergence of parasites resistant to these drugs has been reported (ref. 1).

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In cancer

Non-antifolate drugs. The emergence of drug resistancein cancer cells prompted researchers to discover com-pounds that can reverse resistance to anticancer drugs.This resistance often results from increased expression ofcell-membrane transporters such as P-glycoprotein. Thisleads to an increase in efflux of cytotoxic drugs from thecells, thus lowering their intracellular concentration. Com-

Box 1. Challenges in discovering and developing new

antimalarials.

A lack of funds and interest from the pharmaceutical companies to

invest in antimalarials has been proposed to explain the unavail-

ability of drugs against diseases associated with poverty. However,

in general, the difficulties in meeting criteria for ‘antimalarial for

mass-treatment’ have been underestimated. A great effort has been

put into the discovery and development of antimalarials. A major

step was taken in creating the Medicines for Malaria Venture (MMV)

in 1999 to discover and develop antimalarials. However, 11 years

later, no new drug has progressed beyond phase-III development

despite this significant effort. As shown by the MMV portfolio, drugs

that have reached this stage are lumefantrine/artemether, piper-

aquine/dihydroartemisinin and pyronaridine/artesunate (see MMV

portfolio 2009, www.mmv.org). These drugs were already proven

antimalarials and had reached at least phase-II development before

1999.

In the pharmaceutical industry, strict timelines are put on the

research and development of each promising product. A 10-year

period constitutes a critical landmark during which progress should

be made, otherwise the project may be deemed a failure. Thus, in

the face of the burgeoning problem of drug resistance, we need to

be more innovative in the search for new drugs, and to develop new

strategies to extend the therapeutic lifetime of existing drugs.

2

pounds that interact with this P-glycoprotein sensitizecells to anticancer agents. The first reported chemosensi-tizers were verapamil (VPM) and the immunosuppressiveagent cyclosporine A [19].

The potential of some of these agents was evaluated inthe clinic, but yielded disappointing results. High doseswere required to reverse resistance, and these dosesproved to be too toxic [20]. The second generation of agentsthat reversed resistance were analogs of previous agents,but they had minimal pharmacological effect and thuscould be used at high doses. These agents included modu-lators such as dexverapamil and valspodar, which areanalogs of VPM and cyclosporine A, respectively. Althoughthey showed improved efficiency in reversing resistance,these second-generation agents increased toxicity to antic-ancer drugs, mainly because they decreased metabolism ofthe anticancer drug through interaction with cytochromeoxidases in the liver [20,21]. A third generation of chemo-sensitizers has been developed. These agents are com-pounds with new structures and are designed to haveless interaction with anticancer drugs. Some of them havereached phase II/III trials in humans, although datareported so far show limited efficacy [22,23]. However, thisarea remains an important research topic in cancer che-motherapy [19].

Interestingly, antimalarials such as pyronaridine,mefloquine, quinine (and its congener quinidine), chloro-quine and primaquine have been proved to reverse antic-ancer resistance in vitro [19]. In the case of quinine,promising results have been reported in humans [24],although another study did not support these findings [25].

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Antifolate drugs. The antifolate methotrexate (MTX),an inhibitor of dihydrofolate reductase (DHFR), has beenused in the treatment of several malignancies. Some of themechanisms of MTX resistance are associated with over-expression of multi-resistant proteins (which are primarilytransporters of organic anions). Probenecid (PBN), a drugdeveloped to increase the bioavailability of penicillin andwhich is used for the management of gout [26], has beenshown to reverse MTX resistance in cancer by blockingmulti-resistant proteins; in addition, PBN decreases theuptake of folate derivatives into cells, leading to anincrease in antifolate activity [27]. PBN has been usedas a modulator of efficacy of pralatrexate, an analog ofmethotrexate in humans [28]. Thus, the potential exists toincrease the efficacy of antifolate anticancer drugs in vivo.

In bacterial infection

The best example of chemosensitization has been reportedwith the b-lactam derivatives, antibiotic penicillins. Sev-eral penicillin derivatives have been produced, and one ofthe most widely used is amoxicillin (AMX). Resistance topenicillin is common and is associated with overexpressionof the enzyme b-lactamase, which degrades the b-lactamring of penicillins. Clavulanic acid is a potent inhibitor ofthese b-lactamases, thus restoring AMX activity [29]. Acombination of clavulanic acid and AMX (AugmentinTM)has been developed and is now the drug of choice in thetreatment of infections of the respiratory tract [30].

Other inhibitors of b-lactamase have been developed,including sulbactam and tazobactam, which have beencombined with ampicillin and piperacillin, respectively[31,32]. Sulbactam has also been combined with the cepha-losporin derivative cefoperazone [33]. These combinationsare archetypal examples of the use of modulators of efficacyin human chemotherapy.

Can this concept be used against malaria?

The observation that resistance in multidrug-resistanttumor cell lines can be reversed by agents such as VPMand VPM-related compounds has prompted studies on theuse of such agents to reverse resistance to chloroquine (CQ)in Plasmodium falciparum. The first modulation of CQ-resistance was reported with VPM and desipramine[34,35]. This has been extended, among others, to drugssuch as calmodulin inhibitors, histamine receptorantagonists and antidepressant agents (reviewed in [36]).

Clinical evaluation of some of these agents has beencarried out in West Africa in areas of low-to-moderate CQ-resistance, some with interesting results [37–40], but noattempt has been made in area of high CQ-resistance. Aswith the first generation of resistance-reversing agents incancer, these agents are pharmacologically active withsystemic effects that can result in various adverse effects.In addition, the minimum concentrations of these agentsneeded to chemosensitize CQ-resistance (usually>1 mM offree drug) [41–43] is not achievable in vivo if normal dosesare used. High doses therefore have to be used, with all theattendant risks of toxicity. These pharmacological limita-tions and the absence of efficacy data in areas of high CQ-resistancemight explain why the reversal of CQ-resistancehas not attained widespread application in malaria.

Our research team has explored the potential of theuricosuric agent PBN as an enhancer of antimalarialactivity [44,45]. We demonstrated that PBN increasesthe in-vitro activity of antimalarial antifolates and thatof CQ [44–46].We recently provided evidence that PBN canalso chemosensitize the malaria parasite to piperaquine[47].

PBN was developed to increase antibiotic bio-availabil-ity, and it has become an important drug in the treatmentof gout [26]. Unlike most resistance-reversing agents, PBNhas a better safety profile and pharmacokinetic properties.The concentrations required to reverse antimalarial resist-ance can be readily achieved if normal doses are used. Forinstance, 3 g of PBN is the highest recommended dose inadults, and 2 g (normal adult dose) [25–30 mg/kg] yieldsplasma concentrations ranging between 500 mM and700 mM [26], values that are at least fivefold higher thatthe concentration required to reverse resistance (100 mM).On the basis of this information, we proposed that PBNwould be a potent modulator of antimalarial efficacy.

This hypothesis has been borne out by clinical evalu-ations indicating that 20–25 mg/kg of PBN, in combinationwith a normal dose of the antifolate pyrimethamine–sul-fadoxine, is more efficacious in the treatment of malaria inchildren as compared with pyrimethamine–sulfadoxinealone [48–50]. Thus, the possibility to modulate antima-larial chemosensitivity in malaria does exist.

Why is this concept not exploited in malaria?

As discussed above, the use of modulators of efficacy is animportant tool in human chemotherapy. In cancer che-motherapy, the disappointing results achieved with thefirst generation of chemosensitizers did not dampen effortsto exploit this concept. On the contrary, new syntheticmolecules with better pharmacological properties weregenerated, and some have been taken through the tediousand expensive process of drug development (from precli-nical to phase I–III studies). Likewise, in the treatment ofbacterial infection, effort has been dedicated to developingclavulanic acid and analogs as chemosensitizers of b-lac-tam drugs, with excellent results. The question remains‘why has this concept not been fully exploited in malaria?’

Several attempts by our research team to raise fundsfrom sponsors to study PBN as a modulator of pyrimetha-mine–sulfadoxine efficacy have been unsuccessful on thepremise that it is a ‘failed drug’, so it is not worth spendingfunds on it. Our opinion is to the contrary: why shouldfailing drugs be revived in cancer and bacterial infectionbut not in malaria? Because of the paucity of new, effectiveand affordable antimalarial drugs, one would expect thisconcept to be exploited more in malaria than in cancer andin bacterial infection, the two disciplines in which newdrugs are constantly being generated by the pharmaceu-tical industry. There is evidence that the malaria parasitecan be chemosensitized to antimalarials, so there is noreason why this concept cannot be applied to malaria.

Modulators of toxicityMolecules that modulate drug toxicity have been used toincrease drug therapeutic indices or safety margins, allow-ing administration of drugs at higher doses against some

3

Figure 1. Modulation of toxicity with the anticancer agent 5-fluorouridine (5FU). (a) Toxicity in the systemic circulation is due to conversion of 5FU to 5-fluoro-b-alanine by

the enzyme dihydropyrimidine dehydrogenase (DPD). Inhibition of this enzyme by eniluracil (ENU), K-oxonate (KOX) and uracil lowers 5FU toxicity, which has led to the

development of the combinations 5FU+ENU and 5FU+KOX for the treatment of cancer. (b) In the gastrointestinal (GI) tract, 5FU is converted to 5F-uridine monophosphate

(FdUMP), a compound that is toxic to epithelial cells, by the enzyme orotate phosphoribosyltransferase enzyme (OPRT). The action of this enzyme can be inhibited by

chlorodihydroxypyridine (CDHP), negating the toxicity of 5FU in the GI tract.

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diseases (or even introducing the drugs against otherdiseases). The approach has been used in the treatmentof cancer, arthritis and infection by Pneumocystis jiroveci.

In cancer and arthritis

In the treatment of cancer, MTX is usually employed athigh doses of up to 12 g/adult for several weeks. At thisdose, if used alone, the drug would be fatal, but its toxicityis lowered by the addition of folate derivatives [51].

MTX and its analogs are inhibitors of DHFR and there-fore block the conversion of the dihydrofolate to tetrahy-drofolate, leading to the inhibition of pyrimidine synthesisand cell death. Mitotic divisions are more frequent incancer cells than in normal cells. Antifolates will thereforepredominantly inhibit the growth of cancer cells, but nor-mal cells are also inhibited, explaining why the inhibitionof rapidly dividing cells such as bone-marrow cells is thesalient toxic effect of such drugs [51]. Addition of folatederivatives restores the synthesis of pyrimidine, hence thegrowth of cancer cells and normal cells. As a result, for amaximal anticancer effect, antifolates are used alone forseveral hours, and the addition of folate restores cellgrowth, thereby lowering toxicity without affecting effica-cy. Folic acid and folinic acid (FA/FNA) are the commonlyused modulators.

MTX is also used at low dose (LD-MTX) in the treatmentof several immune diseases, including rheumatoid arthri-tis and juvenile arthritis (RJA) as well as psoriasis forperiods of several months or even years [52,53]. Chronicuse of LD-MTX is also associated with toxicity and, as incancer, a folate derivative is administered (several hoursafter drug administration) to prevent toxicity. Use of themodulators FA/FNA has enabled MTX to be administeredweekly for several years, and hasmade it the drug with thelongest anchorage period in the treatment of RJA [54].

Other examples of modulators of toxicity are the fluor-opyrimidine analog 5-fluorouridine (5FU) and its pro-drugtetrahydrofuryl-5-fluorouracil (Tegafur1). 5FU is a potentinhibitor of thymidylate synthase, which is used in the

4

treatment regimens of several types of cancer. 5FU toxicityis primarily due to its conversion to 5F-b-alanine, an agentcausing hand and foot syndrome, neurotoxicity and cardi-otoxicity [55,56] (Figure 1). Synthesis of 5F-b-alanine iscontrolled by the enzyme dihydropyrimidine dehydrogen-ase (DPD). Thus, 5FU toxicity can be reduced by blockingthe action of this enzyme. Eniluracil (ENU) and potassiumoxonate (KOX), which are potent inhibitors of DPD, havebeen developed as modulators of toxicity of 5FU, and thecombination of 5FU (or its pro-drug Tegafur1) with theseinhibitors has been used in the clinic with success [56]. Thenaturally occurring pyrimidine uracil has also been shownto reduce DPD activity [56].

5FU has poor intestinal absorption and can causetoxicity in the gastrointestinal (GI) tract as a result ofthe synthesis of fluoro-deoxy-uridine monophosphate from5FU by orotate phosphoribosyl transferase in the GI tract.This enzyme can be inhibited by chlorodihydroxypyridine(CDHP). The need to use 5FU for cancer outpatients hasled to the development of a safe oral formulation of 5FU (orTegafur)/KOX/CDHP (known as S-1). This combinationhas now entered phase-III clinical evaluation [55].

In infection by P. jiroveci

The concept of toxicity modulation has been exploited withthe use of trimetrexate (TMX) in the treatment of infectionby P. jiroveci (an opportunistic disease commonly associ-ated with infection by human immunodeficiency virus).TMX is used in the treatment of cancer, and this drug isalso potent against P. jiroveci. This microorganism cannottransport folate derivatives; as a result, the combination ofTMX/folate derivatives is as potent as TMX alone [57].These observations led to the use of the combination ofTMX/folate derivatives to treat this type of infection.Folate derivatives protect the host against drug toxicitywhile maintaining drug activity. In cancer, an adult TMXdose of 100 mg/day is used for several weeks, and thisregimen is associated with toxicity. In infection by P.jiroveci, the same dose is used (100 mg/day/28 days), but

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the addition of 100–200 mg of FNA completely reversesTMX toxicity, making this combination the drug of choicein the treatment of this type of infection [58].

Can anticancer antifolates be combined with folate

derivatives to treat malaria?

Wehave provided evidence that the anticancer drugsMTX,aminopterin (AMP) and TMXare potent agents against themalaria parasite, including against parasites that arehighly resistant to the antifolate pyrimethamine (i.e. para-sites harboring the 164-Leu mutation in DHFR) [46,59].These findings have led us to propose that low and safedoses of these drugs could potentially be used in thetreatment of malaria. In the case of MTX, experience ofits use at low dose in the treatment of RJA and psoriasissuggests that it might also be safe in the treatment ofmalaria. We have initiated a phase-I evaluation of thisdrug in humans (www.clinicaltrials.com; NCT 00791531),a step toward its development as an antimalarial.

We have also demonstrated that 5-methyl-tetrahydrofo-late (Me-THF), themost dominant circulating form of folatein humans, does not reduce the antimalarial activity ofstandard antifolates used against malaria (e.g. pyrimetha-mine, chlorcycloguanil, dapsone, sulfadoxine) or those usedin cancer (e.g. MTX, AMP, TMX) [38]. In addition, we haveshown that FA and FNA, the two modulators of antifolatetoxicity commonly used in cancer and arthritis, do notreduce the antimalarial activity ofMTX and AMP [46]. Thisinformation indicates that therapeutic indices of antifolatescan be increased byMe-THF or FA/FNA in the treatment ofmalaria, ashasbeenshownwithTMX/FNAinthe treatmentof infection by P. jiroveci. Many antifolates against malariahave been abandoned because of toxicity in humans as aresult of folate inhibition. This concept could therefore alsolead to the re-evaluation of such antifolates. New antima-larials could therefore be discovered by using folate deriva-tives as modulators of toxicity.

Limitations

Although the use of the combination of antifolates withfolate derivatives has proved to be useful in the treatmentof cancer, arthritis and infection by P. jiroveci, there mightbe some importation limitations in malaria. In all of theaforementioned diseases in which modulators of safety areused, drugs are obtained under prescription and, in thecase of cancer and P. jiroveci infection, drugs are mainlyadministered parenterally under medical supervision. Ifthis concept is to be extended to malaria, the combinationsshould be sufficiently safe as oral formulations (tablets orsyrups) in the population so as to become mass-treatmentdrugs (over-the-counter drugs).

The main concern is what would happen if absorption ofthe modulator is impaired because of development of thedrugs as oral formulations. Humans cannot synthesizefolate de novo: it is obtained from our diet and it is absorbedthrough intestinal epithelial cells. To the best of our knowl-edge, there are no complications associated with impair-ment of folate transport in epithelial cells in infants andchildren. Such impairment, as a result of inefficiency totransport folate, is associated with being elderly and iscorrected by daily intake of folate tablets [60]. Thus, the

addition of folate would lead to an increase in folate intake,particularly in children.

In addition, it is estimated that >1 million patientsworldwide receive MTX weekly; thus, the same numberalso take oral folate weekly. To the best of our knowledge,there has not been a report on an increase in MTX toxicityas a result of poor absorption of folate. Thus, folate could bea good modulator of low doses of anticancer agents inmalaria treatment, and the use of high doses of folatemay improve drug therapeutic indices.

Concluding remarksThe discovery and development of new antimalarialsrequires not only financial resources, but also innovativeapproaches and strategies. Active research is being con-ducted on the chemosensitization of cancer cells to failinganticancer drugs. The development of inhibitors of b-lac-tamase in the treatment of bacterial infection is the bestexample of this approach. Here we have demonstrated howthe same concept could be extended to antimalarial drugs.Because of the paucity of available antimalarials, onewould expect this concept to be exploited more in malariathan in cancer or bacterial infection, the two fields in whichnew drugs are constantly being generated by the pharma-ceutical industry, because of the considerable financialbenefits.

The concept of modulation of safety has been central tothe treatment of cancer, arthritis and infection by P.jiroveci through the use of antifolates and folate deriva-tives. For instance, antifolates would not have been used athigh doses in the treatment of cancer without the use offolate derivatives asmodulators. Likewise, the use of folatederivatives has made MTX the drug with the longestanchorage period in the treatment of arthritis. Anothergood example is with 5FU, one of themost important drugsin the treatment of solid tumors. We have provided evi-dence that the modulation of toxicity by folate derivativescould also lead to the development of new antimalarialagents. Thus, modulation of efficacy and safety could alsobe part of strategies to counterbalance the burgeoningproblem of drug resistance in malaria.

Conflict of interestThe authors declare no conflict of interest.

AcknowledgmentsWe thank the Director of the Kenya Medical Research Institute forpermission to publish this review. This study was supported by theEuropean Developing Countries Clinical Trials Partnership (EDCTP).

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