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Should chloroquine be laid to rest?
Acta Tropica 96 (2005) 16–23
Should chloroquine be laid to rest? Hagai Ginsburg ∗ Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel Received 3 May 2005; received in revised form 28 June 2005; accepted 28 June 2005 Available online 27 July 2005
Abstract Chloroquine (CQ) has been the front line antimalarial drug due to its efficacy, low cost and scanty side effects, until resistance has evolved. Although its use has been officially discontinued in most malaria-affected countries, it is still widely used. Practical and pharmacological considerations indicate that it could be still used in semi-immune adults and that more efficient treatment protocols could be devised to treat even patients infected with CQ-resistant parasite strains. Since its antimalarial activity is pleiotropic, drug resistance may be due to different mechanisms, each amenable to reversal by drug combination. © 2005 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Chloroquine; Drug resistance; Chemosensitization
1. Introduction Chloroquine (CQ) was laid to rest some time ago (Hastings et al., 2002) and its use has been discontinued in many countries. Is this total termination justified? If so, why are so many efforts are still devoted to understanding the mechanism(s) developed by the parasite to resist this drug? In this short essay, I would like to discuss “CQ today” from a somewhat personal perspective. CQ was (re-)developed during WWII in response to the shortage of quinine that resulted from the Japanese occupation of the cinchona plantations in Southeast Asia. It was soon identified as a cheap, safe and very efficacious drug for both radical cure and prophylaxis. ∗
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Due to worldwide massive deployment of CQ during the post-war period, an inadvertent development of drug resistance ensued, much too soon for those countries who have benefited so much from the drug. Since its first independent appearance in South America and Southeast Asia (Payne, 1987), resistance was soon spotted in other malarious areas, and current molecular information suggests that resistance developed independently in different geographic regions (Wootton et al., 2002). Nowadays, it is estimated that about 80% of the worldwide parasite population is resistant to the drug (Ridley, 2002).
2. Chloroquine and premunition However, CQ is still in quite active use in several African countries such as Togo (Gbadoe et al., 1999),
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Tanzania (Goodman et al., 2004), Ghana (Abuaku et al., 2004), Sierra Leone (Checchi et al., 2005), Ivory Coast (Adoubryn et al., 2004), Burkina Faso (Mueller et al., 2004) and in Pakistan (Rana and Tanveer, 2004). In other countries, in spite of governmental decisions to replace CQ with more effective drugs, CQ (mostly of very low quality) is freely accessible in many African street-markets (Goodman et al., 2004). And the Africans use it regularly. An inhabitant of a place where malaria is holoendemic immediately recognizes a bout of symptomatic disease from her/his past experience—fever, dizziness, muscular fatigue. He/she then buys a tablet and within a few hours after its absorption, the symptoms are mostly over and the sufferers can get back to their daily chores. The health worker or the scientist working in the same place will produce much evidence (mostly from ex vivo drug susceptibility tests) that most of the parasites are resistant to CQ (see for example, Myint et al., 2004). To back their contention, they will provide evidence that CQ does not protect at all the young children in the same area. How can this apparent paradox be explained? The inhabitants of holoendemic areas are partially protected from symptomatic disease by their ability to develop premunition (Druilhe and Perignon, 1994). Thus, although premunition (defined as relative immunity to severe infection by a particular pathogen as a result of a chronic low-grade infection by the same pathogen) cannot clear all parasites from the body, it can keep their numbers at very low levels, around or below those that can cause symptoms (Struik and Riley, 2004). The effect of the drug is in essence a combination of a first order process of parasite killing and the parasite growth rate which is demonstrably lower in immune persons (White, 1997; Hoshen et al., 1998). Thus, tolerable drug doses, even with resistant strains, reduce the parasite population by several orders of magnitude, relieving the individual from the symptoms and giving her/him several quiet days before parasite numbers climb up again to the unstable level where a stressed immune system can allow their unrestricted rise in parasite numbers to cause symptomatic disease. Indeed, it has been repeatedly observed in the clinic that drug treatment that starts at low parasitemia provides faster and more efficient cure (Filippov and Glazunova, 1989; Gernaat et al., 1990; Lokman-Hakim et al., 1996), and we have modeled this effect quite accurately (Hoshen et al., 1998). Premunition cannot
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be achieved in young children and takes time to develop in non-immune visitors (Smith et al., 1999). These considerations lead us to propose that CQ can still be used differentially for adults in regions where malaria is holoendemic. Other drugs should be used in these areas for children and visitors and probably pregnant women. The low price of CQ should certainly encourage policy makers to consider such differential drug use in order to ease the economic burden of impoverished African countries.
3. Drug resistance This approach can be criticized for two reasons: first, the use of CQ in a setting of partial or complete resistance cannot lead to disease eradication through the elimination of the parasite reservoir. True, but what alternative plans exists to achieve such a goal? Are such plans really feasible given the inadequate infrastructures and other health problems (HIV/AIDS), probably requiring greater investments, in the plagued countries? Eradication could be probably achieved (even with a poor infrastructure) through the use of a very efficient vaccine, but even the best available ones today provide only temporary protection (Struik and Riley, 2004). Until a long lasting vaccine is available (and affordable) protection against malaria will have to rely on chemotherapy. And then, even the incurable optimists would be content with containment rather than with eradication. Shouldn’t CQ be left in the pharmacopoeia for those who can benefit from it? The second, a protracted use of CQ will further increase parasite resistance to the point that even relief of symptoms for the immune person will require toxic doses. Is this true? To the best of my knowledge, since the first discovery of resistance to CQ there has been only a very slow increase in the prevailing IC90 . High levels of resistance have been around for a rather long time, with no indications for further gradual increase as would be expected from the dialectics of mutations and selection. How can this be explained? In many cases, drug resistance is known to reduce the viability of the organism, because metabolic resources have to be diverted to counteract the drug effects rather than be used for anabolism and reproduction (we shall return to this point briefly when considering the mechanism(s) of drug resistance). One can, therefore, envisage a situation where such a drain
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of resources is so detrimental that the organism, here the parasite cannot thrive anymore. Hence, extended use of CQ might pose no danger for further or even a total loss of drug response. Indeed, evidence that resistant mutants are less fit than wild-type forms has been reported. Thus, surveys performed following the withdrawal of CQ, have found that drug resistance in field isolates of Plasmodium falciparum has declined in China (Liu et al., 1995), in Thailand (Thaithong et al., 1988), in Vietnam (Huong et al., 2001; Thanh et al., 2001; Nguyen et al., 2003), in Gabon (Schwenke et al., 2001) and more recently in Malawi (Kublin et al., 2003).
4. Alternative treatment protocols The next topic I would like to discuss is that of efficient drug use. The introduction of a new drug requires the development of optimal chemotherapeutic protocols in terms of dosage and spacing of treatments for achieving radical cure. These are usually formulated empirically based on the suitability of a given protocol to achieve clinical success. Alternatively, pharmacokinetic (PK) and pharmacodynamic (PD) modeling based on accurate knowledge of the relevant parameters can be used to achieve even better protocols. PK–PD modeling has never been applied to any new antimalarial drug prior to its deployment, but such exercises have been entertained a posteriori, yielding at times some unexpected results. In the context of CQ, we have devised such a model that successfully simulated a variety of clinical observations such as the half-time of parasite elimination, sub-curative dosing, counteracting resistance with increased doses, drug efficacy and immunity, the selection of resistant strains from a mixed population, etc. (Hoshen et al., 1998). The most significant implication of the model is that retreatment of the patient with a second full dose can result in radical cure. This protocol was previously, perhaps prematurely, discarded on theoretical grounds (White, 1992) and as far as we know, has not been tested in a controlled manner in the field, although such a trial has been suggested (Filippov and Glazunova, 1989). Although such a treatment protocol would suit only those patients who have some level of response, and would require a very high level of compliance, its ability to deal even with CQ-resistant parasites calls for its
serious consideration. In the meantime, it may be relevant to notice that a recent report from Ghana indicates that the success of CQ therapy in pediatric patients depended on the blood levels of CQ acquired during pre-hospital treatment, measured upon hospitalization (Quashie et al., 2005): high pretreatment blood CQ was found to assist the elimination of parasites during subsequent treatment with a full dose of CQ. An inverse relationship was observed between pretreatment blood CQ concentration and the degree of resistance in this study. Authors conclude that pre-treatment “plays a role in the observed reduced proportion of RIII-type resistance in Ghana”. This finding essentially confirms the model’s prediction that re-treatment within a short time could deal with resistant parasite strains, since the blood levels are indicative of the time lapsed between pre-treatment, and subsequent treatment.
5. The action of chloroquine is pleiotropic evoking different mechanisms of resistance The usability of CQ can also be rescued by attempting to reverse the resistance, which is to co-administer CQ with another drug that chemosensitizes the parasite to its effect. For this purpose, some aspects of resistance of the parasite to CQ have to be understood. It is commonly accepted that resistant parasites accumulate less drug than their drug sensitive counterparts. Whether this is due to binding to the ferriptoroporphyrin IX (FPIX) that is generated in the food vacuole during the digestion of ingested host cell hemoglobin (Bray et al., 1999; Hawley et al., 1998), or due to active efflux of the drug (Krogstad et al., 1987; Sanchez et al., 2003) related to expression or mutations in the CQ transporter (Waller et al., 2003; Sidhu et al., 2002) is a matter of hot debate. It is also generally accepted that the accumulated drug inhibits the polymerization of FPIX into non-toxic hemozoin. Thus, free FPIX is available to intoxicate the parasite but how does it perform its act? FPIX is known to dissolve into membranes and permeabilize them. But the first membrane encountered by free FPIX that of the food vacuole, is probably not affected in as much as the pH of the food vacuole is not altered at therapeutic CQ concentrations (Ginsburg et al., 1989). On the other hand, FPIX has been shown to exit the food vacuole and to bind to membranes (Zhang et al., 1998); thus, explaining the
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depletion in infected cells of their potassium and its replacement by sodium (Lee et al., 1988), and to bind to proteins (Famin and Ginsburg, 2003) some of which are enzymes whose activities are reduced (Campanale et al., 2003). These effects are probably the final targets of CQ action. In all these actions, it could be shown that glutathione was able to degrade the membrane- and/or protein-associated FPIX and that this process was competitively inhibited by CQ (Ginsburg et al., 1998; Famin et al., 1999; Famin and Ginsburg, 2003). These observations imply that resistance to CQ can be achieved by high levels of glutathione and that drug-sensitive parasite strains should have low levels of the tripeptide. This was indeed observed experimentally and the biochemical basis of this discrepancy has been recently provided: whereas in the CQ-sensitive 3D7 strain the rate limiting enzyme responsible for low glutathione concentrations is glutathione reductase, de novo synthesis is responsible for the higher glutathione level in the CQ-resistant strain Dd2 (Meierjohann et al., 2002). These results indicate that CQ’s action may be pleiotropic: it inhibits both the polymerization of FPIX and the degradation of free FPIX by glutathione. Pleiotropic action suggests various mechanisms of resistance and different ways to counteract them.
of mutations in the mdr1 gene of P. falciparum which is also involved in resistance to CQ (Duraisingh et al., 2000; Babiker et al., 2001) that confer CQ resulted in a considerable fitness cost in the asexual blood stage of the parasite. The calculated fitness cost of 25% is much lower than the 10% reduction in the viability of resistant mutants that was found by modeling to be sufficient to insure the survival of mutant genes in the parasite pool, even where drug pressure is still being applied (Mackinnon, 1997; Mackinnon and Hastings, 1998; Hastings and D’Alessandro, 2000). The fitness cost may be due to mutations in other genes rather than in the gene that confers the specific resistance to a given drug. Thus, several glycolytic enzymes were found to be up-regulated in the presence of artemether and down-regulated in the presence of lumefantrine (Makanga et al., 2005) attesting the presence of compensatory mutations. A study on altered transcription of a limited sub-set of genes in CQ-exposed parasites has failed to reveal a significant effect on the metabolic transcript profile of 3D7 strain (Gunasekera et al., 2003). However, a marked reduction of mitochondrial rRNA expression was observed that could lead to reduced mitochondrial metabolism; thus, compromising the viability of the parasite.
6. Drug resistance and fitness cost
7. Reversal of resistance
Interestingly, the generation of CQ resistance under drug pressure in Plasmodium berghei resulted in a marked increase in glutathione concentration (Platel et al., 1999) and a higher expression of ␥glutamyl–cysteine synthetase, the first enzyme in the glutathione biosynthetic pathway (Perez-Rosado et al., 2002). These correlates, however, were not found in Plasmodium chabaudi (Ferreira et al., 2004), but, for that matter, neither mdr1 and crt homologues could be found in this species (Ferreira et al., 2004; Hunt et al., 2004). Both CQ accumulation, irrespective of the precise mechanism, and increased synthesis of glutathione, require metabolic energy. When these processes become excessively demanding, energy shortage may develop, thereby restricting parasite viability. This could well be the reason why resistance to CQ cannot increase indefinitely, as mentioned above. In this respect, it is also worth mentioning a recent study (Hayward et al., 2005) in which the effect
Since glutathione-dependent drug resistance is also known in some types of cancer, depletors of glutathione have been tested and currently considered as “reversers” of this type of resistance (Borst et al., 2000). Similarly, combining a drug known for its ability to reduce the concentration of glutathione in vivo should chemosensitize the antimalarial action of CQ, and thus hopefully reinstates it in the pharmacopoeia of antimalarial drugs. The achievability of this advance was first demonstrated in vivo in P. berghei-infected mice by combining CQ with buthionine sulfoximine, and inhibitor of ␥-glutamyl–cysteine synthetase (Dubois et al., 1995). Subsequently, some over-the-counter drugs such as indomethacin, disulfiram and paracetamol, known for their ability to reduce cellular glutathione directly or indirectly, were shown to chemosensitize the antimalarial action of CQ in murine malaria models in vivo (Deharo et al., 2003). These drugs were even more potent at enhancing the action of the CQ
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congener amodiaquine. Since combination antimalarial chemotherapy is riding high these days (Kremsner and Krishna, 2004), should not these combinations be considered as well? If so, it should be stressed that the concentrations required for potentiation by paracetamol or disulfiram are well within the doses tolerated by humans, but the concentrations of indomethacin are too high to be permissible. A different approach has been attempted by the synthesis of a double-headed prodrug. When it penetrates the parasite, it is hydrolyzed to an inhibitor of glutathione reductase (that caused a marked reduction in glutathione), and also to a CQ-mimic 4aminoquinoline. The compound displayed an IC50 similar to that of CQ against sensitive strains of P. falciparum (20–30 nM), both in sensitive and resistant strains. The drug was not toxic to mice and had a considerable antimalarial activity against P. berghei (Davioud-Charvet et al., 2001). Except for these very few sideways inspections of alternative chemosensitizers, all other attempts have been centered around compounds that could enhance the accumulation of CQ (Peters et al., 1990; Kyle et al., 1993; Chandra et al., 1993; Oduola et al., 1998; Singh and Puri, 2000). Only one combination has been tested in human patients with some success, that of CQ + chlorpheniramine, a histamine H1 receptor antagonist (Sowunmi and Oduola, 1997; Sowunmi et al., 1997, 1998a,b). This is probably why so many efforts are still invested in the deciphering of this process—since understanding it may divulge directions for its inhibition. But there is certainly life after accumulation that is worth contemplating. In this context, the recent publication showing up-regulation of scores of ABC-transporter genes in CQ-resistant strains (Mu et al., 2003) is more than an understated hint for a possibly promising direction.
8. Wrapping up In conclusion, although CQ is ineffective in most parts of Africa and the onset of resistance has at least doubled childhood malaria death risk, we suggest that it is too premature to lay CQ to rest. Differential use of CQ in holoendemic areas that precludes children and pregnant women could still confer an efficacious protection for semi-immune adults. The recent introduction of
artemisinine combination therapy as a chemotherapeutic strategy is certainly justified but at the same time it means a 10-fold increase in the price of treatment, a financial burden that cannot be shouldered by most inhabitants of the African continent. Sequential treatment with CQ and its combination with glutathionedepleting drugs is worthy of a serious systematic testing since it could be useful even for treatment of children and adult patients in meso- or hypo-endemic areas where premunition is minimal and is affected seasonally by transmission intensity. Not less important is the fact that CQ is still an efficient drug against Plasmodium vivax infections in most countries (Vijaykadga et al., 2004; Baird, 2004) although even there resistance seems to be spreading.
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Lokman-Hakim, S., Sharifah Roohi, S.W., Zurkurnai, Y., Noor, R.A., Mansor, S.M., Palmer, K., Navaratnam, V., Mak, J.W., 1996. Plasmodium falciparum: increased proportion of severe resistance (RII and RIII) to chloroquine and high rate of resistance to sulfadoxine-pyrimethamine in Peninsular Malaysia after two decades. Trans. R. Soc. Trop. Med. Hyg. 90, 294–297. Mackinnon, M.J., 1997. Survival probability of drug resistant mutants in malaria parasites. Proc. R. Soc. Lond. [Biol]. 264, 53–59. Mackinnon, M.J., Hastings, I.M., 1998. The evolution of multiple drug resistance in malaria parasites. Trans. R. Soc. Trop. Med. Hyg. 92, 188–195. Makanga, M., Bray, P.G., Horrocks, P., Ward, S.A., 2005. Towards a proteomic definition of CoArtem action in Plasmodium falciparum malaria. Proteomics 5, 1849–1858. Meierjohann, S., Walter, R.D., Muller, S., 2002. Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine sensitive and chloroquine resistant Plasmodium falciparum. Biochem. J. 368, 761–768. Mu, J., Ferdig, M.T., Feng, X., Joy, D.A., Duan, J., Furuya, T., Subramanian, G., Aravind, L., Cooper, R.A., Wootton, J.C., Xiong, M., Su, X.Z., 2003. Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol. Microbiol. 49, 977–989. Mueller, O., Razum, O., Traore, C., Kouyate, B., 2004. Community effectiveness of chloroquine and traditional remedies in the treatment of young children with falciparum malaria in rural Burkina Faso. Malar. J. 3, 36. Myint, H.Y., Tipmanee, P., Nosten, F., Day, N.P., Pukrittayakamee, S., Looareesuwan, S., White, N.J., 2004. A systematic overview of published antimalarial drug trials. Trans. R. Soc. Trop. Med. Hyg. 98, 73–81. Nguyen, M.H., Davis, T.M., Cox-Singh, J., Hewitt, S., Tran, Q.T., Tran, B.K., Nguyen, T.H., Vo, N.P., Doan, H.N., Le, D.C., 2003. Treatment of uncomplicated falciparum malaria in southern Vietnam: can chloroquine or sulfadoxine-pyrimethamine be reintroduced in combination with artesunate? Clin. Infect. Dis. 37, 1461–1466. Oduola, A.M.J., Sowunmi, A., Milhous, W.K., Brewer, T.G., Kyle, D.E., Gerena, L., Rossan, R.N., Salako, L.A., Schuster, B.G., 1998. In vitro and in vivo reversal of chloroquine resistance in Plasmodium falciparum with promethazine. Am. J. Trop. Med. Hyg. 58, 625–629. Payne, D., 1987. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol. Today 3, 241–246. Perez-Rosado, J., Gervais, G.W., Ferrer, R., guez, I., Peters, W., Serrano, A.E., 2002. Plasmodium berghei: analysis of the gammaglutamylcysteine synthetase gene in drug-resistant lines. Exp. Parasitol. 101, 175–182. Peters, W., Ekong, R., Robinson, B.L., Warhurst, D.C., 1990. The chemotherapy of rodent malaria. XLV. Reversal of chloroquine resistance in rodent and human Plasmodium by antihistaminic agents. Ann. Trop. Med. Parasitol. 84, 541–551. Platel, D.N., Mangou, F., Tribouley, D.J., 1999. Role of glutathione in the detoxification of ferriprotoporphyrin IX in chloroquine resistant Plasmodium berghei. Mol. Biochem. Parasitol. 98, 215– 223.
Quashie, N.B., Akanmori, B.D., Goka, B.Q., Ofori-Adjei, D., Kurtzhals, J.A., 2005. Pretreatment blood concentrations of chloroquine in patients with malaria infection: relation to response to treatment. J. Trop. Pediatr. 51, 149–153. Rana, M.S., Tanveer, A., 2004. Chloroquine resistance and Plasmodium falciparum in Punjab, Pakistan during 2000–2001. Southeast Asian J. Trop. Med. Public Health 35, 288–291. Ridley, R.G., 2002. Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 415, 686–693. Sanchez, C.P., Stein, W., Lanzer, M., 2003. Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum. Biochemistry 42, 9383–9394. Schwenke, A., Brandts, C., Philipps, J., Winkler, S., Wernsdorfer, W.H., Kremsner, P.G., 2001. Declining chloroquine resistance of Plasmodium falciparum in Lambarene, Gabon from 1992 to 1998. Wiener Klin. Wochensch 113, 63–64. Sidhu, A.B., Verdier-Pinard, D., Fidock, D.A., 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298, 210–213. Singh, N., Puri, S.K., 2000. Interaction between chloroquine and diverse pharmacological agents in chloroquine resistant Plasmodium yoelii nigeriensis? Acta Trop. 77, 185–193. Smith, T., Felger, I., Tanner, M., Beck, H.P., 1999. The epidemiology of multiple Plasmodium falciparum infections—11. Premunition in Plasmodium falciparum infection: insights from the epidemiology of multiple infections. Trans. R. Soc. Trop. Med. Hyg. 93 (Suppl. 1), S59–S64. Sowunmi, A., Oduola, A.M.J., 1997. Comparative efficacy of chloroquine/chlorpheniramine combination and mefloquine for the treatment of chloroquine-resistant Plasmodium falciparum malaria in Nigerian children. Trans. R. Soc. Trop. Med. Hyg. 91, 689–693. Sowunmi, A., Oduola, A.M.J., Ogundahunsi, O.A.T., Falade, C.O., Gbotosho, G.O., Salako, L.A., 1997. Enhanced efficacy of chloroquine-chlorpheniramine combination in acute uncomplicated falciparum malaria in children. Trans. R. Soc. Trop. Med. Hyg. 91, 63–67. Sowunmi, A., Fehintola, F.A., Ogundahunsi, O.A., Oduola, A.M., 1998a. Comparative efficacy of chloroquine plus chlorpheniramine and halofantrine in acute uncomplicated falciparum malaria in Nigerian children. Trans. R. Soc. Trop. Med. Hyg. 92, 441–445. Sowunmi, A., Oduola, A.M.J., Ogundahunsi, O.A.T., Salako, L.A., 1998b. Comparative efficacy of chloroquine plus chlorpheniramine and pyrimethamine/sulfadoxine in acute uncomplicated falciparum malaria in Nigerian children. Trans. R. Soc. Trop. Med. Hyg 92, 77–81. Struik, S.S., Riley, E.M., 2004. Does malaria suffer from lack of memory? Immunol. Rev. 201, 268–290. Thaithong, S., Suebsaeng, L., Rooney, W., Beale, G.H., 1988. Evidence of increased chloroquine sensitivity in Thai isolates of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg. 82, 37–38. Thanh, N.V., Cowman, A.F., Hipgrave, D., Kim, T.B., Phuc, B.Q., Cong, L.D., Biggs, B.A., 2001. Assessment of susceptibility of Plasmodium falciparum to chloroquine, quinine, mefloquine,
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White, N.J., 1992. Antimalarial pharmacokinetics and treatment regimens. Br. J. Pharmacol. 34, 1–10. White, N.J., 1997. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrob. Agents Chemother. 41, 1413–1422. Wootton, J.C., Feng, X., Ferdig, M.T., Cooper, R.A., Mu, J., Baruch, D.I., Magill, A.J., Su, X.Z., 2002. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum. Nature 418, 320–323. Zhang, J., Krugliak, M., Ginsburg, H., 1998. The fate of ferriprotoporphyrin IX in malaria infected erythrocytes in conjunction with the mode of action of antimalarial drugs. Mol. Biochem. Parasitol. 99, 129–141.
Should chloroquine be laid to rest? Hagai Ginsburg ∗ Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel Received 3 May 2005; received in revised form 28 June 2005; accepted 28 June 2005 Available online 27 July 2005
Abstract Chloroquine (CQ) has been the front line antimalarial drug due to its efficacy, low cost and scanty side effects, until resistance has evolved. Although its use has been officially discontinued in most malaria-affected countries, it is still widely used. Practical and pharmacological considerations indicate that it could be still used in semi-immune adults and that more efficient treatment protocols could be devised to treat even patients infected with CQ-resistant parasite strains. Since its antimalarial activity is pleiotropic, drug resistance may be due to different mechanisms, each amenable to reversal by drug combination. © 2005 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Chloroquine; Drug resistance; Chemosensitization
1. Introduction Chloroquine (CQ) was laid to rest some time ago (Hastings et al., 2002) and its use has been discontinued in many countries. Is this total termination justified? If so, why are so many efforts are still devoted to understanding the mechanism(s) developed by the parasite to resist this drug? In this short essay, I would like to discuss “CQ today” from a somewhat personal perspective. CQ was (re-)developed during WWII in response to the shortage of quinine that resulted from the Japanese occupation of the cinchona plantations in Southeast Asia. It was soon identified as a cheap, safe and very efficacious drug for both radical cure and prophylaxis. ∗
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Due to worldwide massive deployment of CQ during the post-war period, an inadvertent development of drug resistance ensued, much too soon for those countries who have benefited so much from the drug. Since its first independent appearance in South America and Southeast Asia (Payne, 1987), resistance was soon spotted in other malarious areas, and current molecular information suggests that resistance developed independently in different geographic regions (Wootton et al., 2002). Nowadays, it is estimated that about 80% of the worldwide parasite population is resistant to the drug (Ridley, 2002).
2. Chloroquine and premunition However, CQ is still in quite active use in several African countries such as Togo (Gbadoe et al., 1999),
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Tanzania (Goodman et al., 2004), Ghana (Abuaku et al., 2004), Sierra Leone (Checchi et al., 2005), Ivory Coast (Adoubryn et al., 2004), Burkina Faso (Mueller et al., 2004) and in Pakistan (Rana and Tanveer, 2004). In other countries, in spite of governmental decisions to replace CQ with more effective drugs, CQ (mostly of very low quality) is freely accessible in many African street-markets (Goodman et al., 2004). And the Africans use it regularly. An inhabitant of a place where malaria is holoendemic immediately recognizes a bout of symptomatic disease from her/his past experience—fever, dizziness, muscular fatigue. He/she then buys a tablet and within a few hours after its absorption, the symptoms are mostly over and the sufferers can get back to their daily chores. The health worker or the scientist working in the same place will produce much evidence (mostly from ex vivo drug susceptibility tests) that most of the parasites are resistant to CQ (see for example, Myint et al., 2004). To back their contention, they will provide evidence that CQ does not protect at all the young children in the same area. How can this apparent paradox be explained? The inhabitants of holoendemic areas are partially protected from symptomatic disease by their ability to develop premunition (Druilhe and Perignon, 1994). Thus, although premunition (defined as relative immunity to severe infection by a particular pathogen as a result of a chronic low-grade infection by the same pathogen) cannot clear all parasites from the body, it can keep their numbers at very low levels, around or below those that can cause symptoms (Struik and Riley, 2004). The effect of the drug is in essence a combination of a first order process of parasite killing and the parasite growth rate which is demonstrably lower in immune persons (White, 1997; Hoshen et al., 1998). Thus, tolerable drug doses, even with resistant strains, reduce the parasite population by several orders of magnitude, relieving the individual from the symptoms and giving her/him several quiet days before parasite numbers climb up again to the unstable level where a stressed immune system can allow their unrestricted rise in parasite numbers to cause symptomatic disease. Indeed, it has been repeatedly observed in the clinic that drug treatment that starts at low parasitemia provides faster and more efficient cure (Filippov and Glazunova, 1989; Gernaat et al., 1990; Lokman-Hakim et al., 1996), and we have modeled this effect quite accurately (Hoshen et al., 1998). Premunition cannot
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be achieved in young children and takes time to develop in non-immune visitors (Smith et al., 1999). These considerations lead us to propose that CQ can still be used differentially for adults in regions where malaria is holoendemic. Other drugs should be used in these areas for children and visitors and probably pregnant women. The low price of CQ should certainly encourage policy makers to consider such differential drug use in order to ease the economic burden of impoverished African countries.
3. Drug resistance This approach can be criticized for two reasons: first, the use of CQ in a setting of partial or complete resistance cannot lead to disease eradication through the elimination of the parasite reservoir. True, but what alternative plans exists to achieve such a goal? Are such plans really feasible given the inadequate infrastructures and other health problems (HIV/AIDS), probably requiring greater investments, in the plagued countries? Eradication could be probably achieved (even with a poor infrastructure) through the use of a very efficient vaccine, but even the best available ones today provide only temporary protection (Struik and Riley, 2004). Until a long lasting vaccine is available (and affordable) protection against malaria will have to rely on chemotherapy. And then, even the incurable optimists would be content with containment rather than with eradication. Shouldn’t CQ be left in the pharmacopoeia for those who can benefit from it? The second, a protracted use of CQ will further increase parasite resistance to the point that even relief of symptoms for the immune person will require toxic doses. Is this true? To the best of my knowledge, since the first discovery of resistance to CQ there has been only a very slow increase in the prevailing IC90 . High levels of resistance have been around for a rather long time, with no indications for further gradual increase as would be expected from the dialectics of mutations and selection. How can this be explained? In many cases, drug resistance is known to reduce the viability of the organism, because metabolic resources have to be diverted to counteract the drug effects rather than be used for anabolism and reproduction (we shall return to this point briefly when considering the mechanism(s) of drug resistance). One can, therefore, envisage a situation where such a drain
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of resources is so detrimental that the organism, here the parasite cannot thrive anymore. Hence, extended use of CQ might pose no danger for further or even a total loss of drug response. Indeed, evidence that resistant mutants are less fit than wild-type forms has been reported. Thus, surveys performed following the withdrawal of CQ, have found that drug resistance in field isolates of Plasmodium falciparum has declined in China (Liu et al., 1995), in Thailand (Thaithong et al., 1988), in Vietnam (Huong et al., 2001; Thanh et al., 2001; Nguyen et al., 2003), in Gabon (Schwenke et al., 2001) and more recently in Malawi (Kublin et al., 2003).
4. Alternative treatment protocols The next topic I would like to discuss is that of efficient drug use. The introduction of a new drug requires the development of optimal chemotherapeutic protocols in terms of dosage and spacing of treatments for achieving radical cure. These are usually formulated empirically based on the suitability of a given protocol to achieve clinical success. Alternatively, pharmacokinetic (PK) and pharmacodynamic (PD) modeling based on accurate knowledge of the relevant parameters can be used to achieve even better protocols. PK–PD modeling has never been applied to any new antimalarial drug prior to its deployment, but such exercises have been entertained a posteriori, yielding at times some unexpected results. In the context of CQ, we have devised such a model that successfully simulated a variety of clinical observations such as the half-time of parasite elimination, sub-curative dosing, counteracting resistance with increased doses, drug efficacy and immunity, the selection of resistant strains from a mixed population, etc. (Hoshen et al., 1998). The most significant implication of the model is that retreatment of the patient with a second full dose can result in radical cure. This protocol was previously, perhaps prematurely, discarded on theoretical grounds (White, 1992) and as far as we know, has not been tested in a controlled manner in the field, although such a trial has been suggested (Filippov and Glazunova, 1989). Although such a treatment protocol would suit only those patients who have some level of response, and would require a very high level of compliance, its ability to deal even with CQ-resistant parasites calls for its
serious consideration. In the meantime, it may be relevant to notice that a recent report from Ghana indicates that the success of CQ therapy in pediatric patients depended on the blood levels of CQ acquired during pre-hospital treatment, measured upon hospitalization (Quashie et al., 2005): high pretreatment blood CQ was found to assist the elimination of parasites during subsequent treatment with a full dose of CQ. An inverse relationship was observed between pretreatment blood CQ concentration and the degree of resistance in this study. Authors conclude that pre-treatment “plays a role in the observed reduced proportion of RIII-type resistance in Ghana”. This finding essentially confirms the model’s prediction that re-treatment within a short time could deal with resistant parasite strains, since the blood levels are indicative of the time lapsed between pre-treatment, and subsequent treatment.
5. The action of chloroquine is pleiotropic evoking different mechanisms of resistance The usability of CQ can also be rescued by attempting to reverse the resistance, which is to co-administer CQ with another drug that chemosensitizes the parasite to its effect. For this purpose, some aspects of resistance of the parasite to CQ have to be understood. It is commonly accepted that resistant parasites accumulate less drug than their drug sensitive counterparts. Whether this is due to binding to the ferriptoroporphyrin IX (FPIX) that is generated in the food vacuole during the digestion of ingested host cell hemoglobin (Bray et al., 1999; Hawley et al., 1998), or due to active efflux of the drug (Krogstad et al., 1987; Sanchez et al., 2003) related to expression or mutations in the CQ transporter (Waller et al., 2003; Sidhu et al., 2002) is a matter of hot debate. It is also generally accepted that the accumulated drug inhibits the polymerization of FPIX into non-toxic hemozoin. Thus, free FPIX is available to intoxicate the parasite but how does it perform its act? FPIX is known to dissolve into membranes and permeabilize them. But the first membrane encountered by free FPIX that of the food vacuole, is probably not affected in as much as the pH of the food vacuole is not altered at therapeutic CQ concentrations (Ginsburg et al., 1989). On the other hand, FPIX has been shown to exit the food vacuole and to bind to membranes (Zhang et al., 1998); thus, explaining the
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depletion in infected cells of their potassium and its replacement by sodium (Lee et al., 1988), and to bind to proteins (Famin and Ginsburg, 2003) some of which are enzymes whose activities are reduced (Campanale et al., 2003). These effects are probably the final targets of CQ action. In all these actions, it could be shown that glutathione was able to degrade the membrane- and/or protein-associated FPIX and that this process was competitively inhibited by CQ (Ginsburg et al., 1998; Famin et al., 1999; Famin and Ginsburg, 2003). These observations imply that resistance to CQ can be achieved by high levels of glutathione and that drug-sensitive parasite strains should have low levels of the tripeptide. This was indeed observed experimentally and the biochemical basis of this discrepancy has been recently provided: whereas in the CQ-sensitive 3D7 strain the rate limiting enzyme responsible for low glutathione concentrations is glutathione reductase, de novo synthesis is responsible for the higher glutathione level in the CQ-resistant strain Dd2 (Meierjohann et al., 2002). These results indicate that CQ’s action may be pleiotropic: it inhibits both the polymerization of FPIX and the degradation of free FPIX by glutathione. Pleiotropic action suggests various mechanisms of resistance and different ways to counteract them.
of mutations in the mdr1 gene of P. falciparum which is also involved in resistance to CQ (Duraisingh et al., 2000; Babiker et al., 2001) that confer CQ resulted in a considerable fitness cost in the asexual blood stage of the parasite. The calculated fitness cost of 25% is much lower than the 10% reduction in the viability of resistant mutants that was found by modeling to be sufficient to insure the survival of mutant genes in the parasite pool, even where drug pressure is still being applied (Mackinnon, 1997; Mackinnon and Hastings, 1998; Hastings and D’Alessandro, 2000). The fitness cost may be due to mutations in other genes rather than in the gene that confers the specific resistance to a given drug. Thus, several glycolytic enzymes were found to be up-regulated in the presence of artemether and down-regulated in the presence of lumefantrine (Makanga et al., 2005) attesting the presence of compensatory mutations. A study on altered transcription of a limited sub-set of genes in CQ-exposed parasites has failed to reveal a significant effect on the metabolic transcript profile of 3D7 strain (Gunasekera et al., 2003). However, a marked reduction of mitochondrial rRNA expression was observed that could lead to reduced mitochondrial metabolism; thus, compromising the viability of the parasite.
6. Drug resistance and fitness cost
7. Reversal of resistance
Interestingly, the generation of CQ resistance under drug pressure in Plasmodium berghei resulted in a marked increase in glutathione concentration (Platel et al., 1999) and a higher expression of ␥glutamyl–cysteine synthetase, the first enzyme in the glutathione biosynthetic pathway (Perez-Rosado et al., 2002). These correlates, however, were not found in Plasmodium chabaudi (Ferreira et al., 2004), but, for that matter, neither mdr1 and crt homologues could be found in this species (Ferreira et al., 2004; Hunt et al., 2004). Both CQ accumulation, irrespective of the precise mechanism, and increased synthesis of glutathione, require metabolic energy. When these processes become excessively demanding, energy shortage may develop, thereby restricting parasite viability. This could well be the reason why resistance to CQ cannot increase indefinitely, as mentioned above. In this respect, it is also worth mentioning a recent study (Hayward et al., 2005) in which the effect
Since glutathione-dependent drug resistance is also known in some types of cancer, depletors of glutathione have been tested and currently considered as “reversers” of this type of resistance (Borst et al., 2000). Similarly, combining a drug known for its ability to reduce the concentration of glutathione in vivo should chemosensitize the antimalarial action of CQ, and thus hopefully reinstates it in the pharmacopoeia of antimalarial drugs. The achievability of this advance was first demonstrated in vivo in P. berghei-infected mice by combining CQ with buthionine sulfoximine, and inhibitor of ␥-glutamyl–cysteine synthetase (Dubois et al., 1995). Subsequently, some over-the-counter drugs such as indomethacin, disulfiram and paracetamol, known for their ability to reduce cellular glutathione directly or indirectly, were shown to chemosensitize the antimalarial action of CQ in murine malaria models in vivo (Deharo et al., 2003). These drugs were even more potent at enhancing the action of the CQ
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congener amodiaquine. Since combination antimalarial chemotherapy is riding high these days (Kremsner and Krishna, 2004), should not these combinations be considered as well? If so, it should be stressed that the concentrations required for potentiation by paracetamol or disulfiram are well within the doses tolerated by humans, but the concentrations of indomethacin are too high to be permissible. A different approach has been attempted by the synthesis of a double-headed prodrug. When it penetrates the parasite, it is hydrolyzed to an inhibitor of glutathione reductase (that caused a marked reduction in glutathione), and also to a CQ-mimic 4aminoquinoline. The compound displayed an IC50 similar to that of CQ against sensitive strains of P. falciparum (20–30 nM), both in sensitive and resistant strains. The drug was not toxic to mice and had a considerable antimalarial activity against P. berghei (Davioud-Charvet et al., 2001). Except for these very few sideways inspections of alternative chemosensitizers, all other attempts have been centered around compounds that could enhance the accumulation of CQ (Peters et al., 1990; Kyle et al., 1993; Chandra et al., 1993; Oduola et al., 1998; Singh and Puri, 2000). Only one combination has been tested in human patients with some success, that of CQ + chlorpheniramine, a histamine H1 receptor antagonist (Sowunmi and Oduola, 1997; Sowunmi et al., 1997, 1998a,b). This is probably why so many efforts are still invested in the deciphering of this process—since understanding it may divulge directions for its inhibition. But there is certainly life after accumulation that is worth contemplating. In this context, the recent publication showing up-regulation of scores of ABC-transporter genes in CQ-resistant strains (Mu et al., 2003) is more than an understated hint for a possibly promising direction.
8. Wrapping up In conclusion, although CQ is ineffective in most parts of Africa and the onset of resistance has at least doubled childhood malaria death risk, we suggest that it is too premature to lay CQ to rest. Differential use of CQ in holoendemic areas that precludes children and pregnant women could still confer an efficacious protection for semi-immune adults. The recent introduction of
artemisinine combination therapy as a chemotherapeutic strategy is certainly justified but at the same time it means a 10-fold increase in the price of treatment, a financial burden that cannot be shouldered by most inhabitants of the African continent. Sequential treatment with CQ and its combination with glutathionedepleting drugs is worthy of a serious systematic testing since it could be useful even for treatment of children and adult patients in meso- or hypo-endemic areas where premunition is minimal and is affected seasonally by transmission intensity. Not less important is the fact that CQ is still an efficient drug against Plasmodium vivax infections in most countries (Vijaykadga et al., 2004; Baird, 2004) although even there resistance seems to be spreading.
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