The prevention of antimalarial drug resistance

Pharmac. Ther.Vol. 47, pp. 499-508, 1990 Printed in Great Britain. All rights reserved

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Specialist Subject Editor: D. C. WARHURST

THE PREVENTION OF ANTIMALARIAL D R U G RESISTANCE WALLACE PETERS Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, U.K. Abstract--The deployment of antiprotozoal drugs on a large scale for prophylaxis or monotherapy inevitably results in the selection of drug-resistance. The use of appropriately selected drug combinations may impede this process. Point mutations underlie resistance to dihydrofolate reductase inhibitors such as pyrimethamine. Potentiating combinations of such compounds with sulfonamides or sulfones have effectively delayed resistance to them. The use of triple combinations may be of value in protecting such compounds as chloroquine and mefloquine, resistance to which is associated in some cases with gene amplification. It is essential to seek partner compounds for any new antimalarials, e.g. artemisinin. Past experience with existing compounds is discussed and the need to make use of all available means of interrupting malaria transmission is stressed, rather than depending entirely on drugs.

CONTENTS I. Introduction 1.1. Rate of development of resistance 1.2. Experience in organisms other than Plasmodium 1.3. Procedures that may impede the emergence of drug resistance 2. The Use of Drug Pairs 2.1. Potentiating combinations 2.1.1. Antifols with sulfonamides or sulfones 2.1.2. Sesquiterpene lactones with aminoalcohols 2.2. Compounds acting at different stages of the malaria life cycle 2.2.1. 4-Aminoquinolines with primaquine 2.2.2. Mefloquine with primaquine 2.3. Compounds acting in different ways on asexual blood stages 2.3.1. 4-Aminoquinolines with pyrimethamine 2.3.2. Chloroquine with calcium channel or calmodulin blockers 3. Multiple Drug Combinations 4. Other Control Methods in Preventing Antimalarial Resistance Acknowledgements References

1. I N T R O D U C T I O N

1.1. RATE OF DEVELOPMENTOF RESISTANCE Experimental and clinical field experience has shown that a given species of Plasmodium may develop resistance to various antimalarials at different rates when exposed to a specific type of selection pressure. Moreover, other strains of that species, as well as separate species may become resistant at yet different rates. In spite of these variables certain patterns have become apparent in reviewing the problem as a whole. Resistance to the dihydrofolate reductase inhibitors cycloguanil (the active metabolite of proguanil) and pyrimethamine may emerge after a single exposure to a large drug dose both in vitro and in vivo. The phenomenon has been documented in the rodent

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parasite Plasmodium berghei, the simian species P. cynomolgi and three species in h u m a n volunteers, P. falciparurn, P. vivax and P. malariae. It has also been shown that P. berghei very rapidly becomes resistant to sulfadiazine when exposed to large doses in vivo in mice. On the contrary, resistance to compounds such as mepacrine and chloroquine requires more prolonged drug exposure and appears either after a very long exposure or not at all in some species. Thus chloroquine resistance has not yet been documented unequivocally in P. vivax (although the suspicion that it is emerging has now been raised in Papua New G u i n e a - - R i e c k m a n n et al., 1989; Schuurkamp et al., 1989; Whitby et al., 1989) and the avian parasite P. gallinaceum required many passages in vivo in chicks to reach a significant level (see review in Peters, 1987). Moreover, different types of resistance may develop in P. berghei and other rodent malaria parasites depending upon the way in which 499

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chloroquine selection pressure is applied, the highly resistant RC-type strains showing very different characteristics from the moderately resistant NS-type parasites (which appear to be a better model for the type of chloroquine resistance seen in P. falciparum of man). 1.2 EXPERIENCE IN ORGANISMS OTHER THAN PLASMODIUM

A similar picture can be seen in the development of resistance to anticoccidial agents, that to sulfonamides, for example, appearing rapidly compared with resistance to certain other compounds such as monensin that do not act as primary antimetabolites (Peters, 1985). If one compares the situation among prokaryotic organisms or even in mammalian neoplastic cells, an equally great diversity is seen in the rate at which resistance to single drugs emerges. Broad experience of resistance to insecticides also has much to teach us about resistance in eukaryotic protozoa. It is salutary to note the conclusion that: "The development of resistance is . . . dependent on genetic variability already present in a population [of insects], or arising during the period of selection." "The tolerance resulting from induction is not transferred to offspring" (Oppenoorth, 1985). 1.3. PROCEDURES THAT MAY IMPEDE THE EMERGENCE OF DRUG RESISTANCE

One of the principal means that has been deployed in an attempt to impede, if not totally prevent, the emergence of drug resistance, not only in microorganisms but also in neoplastic cells, has been the use of drug combinations. So far these have been, to a large extent, empirically rather than rationally based. They have taken various forms such as known potentiating combinations, mixtures of compounds believed to act on the target cells at different sites, sequential therapy with two or more drugs etc. Only recently has a deeper insight into the genetic mechanisms underlying drug resistance begun to open the way to more rational approaches. Examples of these approaches as they have been applied in the prevention and treatment of malaria are discussed in this review. It should be obvious that any scheme to impede or prevent the development of resistance to antimalarials should be based on an understanding, first, of the ways in which particular drugs exert their noxious effect on the parasites and, second, the mechanisms by which the parasites overcome these effects and the genetic basis of the mechanisms. Unfortunately our knowledge on these points is still far from complete although recent investigations of the biochemical and molecular genetic basis of resistance to several widely used antimalarials have begun to throw light on the problem. So far, however, the few efforts that have been made to impede the development of resistance to antimalarials have been based on hypothetical or experimental bases that are open to criticism. Indeed some early, empirical attempts in this direction were shown post hoc to have been doomed to failure from the start.

2. THE USE OF D R U G PAIRS

2.1. POTENTIATING COMBINATIONS 2.1.1. Antifols with Sulfonamides or Sulfones 2.1.1.1. Mechanisms o f action. Mammalian tissues can utilize preformed folate in the production of the tetrahydrate cofactors for the synthesis of pyrimidines. They may also utilize exogenous pyrimidines since they possess suitable salvage pathways. Generally speaking bacteria and malaria parasites cannot. Soon after the antibacterial action of sulfonamides was described it was shown that these compounds block the synthesis by the organisms of dihydropteroic acid which is essential for dihydrofolate synthesis, thus making them highly selective against such organisms. Before long other compounds were synthesized that block the enzyme, dihydrofolate reductase, which mediates the final step in tetrahydrofolate synthesis. Proguanil (through its triazine metabolite, cycloguanil) and pyrimethamine proved to act in this way. The potentiating effect of combining a wide variety of such compounds which block consecutive steps in folate synthesis of either prokaryotes or eukaryotes, has been extensively documented (see review by Peters, 1987). 2.1.1.2. Practical applications in malaria. In malaria parasites the effect was first reported in 1948 by Greenberg et al. who used a combination of proguanil and sulfadiazine against P. gallinaceum. It was confirmed with pyrimethamine and sulfadiazine against the same infection in chicks by Goodwin (1952). In 1959 Hurly demonstrated the value of this mixture for the treatment of P. falciparum infections in man in West Africa and this was later confirmed by McGregor et al. (1963), but it was only when long-acting sulfonamides, particularly sulfadoxine, were introduced that practical use was made of such a combination. DeGowin and Powell (1964) successfully treated nonimmune patients with chloroquine-resistant falciparum infections in Vietnam with pyrimethamine and sulfadiazine. However, pyrimethamine plus sulfadoxine (under the name of Fansidar ~) proved to be greatly superior in this indication and its value as a single-dose treatment of such infections was demonstrated in Vietnam (Bartelloni et al., 1967), in Thailand (Harinasuta et al., 1967) and in Brazil (Walker and LopezAntufiano, 1968). Subsequently the rapid curative action of Fansidar ~ was confirmed even against strains of this parasite that showed resistance not only to chloroquine but also to pyrimethamine. Following this encouraging experience, the combination was marketed, originally as a prophylactic with the express objective of impeding the emergence of further resistance to pyrimethamine which, used alone, was already unable to protect against many strains of the malaria parasites of man, including all the species except P. ovale. Experimental data based on studies with P. berghei in mice to support this stand were provided by Richards (1968, quoted in Peters, 1987). He showed also that dapsone had a similar effect to sulfadoxine in impeding the emergence of resistance to pyrimethamine and a

Prevention of antimalarial drug resistance combination of the two was soon made available for prophylaxis under the name of Maloprim ~. Fansidar" came to be deployed extensively for both therapy and prophylaxis in countries where multiple drug-resistant P. falciparum had become a problem but also, unfortunately, in other countries where this parasite was still chloroquine-sensitive. Maloprim ~ proved to be less popular and was, in any case, not used for treatment. Practical experience proved that Fansidar ~ did, as anticipated, remain effective far longer than pyrimethamine (or indeed a sulfonamide) would have done had it been used alone. Resistance to Fansidar ~ began to emerge on a serious scale in Thailand in the 1970s. The cure rate of a standard dose fell from virtually 100% in 1966 to 82% in 1975 and 42% in 1979 (Harinasuta et al., 1981, quoted by Brockelman and Tan-Ariya, 1982). Subsequently, although on a less serious scale, Fansidar ~ resistance was reported from other countries including some in which chloroquine resistance had not yet evolved, the latter demonstrating the folly of using 'fall-back' antimalarials before they are really needed. Since the mid 1980s there have been increasing numbers of reports also of the failure of Maloprim ~ as a prophylactic from regions as far apart as East Africa and Papua New Guinea. Other antifol-sulfonamide/sulfone combinations have been shown to be effective in the prevention or treatment of multiple drug-resistant P. falciparum. With the aim of preventing the development of drug resistance, extensive laboratory and clinical investigations were carried out by Thompson and his colleagues (see reviews by Thompson and Werbel, 1972 and Peters, 1987) on an injectable, repository formulation containing a mixture of the poorly soluble pamoate of cycloguanil, together with diacetyl dapsone which is of similarly low solubility. Unfortunately this project failed for several reasons that included a lack of tissue tolerance of the injection under field conditions and a high background of resistance to proguanil in some areas where clinical trials were being conducted. Interest is, however, reviving in this type of combination. Watkins et al. (1988) have found that a combination of chlorproguanil with dapsone is effective in areas of Kenya where a high level of resistance exists in P.falciparum against pyrimethamine and against proguanil, as well as chloroquine, and where a low level of resistance has already been reported to Fansidar ~. Cross-resistance between pyrimethamine and proguanil (i.e. its triazine metabolite) is widespread but inconsistent, but it remains to be seen if, and for how long, this new combination will retain its effectiveness. 2.1.1.3. The genetic basis o f resistance to antifols. Walliker (1983) and Peters (1987), reviewing a large number of reports on the genetic basis of resistance to pyrimethamine in several species of Plasmodium, concluded that the factor responsible for such resistance in nature is the selection of a clone of the parasites which has a mutant form of the enzyme dihydrofolate reductase (DHFR), the coding for which is linked with that of thymidylate synthase on a single gene. The mutant enzyme has a lower affinity for the drug, probably by virtue of changes in one to three aminoacids (Inselburg et al., 1988; Hyde, 1989).

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Pyrimethamine resistance can be selected in a single step under appropriate experimental conditions and the mutant gene has been shown to be transmissible in typical Mendelian fashion during the sexual cycle in the anopheline vector. Walliker et al. (1987) have pointed to the ease with which novel genetic recombinations of this type occur in P. falciparum and the implications for the spread of drug resistance in the field. Resistance to the triazine metabolites of proguanil and chlorproguanil (Lapudrine~), namely cycloguanil and chlorcycloguanil, is probably due to a similar gene mutation although this has yet to be proved. Cross-resistance between these compounds and pyrimethamine is common but by no means consistent. Although the precise location on the gene or the nucleotides concerned have not yet been determined it seems likely that, at least in some cases, they are not identical to those concerned in pyrimethamine resistance, even though the same gene appears to be involved (see Hyde, 1990). In none of these types of antifol resistance does gene amplification appear to play a significant role in field isolates. The genetic basis of resistance to sulfonamides and sulfones in Plasmodium is less certain but several mechanisms appear to be involved in P. falciparum. Dieckmann and Jung (1986) found that resistance parasites take up only ~ of the quantity of [35S] sulfadoxine compared with drug-sensitive parasites in red cells, that metabolism of the compound to a toxic dihydropteroate analog is reduced and that the resistant organisms may be able to synthesise p-aminobenzoic acid de novo, unlike the sensitive ones. It is possible that these three observations are the consequence of one change but the drug sensitivity of dihydropteroate synthase in a resistant strain has not been studied to date, although a high level of resistance can readily be selected by exposure to a single, high drug dose in vivo. Resistance is transmitted, like that to pyrimethamine, in Mendelian fashion during sexual reproduction in the vector. Since most natural infections contain a mixture of parasite clones (Graves et al., 1984), it is not surprising, by genetic recombination, that organisms can be produced that show resistance to sulfonamides and to the antifols. Nevertheless, there is as described above strong evidence that the use of a potentiating sulfonamide-pyrimethamine combination such as Fansidar ~ does impede the emergence of resistance to these compounds both in experimental models such as P. berghei and under natural conditions in P. falciparum, even though parasites resistant to the combination do become selected in time (reviewed by Peters, 1987). 2.1.2. Sesquiterpene Lactones with Aminoalcohols 2.1.2.1. Mechanisms o f action. Peroxide-containing sesquiterpenes of which artemisinin (Qinghaosu, a component of the plant Artemisia annua Linn., Compositae) is the lead compound, are potent blood schizontocides that are effective against strains of Plasmodium which are resistant to antifols and chloroquine. They are concentrated, for example, dihydroartemisinin some 300 times (Gu et al., 1984),

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inside the intra-erythrocytic parasites in which they are strongly bound to various membranes. Ultrastructural studies have shown damage to various membranes including those surrounding the nuclei (Ellis et al., 1985) and mitochondria (Jiang et al., 1985). While it is known that the compounds rapidly inhibit first protein then nucleic acid synthesis, the molecular basis of these actions has not yet been precisely determined. Artemisinin which is a lactone is rapidly reduced to produce the active dihydro derivative, but further metabolism leads to the loss of the peroxide bridge, the resulting dihydrodesoxyartemisinin being inactive. The antimalarial action of the sesquiterpene lactones is very rapid but their effective half-lives are brief both in rodents and in man (see review by Peters, 1987). Even though artemisinin and its analogs rapidly reduce P. JMciparum or P. Hvax parasitemia in man, recrudescences occur frequently (up to 15 or 20%), the reason for this remaining obscure. In mice infected with chloroquine-resistant P. yoelii resistance can be selected readily by drug pressure (Chawira et al., 1986). Moreover a number of artemisininresistant lines have been developed in P. falciparum in t,itro with the aid of mutagenic agents (Inselburg, 1985). In the light of previous experience of the danger of producing resistance by the large-scale deployment of single antimalarials and the great practical difficulty of developing new antimalarials, it is of obvious importance to seek ways and means of protecting the few novel and potentially valuable new drugs that do appear, such as artemisinin. Chawira et al, (1987) reported that artemisinin displays a significant level of potentiation with the aminoalcohol antimalarial, mefloquine, against a drugsensitive strain of P. berghei. Potentiation was also seen when artemisinin was administered with tetracycline or spiramycin but combinations with antifols or sulfonamides were antagonistic as reported also by Chinese workers. The potentiation between artemisinin and mefloquine was retained against strains of P. yoelii that were already resistant to one or other of the components, Potentiation was also significant between artemisinin and primaquine against strains resistant to one or other of this pair. Subsequently the existence of potentiation between artemisinin and mefloquine, as well as artemisinin and tetracycline was confirmed against a drugsensitive strain of P. falciparum in eitro, as well as antagonism between artemisinin and pyrimethamine (Chawira and Warhurst, 1987). Against a chloroquine-resistant strain potentiation was again seen when artemisinin was administered with mefloquine or tetracycline or, in addition, primaquine. Antagonism was seen between artemisinin and chloroquine against this strain. 2.1.2.2. Practical e,x'perience. No reports have appeared yet of the identification of resistance to artemisinin or its analogs in human malaria infections but none of these compounds has been widely used in man yet outside the People's Republic of China. Resistance to mefloquine used in monotherapy has been identified in several foci in Southeast Asia, Africa and South America and numerous reports of studies in t,itro indicate that mutants of

P. falciparum resistant to this compound are widely distributed, even prior to its large-scale deployment. As mefloquine has a very prolonged half-life in man it would not be logical from a pharmacokinetic point of view to administer a combination with one of the artemisinin series. On the other hand, a combination with the shorter-lived aminoalcohol, halofantrine, could be considered. Like mefloquine, halofantrine has a potentiating action with artemisinin against rodent malaria parasites (Peters and Robinson, unpublished data) and halofantrine resistance is readily developed (Peters et al., 1987). (The aminoalcohols are discussed further below.) To date no clinical studies appear to have been made with any such artemisinin combinations that may possibly be potentiating, nor are there any reports of the effects of such combinations on the emergence of resistance in experimental models.

2.1.2.3. The genetic basis of resistance. Nothing has been published to date on the genetics of resistance to artemisinin beyond the report that resistant mutans could be produced by exposing P.falciparum to mutagenic agents (Inselburg, 1985). Neither the influence of genetic recombination in the vector nor the pattern of inheritance of the mutant parasites after transmission have yet been identified. In discussing the broad pattern of cross-resistance between artemisinin and totally unrelated compounds in rodent malaria, Chawira et al. (1986) suggested that artemisinin resistance may be due to changes in membrane composition. Perhaps these involve the mdr that is discussed in the following section? 2.2. COMPOUNDSACTING AT DIFFERENTSTAGESOF THE MALARIA LIFE CYCLE

2.2.1. 4-Aminoquinolines with Primaquine 2.2.1.1. Mechanisms o f action. The most widely used 4-aminoquinoline by far is chloroquine. Although this compound has been deployed constantly for the last 40 years its mode of action at the molecular level is still uncertain. Observations on its high degree of concentration in parasitized red cells and on its inhibitory action on the digestion of hemoglobin in plasmodial food vacuoles have produced a variety of explanations, as well as controversies. Warhurst (1988), for example, has proposed that monoprotonated chloroquine diffuses from the plasma down a pH gradient into the red cell where it becomes diprotonated. It is then bound by a permease on the parasite membrane to enter the parasite cytoplasm where it again loses a proton to become monoprotonated. An ATP-driven proton pump decreases the pH within the food vacuoles permitting the monoprotonated drug to accumulate in the food vacuole where it again acquires a proton, the diprotonated form inhibiting the proteases that digest ingested host hemoglobin to provide essential nutrients for the parasites. It has long been recognized that less chloroquine is accumulated in chloroquine-resistant Plasmodium than in sensitive parasites. Recently this phenomenon has been shown to involve an active efflux of drug mediated by a protein analogous to the 'multiple drug

Prevention of antimalarial drug resistance resistance protein' (mdr) found in the plasmalemma of certain mammalian neoplastic cells, the net accumulation being a balance between influx and efflux (Krogstad et al., 1987; Foote et al., 1989; Wilson et al., 1989). Having gained access to the parasite cytoplasm, chloroquine may also act as an inhibitor of the calmodulin-Ca 2+ complex which is responsible for the regulation of the enzymes that are essential in many parasite metabolic processes (Scheibel et al., 1987). Resistance to chloroquine emerged as a practical problem in the prevention or treatment of malaria due to P. falciparum almost before resistant strains had been reported in experimental models. The need to contain the problem was rapidly recognized and means were sought to minimize the spread of resistance pending the development of alternative antimalarials. One avenue appeared to be to attack the parasites in man with one compound while attacking them in the vector with another. Two types of compound were available for the latter purpose, the 8-aminoquinolines and the antifols. Although primaquine does have a direct inhibitory action on several metabolic processes of the asexual stages of Plasmodium in the red cells, possibly through an indirect action on host cell enzymes, it also has a potent inhibitory action on the development of gametocytes once they are ingested by vector mosquitoes. Although it has now been recognised that primaquine is rapidly converted in vivo by man and animals to a number of metabolites, it is probable that the gametocytocidal action is due to the parent compound and not one or more of its metabolites (Peters and Robinson, 1987) since it has no direct action on sporogonic development if fed directly to infected mosquitoes. 2.2.1.2. Practical experience. Chloroquine resistance is readily developed in certain rodent malaria parasites (especially P. yoelii, P. chabaudi) but with difficulty in the avian species and only, so far, in P. falciparum among the parasites infecting man. Chloroquine-resistant P. falciparum has spread rapidly since its first appearance in the early 1960s almost simultaneously in Thailand and Colombia. Resistant strains are now present and pose serious practical difficulties in virtually all areas endemic for this parasite. Moreover, many of the parasites display concomitant resistance not only to other 4-aminoquinolines but also to quinine, mefloquine, pyrimethamine, proguanil, sulfonamides and Fansidar ~ (see Peters, 1987). The situation resembles in some ways that which exists in bacteria where multiple drug resistance is commonplace and in cancer cells where the same phenomenon also poses major therapeutic problems (see below). The fact that chloroquine attacks essentially the asexual intraerythrocytic stages while primaquine can help to interrupt transmission through the vectors led to the deployment on a fairly large scale of combinations of chloroquine or amodiaquine (another 4aminoquinoline) with primaquine as one means of combatting malaria in the 1960s during the global malaria eradication campaign in the hope of minimizing the risk of the spread of resistance to chloroquine. This is particularly important in areas where JPT 47~3--L

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chloroquine is still widely used since there is evidence that chloroquine may itself increase the readiness with which chloroquine-resistant strains of Plasmodium are transmitted through the vectors, thus facilitating the ease with which such strains can be disseminated in an endemic area where vector anophelines are still present. The full potential of such a drug combination in blocking the spread of resistance has never been evaluated and it is now almost certainly too late to do so. A hint of its value in reducing the quantum of falciparum malaria in a given area was provided by workers in Nicaragua where a massive campaign covering 70% of the population in 1981 did appear to have a significant, if temporary impact (Garfield and Vermund, 1986). However, adverse experience with various combinations of the 4-aminoquinoline, amodiaquine with primaquine, mainly in Latin America and Papua New Guinea, suggest that such a combination in the long run is likely to have little, if any, effect in slowing down the emergence of drug resistance, partly because of the practical, logistic problems involved in their administration. Moreover, amodiaquine has recently fallen into disfavor because of reports that it can produce serious toxicity when given repeatedly. Mass chemoprophylaxis or chemotherapy has been strongly discouraged since 1984 by malaria experts (WHO, 1984) who finally accepted that the massive distribution of chloroquine, for example in medicated salt during the eradication era, was a major factor in the selection of chloroquineresistant P. falciparum (Payne, 1988). Resistance to primaquine itself has not been recorded in P. falciparum although it has been reported in several species of Plasmodium in laboratory models (Peters, 1987). In all these cases the resistance has been associated with the blood schizontocidal action of this compound. In P. vivax, on the other hand, a low level of resistance to primaquine exists in the hypnozoites of certain strains which are exemplified by the famous Chesson strain of Papua New Guinea. This phenomenon is revealed by the failure of a standard dose of primaquine to bring about a radical cure, i.e. to prevent true relapses of these parasites. If 8-aminoquinolines ever come to be deployed on a large scale alone (and several new analogs that combine activity against both blood and tissue stages are in an advanced stage of development), there is a high risk that resistant mutants of the intraerythrocytic forms of both P. falciparum and P. vivax, as well as the hypnozoites of the latter will surface in a relatively short time. 2.2.1.3. The genetic basis of resistance. It is unusual to be able to obtain an insight into the basis of resistance of a compound before even its mode of action has been established, but such is the case with chloroquine. The demonstration referred to above that the efflux of chloroquine is increased in red cells that contain chloroquine-resistant P. falciparum, was long preceded by reports that the equilibrium concentration of this drug in both rodent (P. berghei-Macomber et al., 1966) and human parasites (P. falciparum--Fitch, 1970) is much less in resistant than in sensitive strains. By analogy with multiple drug resistance (MDR) in mammalian tumor cells

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which is associated with the amplification of the mdr gene coding for an export protein in their plasma membranes (a 170kDa 'P-glycoprotein'), it was hypothesized that a similar phenomenon might underlie M D R in malaria parasites. Martin et al. (1987) demonstrated that verapamil reverses chloroquine resistance in P.falciparum in vitro. Their report was followed by descriptions of other compounds that possessed this property also in vivo, e.g. desipramine (Bitonti et al., 1988) and cyproheptadine (Peters et al., 1989). This lead was taken up by Foote et al. (1989) who demonstrated that an analogous gene, pfmdr is present on chromosome 5 of the P. falciparum genome and that this gene is amplified (up to 5 times) in some (but not in all) chloroquine-resistant strains. Wilson et al. (1989) found two pfmdr genes in three strains of P. falciparum, one of which was chloroquine-resistant and one a clone doubly resistant to mefloquine and chloroquine. One of the genes (pfmdr 1) was found to be amplified two to four times in the mefloquine-resistant clone and, like that associated with chloroquine resistance, it was located on chromosome 5. Earlier genetic studies in the rodent parasite, P. chabaudi had indicated that chloroquine resistance in that parasite was multifactorial and that several genetic changes were responsible (Padua, 1981). The nature of the other factors remains to be determined. While it is an open question whether this insight into the genetics of chloroquine resistance will provide an avenue for the design of drug combinations aimed at blocking them, the topic is discussed further below (Section 3.2). 2.2.2. Mefloquine with Primaquine 2.2.2.1. Mechanisms o f action. The synthetic blood schizontocides of which mefloquine is the best developed at present, retain, like their forerunner quinine, a potent action against chloroquine-resistant Plasmodium, including P. falciparum. The mode of action of quinine, mefloquine and halofantrine clearly differs from that of chloroquine as shown, for example, by the specific changes all three exert on the ultrastructure of the food vesicles in the intraerythrocytic schizonts and the inability of mefloquine to intercalate with D N A (see Peters, 1987). Nevertheless, there is abundant evidence to indicate that parasites which are already resistant to chloroquine readily evolve resistance also to the aminoalcohols. Mefloquine resistance emerging under drug selection pressure in a clone of P. falciparum maintained in continuous culture in vitro was associated with marked chromosomal changes due to the asexual recombination of genes in these haploid organisms with the overgrowth of a mutant gene resistant to this compound. Wellems et al. (1988) found no evidence to associate mefloquine resistance in these parasites with gene amplification, an observation that conflicts with that of Wilson et al. (1989). 2.2.2.2. Practical experience. The principle of combining mefloquine with primaquine for the treatment of chloroquine-resistant P. falciparum infections in the presence of continuing malaria transmission was proposed on the assumption that the first compound

would eliminate the resistant asexual intraerythrocytic stages while the primaquine would sterilize any existing gametocytes, thus blocking further transmission of the resistant parasites. Experiments in mice infected by blood passage with the drug-sensitive P. berghei N strain showed that the rate of emergence of resistance to mefloquine when serial passages were carried out under drug selection pressure was greatly delayed when compared with the rapid emergence of mefloquine resistance in a parallel line exposed to mefloquine selection pressure alone (Peters et al., 1977). It was also observed that resistance to mefloquine, as well as to halofantrine, could arise very rapidly under drug selection pressure in lines of rodent parasites that were already resistant to chloroquine. While serious consideration was given to carrying out pilot field trials in man of such a mefioquine-primaquine combination (WHO, 1984), this objective has not been realized to date. In the meantime, in spite of international guidelines aimed at a limited and rational deployment of mefloquine designed to impede the emergence of resistance, an increasing number of reports has appeared in the past few years of resistance to this compound in P.falciparum, either in vitro (e.g. from West Africa-Van der Kaay et al., 1985) or in vivo (from East Africa--Bygbjerg et al., 1983), even where the compound has never been deployed except for the treatment of a few isolated patients. In areas such as Thailand where M D R strains of this parasite are very common and mefloquine has been rather extensively used, largely on its own, clinical resistance started to occur as early as 1983 (Lapierre et al., 1983) and cross-resistance to halofantrine and enpiroline (a pyridine aminoalcohol) has been demonstrated in man (Cosgriff et al., 1985). Interestingly, this strain appeared to regain its sensitivity to chloroquine. 2.3. COMPOUNDSACTING IN DIFFERENTWAYS ON ASEXUALBLOOD STAGES 2.3.1. 4-Aminoquinolines with Pyrimethamine 2.3.1.1. Mechanisms o f action. The mechanisms of action of 4-aminoquinolines and of pyrimethamine have been discussed above (see Sections 2.1 and !.1). On the assumption that chloroquine and pyrimethamine had fundamentally different modes of action it was believed that, by administering a combination of the two compounds, the emergence of resistance to pyrimethamine (against which it was already known that resistance could be selected rapidly) would be hindered. Consequently a variety of combination schedules were employed, often for mass drug administration, in the early years of the malaria eradication campaign which started in the 1950s. It was hoped, moreover, that the pyrimethamine component, by virtue of the sporontocidal action of this antifol, would also have a blocking effect on the transmission of parasites through the local anopheline vectors. 2.3.1.2. Practical experience. The results of this apparently rational policy, which were horrendous, have been reviewed by Peters (1987). Retrospective

Prevention of antimalarial drug resistance studies in an experimental model using P. berghei clearly showed that this particular drug combination, for reasons still unknown, had no beneficial effect whatsoever and, indeed, the pre-existence of pyrimethamine resistance could even facilitate the selection of resistance to chloroquine in another species, P. vinckei in which chloroquine resistance had not been achieved when an originally chloroquinesensitive strain was made pyrimethamine-resistant and then exposed to chloroquine. In contrast, the exposure of P. berghei to a mixture of chloroquine and a sulfonamide resulted in a very significant delay in the emergence of resistance to either of the two compounds. Moreover, an even longer delay was experienced when the parasites were exposed to a mixture of chloroquine, pyrimethamine and sulfadoxine, the latter two themselves having a delaying effect on the emergence of resistance to each of them. No clinical studies were ever launched with a combination of chloroquine and a sulfonamide or chloroquine and Fansidar ~, partly because of the appearance of mefloquine on the horizon as an alternative to chloroquine and partly because of an increasing fear of the potential toxic effects of sulfadoxine. Indeed the recommendation to take prophylactic chloroquine with Fansidar ~ put forward in the early 1980s to nonimmune travellers to areas where chloroquine-resistant P. falciparum is present was rapidly dropped because of a minor flood of reports of serious toxic reactions to repeated doses of sulfadoxine in that mixture. (Fansidar ~ is currently recommended only for single-dose treatment in individuals who are not known to be sulfonamidesensitive.) 2.3.1.3. The genetic basis o f resistance. Unless the pfmdr gene also confers resistance to pyrimethamine, it is unclear why resistance to chloroquine should be associated with resistance to pyrimethamine unless the former involves some mechanism which is common to both types of drug resistance. It is possible that the genetic changes demonstrated in pyrimethamine resistance, namely point mutations in three aminoacids of the D H F R molecule, also have a bearing on one or other mechanisms of resistance to chloroquine although no such association has yet been mooted. There are, on the other hand, two reports which show that an increase in the synthesis of parasite dihydrofolate reductase can be associated with pyrimethamine resistance (Kan and Siddiqui, 1979; Inselburg et al., 1987), implying that gene amplification may also be a factor responsible for resistance to this compound. (It would be interesting to follow this up to see whether the amplified gene lies, like that for pfmdr on chromosome 5, but this seems unlikely since the normal gene coding for this enzyme lies on chromosome 4--Cowman et al., 1988; Peterson et al., 1988.) 2.3.2. Chloroquine with Calcium Channel or Calmodulin Blockers The principle underlying a possible combination of chloroquine with a calcium channel blocker (e.g. of the verapamil type) or calmodulin antagonist (e.g. desipramine) has already been discussed above (see

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Section 2.1). Up to the time of writing no reports have appeared of clinical trials with any such combination but the new approach indicated by these early experiments has stimulated a search for alternative partners for chloroquine which remains one of the cheapest and safest antimalarials, albeit one which is currently losing much of its ability to prevent or cure falciparum malaria.

3. MULTIPLE D R U G COMBINATIONS The possible use of multiple drug combinations (i.e. of three or more compounds) has been hinted at above in reference to a combination of chloroquine, pyrimethamine and sulfadoxine which Peters (1974) found very effective in protecting each of the components in a rodent malaria model. Because of the very real danger that strains of P.falciparum resistant to mefloquine would rapidly make their appearance if this compound were to be widely deployed in areas where chloroquine resistance was already prevalent, in the presence of ongoing transmission, endeavours were made to select partner compounds for mefloquine that would impede this progress. One important problem would be to detect compounds that would have a similar biological half-life in the human body to that of mefloquine which is exceptionally long. Experiments using two or three compounds were carried out in rodent models by Merkli et al. (1980) and by Peters and Robinson (1984). In addition to primaquine to which reference was made above, it was found that both pyrimethamine and sulfadoxine had a protective effect when either was administered with mefloquine. Puri and Dutta (1989) have confirmed the protective effect of primaquine and shown that dapsone and erythromycin have a similar action. Merkli et al. 0980) and Peters and Robinson (1984) also demonstrated that, when drug selection pressure was exerted with a triple combination containing mefloquine, pyrimethamine and sulfadoxine, resistance developed to all the components far more slowly than when only two compounds were used. A similar observation was made by Tan-Ariya et al. (1984) with P. falciparum exposed to the drugs alone or in combination in vitro. Fortunately both pyrimethamine and sulfadoxine have prolonged half-lives in man, not as long as that of mefloquine but more closely matching than any other antimalarials then available. In addition, pyrimethamine and sulfadoxine had already proven their value in the prevention and treatment of chloroquineresistant falciparum malaria (in the combination known as Fansidar ~) and had been successfully employed for that purpose for over a decade. A thorough study was therefore launched under the auspices of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), in close cooperation with Hoffman La Roche, the manufacturers of mefloquine, of this triple combination. Extensive clinical trials carried out in three continents proved the value of this combination (now known as Fansimef ~) as a single-dose therapeutic agent for M D R P. falciparum, and one that would lend itself to extensive deployment at a peripheral, primary health service level,

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with at least a theoretical chance of minimizing the otherwise inevitable increase in the emergence of mefloquine-resistant parasites (WHO, 1984). Unfortunately, the encouraging results that were received from the pilot field trials were accompanied by reports from other sources of sulfadoxine toxicity, mainly in nonimmune travellers. These exerted a dampening effect on those involved in the clinical studies of Fansimef" the future of which is currently in question. A further question overshadowing all experimental research on drug combinations has been the validity of studies carried out in rodent models which were based largely on the passage of parasites from one host to the next without the intervention of cyclical transmission through an insect vector as would occur in nature (WHO, 1987). While a computer simulation study by Curtis and Otoo (1986) did suggest that an appropriate drug combination could be expected to be of benefit under certain defined conditions of drug deployment, the demonstration by Walliker et al. (1987) has clearly shown that cyclical transmission greatly enhances the appearance of multiple drug resistance through the opportunity this gives of genetic recombination through sexual exchange in the mosquito. In the presence of selection pressure by a mixture of drugs it still remains an open question whether a multiple drug combination will prove valuable in the long run in slowing down or, much less likely, preventing the appearance of resistance to the individual components. While several studies in animal models are currently under way to answer this question, the technical difficulties are considerable and the experiments are, of necessity, of long duration.

4. O T H E R C O N T R O L M E T H O D S IN PREVENTING ANTIMALARIAL RESISTANCE In order to reduce the risk that resistance would develop when mefloquine became available, the writer laid down a number of basic principles for its deployment (Peters, 1982). They were as follows: (i) Strict governmental control should be exerted over its use. (ii) It should only be used in areas where its value is indicated by a high prevalence of P. falciparum resistant both to antifols and 4-aminoquinolines. (iii) A time limit should be set for its use. (iv) The response of the parasites should be monitored by in vitro testing as well as by clinical reporting. (v) It should only be deployed as one tool in an integrated programme o f malaria control using all available methods including vector control, health education and, i f and when it becomes available, vaccination. These principles are still valid. Malaria control has never been achieved anywhere in the world simply by the use of drugs nor, for that matter, even by insecticides used on their own. There is no doubt that the future of malaria control lies in the planning of integrated measures that adapt whatever means are best suited to the local epidemiological and economic

situations of the areas concerned, i.e. programmes tailor-made to suit local conditions. This is the only reliable way to prevent totally the continuing development of resistance to antimalarial drugs. Acknowledgements--Original investigations on antimalarial resistance carried out by the writer and his associates during the past two decades have been sustained by generous support from the Malaria Action Programme of WHO, the CHEMAL component of the TDR programme, from the U.S. Army Research and Development Command and from various pharmaceutical companies, to all of whom he expresses his deep gratitude. The writer wishes particularly to thank his long-time collaborators, Dr D. C. Warhurst and Mr B. L. Robinson and his current technical staff for their unflagging efforts in trying to resolve the problem of drug resistance and its prevention.

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