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Anticancer agents against malaria: time to revisit?

2010; Elsevier BV; Volume: 26; Issue: 3 Linguagem: Inglês

10.1016/j.pt.2009.12.002

1471-5007

Alexis Nzila, John Okombo, Ruy Perez Becker, Roma Chilengi, Trudie Lang, Tim Niehues,

Mosquito-borne diseases and control

The emergence of artemisinin resistance could adversely impact the current strategy for malaria treatment; thus, new drugs are urgently needed. A possible approach to developing new antimalarials is to find new uses for old drugs. Some anticancer agents such as methotrexate and trimetrexate are active against malaria. However, they are commonly perceived to be toxic and thus not suitable for malaria treatment. In this opinion article, we examine how the toxicity of anticancer agents is just a matter of dose or 'only dose makes the poison', as coined in Paracelsus' law. Thus, the opportunity exists to discover new antimalarials using the anticancer pharmacopoeia. The emergence of artemisinin resistance could adversely impact the current strategy for malaria treatment; thus, new drugs are urgently needed. A possible approach to developing new antimalarials is to find new uses for old drugs. Some anticancer agents such as methotrexate and trimetrexate are active against malaria. However, they are commonly perceived to be toxic and thus not suitable for malaria treatment. In this opinion article, we examine how the toxicity of anticancer agents is just a matter of dose or 'only dose makes the poison', as coined in Paracelsus' law. Thus, the opportunity exists to discover new antimalarials using the anticancer pharmacopoeia. The malaria parasite, the cause of one of the most significant infectious diseases, is characterised by an intrinsic ability to select for resistance against drugs. To slow down the pace of resistance selection, it has been recommended that antimalarials be used in combinations of at least two drugs with different modes of action, and the current recommendation is to use artemesinin-based combinations [1Nosten F. White N.J. Artemisinin-based combination treatment of falciparum malaria.Am. J. Trop. Med. Hyg. 2007; 77: 181-192Crossref PubMed Scopus (462) Google Scholar]. However, recent reports indicate that resistance to artemesinin is emerging in Southeast Asia, and there are concerns that the therapeutic life span of artemisinin combinations might be compromised [2Dondorp A.M. et al.Artemisinin resistance in Plasmodium falciparum malaria.N. Engl. J. Med. 2009; 361: 455-467Crossref PubMed Scopus (2520) Google Scholar]. The choices for combination therapy could be reduced and, as a result, drug resistance would remain a limitation to sustaining effective malaria treatment. One approach for discovering new antimalarials is to re-use drugs developed for the treatment of other diseases. The literature is replete with examples of new uses for old drugs. For example, among the few drugs that are available in the treatment of malaria, quinine is used to treat muscle cramps [3Miller T.M. Layzer R.B. Muscle cramps.Muscle Nerve. 2005; 32: 431-442Crossref PubMed Scopus (196) Google Scholar], and chloroquine (CQ) (preferably hydroxyl-chloroquine) is used for the management of rheumatoid arthritis and systemic lupus erythematosus [4Sibilia J. Pasquali J.L. Systemic lupus erythematosus: news and therapeutic perspectives.Presse Med. 2008; 37: 444-459Crossref PubMed Scopus (3) Google Scholar]. Artemisinin is being investigated for the treatment of schistosomiasis and cancer [5Efferth T. Willmar Schwabe Award 2006: Antiplasmodial and antitumor activity of artemisinin – from bench to bedside.Planta Med. 2007; 73: 299-309Crossref PubMed Scopus (253) Google Scholar, 6Utzinger J. et al.Artemisinins for schistosomiasis and beyond.Curr. Opin. Investig. Drugs. 2007; 8: 105-116PubMed Google Scholar]. However, thus far, there is no drug that has been re-used for acute malarial treatment, in spite of the richness of the human pharmacopoeia. Only drugs, such as the antibiotic doxycycline, azithromycin and erythromycin, have been used, or are currently being investigated, for prophylaxis against malaria [7Chico R.M. et al.Azithromycin-chloroquine and the intermittent preventive treatment of malaria in pregnancy.Malar. J. 2008; 7: 255Crossref PubMed Scopus (68) Google Scholar, 8Croft, A.M. (2005) Malaria: prevention in travellers. Clin. Evid. 954–972Google Scholar]. Dapsone, a drug used to treat leprosy and dermatitis herpetiformis [9Wolf R. et al.Dapsone.Dermatol. Online J. 2002; 8: 2PubMed Google Scholar], was combined with chlorproguanil (Lapdap®) [10Nzila A. The past, present and future of antifolates in the treatment of Plasmodium falciparum infection.J. Antimicrob. Chemother. 2006; 57: 1043-1054Crossref PubMed Scopus (137) Google Scholar]; however, this combination has been phased out because of dapsone toxicity. There is evidence that anticancer compounds methotrexate (MTX) and trimetrexate (TMX) are active against both pyrimethamine (PM)-sensitive and PM-resistant laboratory strains and field isolates of Plasmodium falciparum, including those carrying the Ileu164-Leu dhfr codon (dihydrofolate reductase, DHFR), with IC50 < 85 nM (inhibitor concentration that kills 50% of parasitaemia) [11Kiara S.M. et al.In vitro activity of antifolate and polymorphism in dihydrofolate reductase of Plasmodium falciparum isolates from the Kenyan coast: emergence of parasites with Ile-164-Leu mutation.Antimicrob. Agents Chemother. 2009; 53: 3793-3798Crossref PubMed Scopus (45) Google Scholar], and IC90/99 values for these folate antagonist agents fall between 150 and 350 nM; thus, if such a concentration can be achieved in vivo with an acceptable toxicity profile, these compounds could potentially be used as antimalarials. However, anticancer agents in general, and MTX in particular, are perceived to be toxic and therefore not suitable for malaria treatment. Yet the literature is replete with examples of new uses of anticancer agents in the treatment of non-neoplastic diseases (Table 1).Table 1Selected oncologic drugs used in the treatment of non-neoplastic diseasesaAbbreviations: ALL=acute lymphocytic leukaemia, AML=acute myelogenous leukaemia, CLL=chronic lymphocytic leukaemia, CML=chronic myelogenous leukaemia, DLE=discoid lupus erythematosus, HD=Hodgkin's disease, ITP=idiopathic thrombocytopenic purpura, JIA=juvenile idiopathic arthritis, MM=multiple myeloma, NHL=non-Hodgkin's lymphoma, NS=nephritic syndrome, SLE=systemic lupus erythematosus, TTP=thrombotic thrombocytopenic purpura.DrugMechanism of actionNeoplasm (oncology)Non-neoplasmRefsDose rangebAdult doses. and indicationsToxicity profilecGrade 0=no adverse reaction, Grade 1=mild adverse reactions, Grade 2=moderate adverse reactions, Grade 3=severe adverse reactions, Grade 4=life-threatening toxicity.Dose rangebAdult doses. and indicationsToxicity profilecGrade 0=no adverse reaction, Grade 1=mild adverse reactions, Grade 2=moderate adverse reactions, Grade 3=severe adverse reactions, Grade 4=life-threatening toxicity.MTX200–2000 mg per doseGrade 37.5–35 mg per weekGrade 0–119Niehues T. Lankisch P. Recommendations for the use of methotrexate in juvenile idiopathic arthritis.Paediatr. Drugs. 2006; 8: 347-356Crossref PubMed Scopus (39) Google Scholar, 20Swierkot J. Szechinski J. Methotrexate in rheumatoid arthritis.Pharmacol. Rep. 2006; 58: 473-492PubMed Google Scholar, 22Domenech E. et al.Long-term methotrexate for Crohn's disease: safety and efficacy in clinical practice.J. Clin. Gastroenterol. 2008; 42: 395-399Crossref PubMed Scopus (38) Google ScholarFolate antimetabolite, inhibits DNA synthesisALL, breast cancer, head and neck cancer, NHL, lung cancer, osteosarcoma, Trophoblastic neoplasmNeurological, gastrointestinal and dermatological symptoms. Pulmonary, bone marrow, renal and hepatic toxicityCrohn's disease, rheumatoid arthritis, JIA, psoriasis, psoriatic arthritisGastrointestinal symptoms, transient elevation of liver enzymes, liver dysfunctionCyclophosphamideDNA alkylating agent, inhibits DNA synthesis250–1300 mg per doseGrade 375–250 mg per doseGrade 0–151Nannini C. et al.Effects of cyclophosphamide on pulmonary function in patients with scleroderma and interstitial lung disease: a systematic review and meta-analysis of randomized controlled trials and observational prospective cohort studies.Arthritis Res. Ther. 2008; 10: R124Crossref PubMed Scopus (114) Google ScholarALL, breast cancer, Burkitt lymphoma, HD, NHL, MM, ovarian cancer, retinoblastomaGastrointestinal, dermatological and catarrhal symptoms. Bone marrow, renal, hepatic, pulmonary toxicity. Acute haemorrhagic cystitis, infertilityBehcet's syndrome, idiopathic pulmonary fibrosis, ITP, JIA, lupus nephritis, NS, SLE, transplant rejection prophylaxis, multiple sclerosis, Wegener's granulomatosisFatigue, gastrointestinal, catarrhal and dermatological symptoms6-Mercapto-purinePurine antagonist, inhibitor of DNA and RNA synthesis150–350 mg per doseGrade 3100–150 mg per doseGrade 1–252Prefontaine E. et al.Azathioprine or 6-mercaptopurine for maintenance of remission in Crohn's disease.Cochrane Database Syst. Rev. 2009; : CD000067PubMed Google ScholarALL, AML, CMLGastrointestinal and dermatological symptoms. Bone marrow, hepatic and renal toxicityCrohn's disease, ulcerative colitisGastrointestinal symptoms, bone marrow suppression, elevation of liver enzymesThalidomidedNot used in pregnancy because of its effect against foetal growth teratogenicity.Mechanism of action is not completely understood. Selectively reduces levels of TNF200–1200 mg per doseGrade 2–325–100 mg per doseGrade 0–153Wu J.J. et al.Thalidomide: dermatological indications, mechanisms of action and side-effects.Br. J. Dermatol. 2005; 153: 254-273Crossref PubMed Scopus (176) Google ScholarKaposi's sarcoma, MM, malignant glioma, myelodysplastic syndrome, renal cell cancerNeurological and dermatological symptoms. Bone marrow suppression, increased risk of thrombosisBehcet's syndrome, Crohn's disease, SLE, DLENeurological symptomsVincristineVinca alkaloid: inhibitor of microtubule formation, stopping cell division2–3.5 mg per weekGrade 32 mg per monthGrade 0–154Mateos J. et al.Vincristine is an effective therapeutic approach for transplantation-associated thrombotic microangiopathy.Bone Marrow Transplant. 2006; 37: 337-338Crossref PubMed Scopus (9) Google ScholarALL, HD, malignant glioma, neuroblastoma, NHL rhabdomyosarcoma, Wilms' tumourNeurological, gastrointestinal and dermatological symptoms. Bone marrow suppression, neurotoxicityITP,TTPNeurological symptomsDFMOeDFMO (Difluoro-methyl-ornithine) was used as an experimental drug in cancer but was abandoned because efficacy could only be achieved with doses associated with serious life-threatening toxicity. However, the drug was revived for cancer chemoprotection, hirsutism and trypanosomiasis treatment at low dose.Inhibitor of ornithine decarboxylase>10 g per doseeDFMO (Difluoro-methyl-ornithine) was used as an experimental drug in cancer but was abandoned because efficacy could only be achieved with doses associated with serious life-threatening toxicity. However, the drug was revived for cancer chemoprotection, hirsutism and trypanosomiasis treatment at low dose.Grade 40.4–0.8 g per day for a yearGrade 0–155Balasegaram M. et al.Effectiveness of melarsoprol and eflornithine as first-line regimens for gambiense sleeping sickness in nine Medecins Sans Frontieres programmes.Trans. R. Soc. Trop. Med. Hyg. 2009; 103: 280-290Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 56Hickman J.G. et al.Human dermal safety studies with eflornithine HCl 13.9% cream (Vaniqa), a novel treatment for excessive facial hair.Curr. Med. Res. Opin. 2001; 16: 235-244Crossref PubMed Scopus (47) Google ScholarProstate cancerDiarrhoea, abdominal pain, alopecia and ototoxicityChemoprotection against prostate cancer, actinic keratosis>0.4 g per day for several monthsGrade 0–1As a cream Hirsutism (facial hair)>5 g per day for 15 daysGrade 1–2Sleeping sickness (trypanosomiasis)Gastrointestinal symptomsMiltefosinefMiltefosine was initially discovered as an antineoplastic drug; however, few studies were carried out in humans to treat cancer, thus detailed information on its toxicity and dose ranges as an antineoplastic is not available.Inhibitors of phospholipid biosynthesis of cell membranexx1.5–2.5 mg per kg for 28 days LeishmaniasisGrade1–257Berman J.J. Treatment of leishmaniasis with miltefosine: 2008 status.Expert Opin. Drug Metab. Toxicol. 2008; 4: 1209-1216Crossref PubMed Scopus (67) Google ScholarNausea, vomiting, diarrhoeaa Abbreviations: ALL = acute lymphocytic leukaemia, AML = acute myelogenous leukaemia, CLL = chronic lymphocytic leukaemia, CML = chronic myelogenous leukaemia, DLE = discoid lupus erythematosus, HD = Hodgkin's disease, ITP = idiopathic thrombocytopenic purpura, JIA = juvenile idiopathic arthritis, MM = multiple myeloma, NHL = non-Hodgkin's lymphoma, NS = nephritic syndrome, SLE = systemic lupus erythematosus, TTP = thrombotic thrombocytopenic purpura.b Adult doses.c Grade 0 = no adverse reaction, Grade 1 = mild adverse reactions, Grade 2 = moderate adverse reactions, Grade 3 = severe adverse reactions, Grade 4 = life-threatening toxicity.d Not used in pregnancy because of its effect against foetal growth teratogenicity.e DFMO (Difluoro-methyl-ornithine) was used as an experimental drug in cancer but was abandoned because efficacy could only be achieved with doses associated with serious life-threatening toxicity. However, the drug was revived for cancer chemoprotection, hirsutism and trypanosomiasis treatment at low dose.f Miltefosine was initially discovered as an antineoplastic drug; however, few studies were carried out in humans to treat cancer, thus detailed information on its toxicity and dose ranges as an antineoplastic is not available. Open table in a new tab The inhibition of pathways or enzymes in tumour cells also affects normal human cells. Some anticancer agents are directed against cancer-specific targets [e.g. Imatinib mesylate (Gleevec™) targets cancer-specific tyrosine kinases] and thus can be used at doses with a relatively better toxicity profile than most drugs [12Pytel D. et al.Tyrosine kinase blockers: new hope for successful cancer therapy.Anticancer Agents Med. Chem. 2009; 9: 66-76Crossref PubMed Scopus (110) Google Scholar]. However, most anticancer agents are used at doses that also lead to the inhibition of growth of normal cells, in addition to blocking tumourous cells. Most specifically affected are cells that multiply actively, such as bone marrow cells, e.g. leukocytes, cells of the gastrointestinal mucosa and hair follicle cells, explaining why bone marrow suppression, mucositis and alopecia (hair loss) are among the most common side effects of anticancer compounds. However, according to Paracelsus' law, for any drug (including anticancers), there is always a dose range at which a drug is safe. Paracelsus' law states 'Sola dosis facit venenum (only dose makes the poison)', meaning that all substances are poisons and there are none which are not. The right dose differentiates a poison from a remedy; this principle is also known as the 'dose–response effect' [13Langman L.J. Kapur B.M. Toxicology: then and now.Clin. Biochem. 2006; 39: 498-510Crossref PubMed Scopus (49) Google Scholar, 14Rozman K.K. Doull J. Paracelsus, Haber and Arndt.Toxicology. 2001; 160: 191-196Crossref PubMed Scopus (34) Google Scholar] (Figure 1). Thus, a molecule becomes a drug if the dose required to treat a complication is pharmacologically active with minimal toxicity. The example of CQ is noteworthy. CQ is used at 10 mg kg–1 as a starting dose on days 1 and 2, and at 5 mg kg–1 on day 3 [15Taylor W.R. White N.J. Antimalarial drug toxicity: a review.Drug Safety. 2004; 27: 25-61Crossref PubMed Scopus (402) Google Scholar]. At this dose regimen, CQ has an acceptable safety profile. However, a dose of 20 mg kg–1 is considered toxic [15Taylor W.R. White N.J. Antimalarial drug toxicity: a review.Drug Safety. 2004; 27: 25-61Crossref PubMed Scopus (402) Google Scholar], and fatal cases have been reported from doses as low as 30 mg kg–1, only three times higher than the therapeutic dose [16Cann H.M. Verhulst H.L. Fatal acute chloroquine poisoning in children.Pediatrics. 1961; 27: 95-102PubMed Google Scholar, 17Riou B. et al.Treatment of severe chloroquine poisoning.N. Engl. J. Med. 1988; 318: 1-6Crossref PubMed Scopus (199) Google Scholar]. This indicates that a slight dose increase shifts the effect of CQ from the second range (acceptable safety profile with a pharmacological effect) to the third range (life-threatening toxicity) (Figure 1). Thus, CQ has a very low safety margin, and yet it has been used widely (at the correct dose) and is considered to be one of the safest antimalarial agents available. MTX is another interesting example. MTX is used at high dose, up to 5000–12,000 mg per square meter (m2) per week (130–300 mg kg–1) for several weeks for the treatment of cancer, and this dose can yield serum concentrations of >1000 μM, i.e. within the range of concentrations that is associated with MTX life-threatening toxicity [18Chabner B.A. et al.Antineoplastic agents.in: Brunton L. The Pharmacological Basis of Therapeutics. 9th edn. McGraw–Hill, 2006: 1315-1465Google Scholar]. By contrast, a 1000-fold lower dose of MTX (LD–MTX) [0.1–0.4 mg kg–1 (7.5–30 mg per adult)] is used once weekly in the treatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis in children (including infants 7.5 mg per week and several weeks after the first drug administration. The toxicity of MTX is a result of inhibition of the DHFR enzyme in normal cells. To prevent toxicity, a folate derivative is administered several hours after the dose of MTX. The addition of folate derivatives increases either dihydrofolate or tetrahydrofolate (DHFR substrate and product, respectively). In either case, the action of MTX against DHFR will be minimal because of the high folate content, leading to normal synthesis of pyrimidine and the restoration of cell growth. In juvenile arthritis, MTX is used at a higher dose, and there is still debate over the benefit of the addition of a folate derivative because mucositis and GI tract disturbances are rare: this drug is better tolerated by children than by adults [19Niehues T. Lankisch P. Recommendations for the use of methotrexate in juvenile idiopathic arthritis.Paediatr. Drugs. 2006; 8: 347-356Crossref PubMed Scopus (39) Google Scholar], probably as a result of high cell multiplication processes in children. Overall, it is clear that the toxicity of LD–MTX result from chronic use of doses >7.5 mg per week, as has been clearly demonstrated with the use of MTX in the treatment of multiple sclerosis [21Gray O.M. et al.A systematic review of oral methotrexate for multiple sclerosis.Multiple Sclerosis. 2006; 12: 507-510Crossref PubMed Scopus (23) Google Scholar]. MTX is also being evaluated in the treatment of various disease conditions including inflammatory bowel disease [22Domenech E. et al.Long-term methotrexate for Crohn's disease: safety and efficacy in clinical practice.J. Clin. Gastroenterol. 2008; 42: 395-399Crossref PubMed Scopus (38) Google Scholar], urticaria [23Montero Mora P. et al.Autoimmune urticaria. Treatment with methotrexate.Rev. Alerg. Mex. 2004; 51: 167-172PubMed Google Scholar], ankylosing spondylitis [24Chen J. Is methotrexate effective for the treatment of ankylosing spondylitis?.Nat. Clin. Practice. 2007; 3: 490-491Crossref Scopus (3) Google Scholar], idiopathic hypertrophic cranial pachymeningitis [25Bosman T. et al.Idiopathic hypertrophic cranial pachymeningitis treated by oral methotrexate: a case report and review of literature.Rheumatol. Int. 2008; 28: 713-718Crossref PubMed Scopus (64) Google Scholar], chronic cholestatic disorder [26Novak K. Swain M.G. 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Inflamm. 2003; 11: 79-82Crossref PubMed Scopus (6) Google Scholar], haemophagocytic lymphohistiocytosis (HLH), a disease that affects younger children, including infants (<12 months of age) [31Henter J.I. et al.HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis.Pediatr. Blood Cancer. 2007; 48: 124-131Crossref PubMed Scopus (3342) Google Scholar]. Worldwide, it is estimated that 0.5–1 million adults and 50,000–100,000 children receive LD–MTX weekly for the treatment of RA and juvenile idiopathic arthritis, respectively. The drug is now being used in the African population, and its safety profile is similar to that reported in the Western world [32Tahiri L. et al.Therapeutic maintenance level of methotrexate in rheumatoid arthritis.Sante. 2006; 16: 167-172PubMed Google Scholar]. The antimalarial potential of MTX has been established for almost 40 years. Two relatively small clinical trials, involving seven patients, have demonstrated that doses as low as 2.5 mg per day for 3–5 days were effectively treating malaria infection in humans (Plasmodium falciparum and/or Plasmodium vivax) [33Sheehy T.W. Dempsey H. Methotrexate therapy for Plasmodium vivax malaria.J. Am. Med. Assoc. 1970; 214: 109-114Crossref Scopus (25) Google Scholar, 34Wildbolz A. Methotrexate in the therapy of malaria.Ther. Umsch. 1973; 30: 218-222PubMed Google Scholar]. However, MTX has not come into widespread use because of concerns over toxicity [35Ferone R. Methotrexate therapy for P. vivax malaria.J. Am. Med. Assoc. 1971; 215: 117Crossref Scopus (4) Google Scholar, 36Laing A.B. Methotrexate in malaria.Trans. R. Soc. Trop. Med. Hyg. 1972; 66: 518-519Abstract Full Text PDF PubMed Scopus (5) Google Scholar]. At the time of the clinical trials (in the 1970 s), no information was available on the safety of LD–MTX. Indeed, LD–MTX started to be used for the treatment of arthritis from the 1980 s; before then, MTX was used only at high doses, associated with toxicity in cancer treatment. We now have 30 years experience on the safety of LD–MTX in adults and in children, and thus this drug could now be re-evaluated as a potential antimalarial. Unlike its use in immune diseases, MTX would not be used on a chronic basis against malaria; it would be used for 3–5 days only and thus the risk of toxicity would be even lower. The in vivo efficacy of LD–MTX is also supported by pharmacokinetics. Indeed, a daily dose of 5 mg in adults (0.035–0.1 mg kg–1) could yield serum MTX concentrations between 250 and 500 nM [37Chladek J. et al.Low-dose methotrexate pharmacokinetics and pharmacodynamics in the therapy of severe psoriasis.Basic Clin. Pharmacol. Toxicol. 2005; 96: 247-248Crossref PubMed Scopus (32) Google Scholar, 38Chladek J. et al.Pharmacokinetics and pharmacodynamics of low-dose methotrexate in the treatment of psoriasis.Br. J. Clin. Pharmacol. 2002; 54: 147-156Crossref PubMed Scopus (59) Google Scholar], concentrations which exceed the IC90/99 concentrations required to kill the parasite in vitro[11Kiara S.M. et al.In vitro activity of antifolate and polymorphism in dihydrofolate reductase of Plasmodium falciparum isolates from the Kenyan coast: emergence of parasites with Ile-164-Leu mutation.Antimicrob. Agents Chemother. 2009; 53: 3793-3798Crossref PubMed Scopus (45) Google Scholar]. Taken together, the information warrants further investigation of this drug as an antimalarial. TMX is used primarily for the treatment of solid tumours [39Haller D.G. Trimetrexate: experience with solid tumors.Semin. Oncol. 1997; 24 (S18-71-S18-76)PubMed Google Scholar]. Evidence is also available that TMX has good activity against P. falciparum, and the addition of the folate derivative 5-methyl tetrahydrofolate (5-Me-THF) does not reduce TMX activity [40Nduati E. et al.Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer.Parasitol. Res. 2008; 102: 1227-1234Crossref PubMed Scopus (45) Google Scholar]. Thus, this form of folate could be used as an adjuvant, in combination with TMX, to increase its safety margin. 5-Me-THF would protect the host against drug toxicity and it would not negate the antimalarial activity of TMX. The same rationale has been used in the combination TMX + folinic acid (FNA) for the treatment of Pneumocystis jiroveci infection (an opportunistic infection commonly found in association with HIV infection). TMX is active against P. jiroveci and this microorganism does not salvage folate derivatives because of lack of folate receptors and transporters; thus, the addition of folate derivatives does not negate TMX activity. As a result, the combination of TMX + FNA is as active as TMX alone [41Walzer P.D. et al.Activities of antifolate, antiviral, and other drugs in an immunosuppressed rat model of Pneumocystis carinii pneumonia.Antimicrob. Agents Chemother. 1992; 36: 1935-1942Crossref PubMed Scopus (72) Google Scholar]. In cancer, a TMX dose of 100 mg per day is used for several weeks and this is associated with toxicity. In P. jiroveci infection, the same dose is used (100 mg per day for 28 days) but the addition of 200 mg of FNA completely reverses the toxicity of the drug, making the combination TMX + FNA well tolerated. As a result, TMX has become useful against P. jiroveci infection [42Fulton B. et al.Trimetrexate. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of Pneumocystis carinii pneumonia.Drugs. 1995; 49: 563-576Crossref PubMed Scopus (27) Google Scholar]. For the treatment of malaria, a much lower dose of TMX would be required. Indeed, 100 mg of TMX yields plasma concentrations of ∼5000 nM [43Marshall J.L. DeLap R.J. Clinical pharmacokinetics and pharmacology of trimetrexate.Clin. Pharmacokinet. 1994; 26: 190-200Crossref PubMed Scopus (26) Google Scholar] and this is ∼100 times higher than the IC50 value (i.e. <50 nM) of TMX against P. falciparum. Thus, it is reasonable to propose that doses <10–20 mg of TMX would be effective to treat malaria. Such a low dose could be safe and the addition of 5-CH3-THF could even improve the therapeutic index of the drug. Although the potential exists for TMX to become an antimalarial, more work is needed to establish whether this concept could be translated in vivo. We have also shown that the folate antagonist pemetrexate is active against P. falciparum in vitro, with activity in the nanomolar range (IC50 <50 nM) (A. Nzila et al. unpublished). Because the anticancers MTX, aminopterin and pemetrexate, all inhibitors of DHFR enzymes, are active against P. falciparum, other anticancer inhibitors of DHFR, such as edatrexate, pralatrexate, and piritrexim might also be active against P. falciparum. Several other anticancer agents have also been shown to have activity against P. falciparum malaria. For example, the inhibitors of the microtubulin assembly tubulozole, vinblastine, docetaxel, paclitaxel and dolastatin [44Fennell B.J. et al.Effects of the antimitotic natural product dolastatin 10, and related peptides, on the human malarial parasite Plasmodium falciparum.J. Antimicrob. 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J. Med. Sci. Biol. 1994; 47: 241-252PubMed Google Scholar] and the proteasome inhibitor Bortezomib [50Kreidenweiss A. et al.Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon.Malar. J. 2008; 7: 187Crossref PubMed Scopus (89) Google Scholar] are effective antimalarials in vitro. If these drugs were active in vivo at low and tolerable doses, they could potentially become antimalarials. This is all the more possible in view of the fact that many anticancer drugs have already been used in the treatment of non-neoplastic diseases at low dose (Table 1). The burgeoning problem of antimalarial resistance highlights the need to have a strong pipeline of new drugs to treat malaria. Development of new drugs takes time and is associated with significant costs, explaining (at least partly) the paucity of available antimalarials, particularly as this is not a profitable market and thus there is little incentive for the pharmaceutical industry to develop new treatments. The re-use of existing drugs is an attractive strategy to discover new antimalarials as the development costs would be lower and the time to licensure shorter. Moreover, in the case of anticancer agents, the in-human toxicity profile would have already been well documented at much higher doses than could ever be achieved for any other product primarily developed for malaria. Many antineoplastic drugs, including MTX, were shown to be effective against the malaria parasite almost 40 years ago; however, their perceived toxicity has prevented their development as antimalarials. It is now known, and as stated by Paracelsus (more than 400 years ago), that it is 'the dose that makes a drug'; this principle has been exploited in the use of such drugs at lower doses in several non-neoplastic diseases. It is surprising that the antimalarial potential of MTX has not been fully investigated. Exploitation of the available pharmacopoeia could be a cost-effective and efficient channel to develop much needed alternative treatments for challenging diseases such as malaria. We thank the director of the Kenya Medical Research Institute for permission to publish this manuscript. This study was supported by Pfizer-Royal Society Award, UK, the EU Commission under Framework 6 as part of the AntiMal Integrated Project 018834, and the European and Developing Countries Clinical Trials Partnership (EDCPT) to A.N. We also thank Andrea Groth for reading and editing the manuscript.