Prevention of drug resistance in rodent malaria by the use of drug mixtures

Bull. World Health Organ. 1974 51 379-383Bull. Organ. mond. Sante 5 3 Prevention of drug resistance in rodent malaria by the use of drug mixtures W. PETERS 1 Development of resistance to chloroquine in rodent malaria is inhibited by giving this compound together with a potentiating mixture ofpyrimethamine and sulfadoxine but this does not prevent the development of resistance to the last two compounds. The use of drug mixtures should be explored as a means of " protecting " chloroquine or new blood schizontocides intended for mass chemotherapy against human malaria. However, no gen- eral rule can be laid down without testing specific drug mixtures in long-term experiments in a suitable model such as rodent malaria. The danger of selecting drug-resistant organisms by administering potent antimicrobial agents alone has led to the now accepted practice of using several compounds together in the treatment of bacterial disease. Thus, the accepted procedure for the treat- ment of tuberculosis has long been to give, for example, a mixture of streptomycin, PAS, and isoniazid. Once resistance has developed it is some- times possible to overcome this by the judicious use of mixtures of drugs each of which stimulates the action of the others. Bacterial infections that are re- sistant to sulfonamides, for example, often re- spond to a mixture of a sulfonamide with a folate reductase inhibitor, such as the combination of tri- methoprim with sulfamethoxazole. It has long been known that this type of potentiation is also present when sulfonamides in combination with a suitable folate reductase inhibitor, such as pyrimethamine, are given to patients with acute malaria. However, the role that such mixtures may play in preventing the parasites becoming resistant to the individual components of the mixture has been little studied. It is evident that this information must be obtained before a rational decision can be made as to whether this type of mixture should be used for mass drug administration in the control of malaria. The follow- ing experiment was designed to compare the rates at which intraerythrocytic asexual stages of the rodent malaria parasite, Plasmodium berghei, become resist- 1 Honorary Director, WHO Regional Malaria Reference Centre for Screening of Potential Antimalarial Compounds, Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, England. ant to pyrimethamine, sulfadoxine, and chloroquine, given in various combinations. MATERIALS AND METHODS A detailed account of the technique used in this experiment has been published elsewhere (1). The principle employed is to expose parasites in albino mice to increasing doses of the drugs, singly or in predetermined mixtures, passaging the parasites into clean mice once weekly. The drugs used in this experiment were pyrimethamine base and sulfadoxine base as a 1: 3 mixture (PS mixture), and chloro- quine phosphate (C). The dose of the drugs is increased in successive passages as long as the sub- sequent infection is sufficiently high for a further passage to be made. Two series of lines were devel- oped, one starting from the NK65 strain of P. berghei (drug-sensitive) and the other from the slightly chloroquine-resistant NS line (2). The following lines were produced: NK65 series (from P. berghei NK65) NK65 PS exposed to pyrimethamine and sulfadoxine (1 :3) NK65 PSC exposed to a fixed combination of pyrimeth- amine with sulfadoxine (1: 3), and chloro- quine NK65 C exposed to chloroquine NS series (from P. berghei NS) NS PS, NS PSC, and NS C exposed as above. After various intervals, " 4-day suppressive tests " (3), were carried out on all lines to determine the effective doses of each drug to each line, and the corresponding levels of drug resistance. 3280 -379- W. PETERS RESULTS After 52 weekly passages the following levels of antimalarials were tolerated by parasites of the various NK65 lines in passage, the figures shown being the dose given daily for 6 days of each week: NK65 PS pyrimethamine with sulfadoxine (1: 3 mix- ture), up to 20 mg/kg body weight PSC pyrimethamine with sulfadoxine (1: 3), up to 9 mg/kg+chloroquine phosphate, 4 mg/kg C chloroquine phosphate (since the 12th pass- age), 60 mg/kg The NS lines after 45 passages tolerated the following doses: NS PS pyrimethamine with sulfadoxine (1: 3), up to 25 mg/kg PSC pyrimethamine with sulfadoxine (1: 3), up to 4.5 mg/kg+chloroquine phosphate, 3 mg/kg C chloroquine phosphate (since the 7th pass- age), 60 mg/kg The rate at which the dose could be increased in successive passages is illustrated and summarized in Fig. 1. 10 '1AW~~ At the end of 52 passages the drug responses of the NK65 series in the " 4-day suppressive test " were compared with those of the 45th passage of the NS series. The data obtained are summarized in Table 1. A subsequent check of the response to pyrimetha- mine and to the 1: 3 pyrimethamine/sulfadoxine mixture showed that, by the 63rd passage, the ID90 values of the NK65 series had increased markedly. The values were as follows: NK65 PS NK65 PSC pyrimethamine PS mixture 282 242 1000 400 In order to follow the extent of the changes a final check was made with the " 4-day suppressive test " when the NK65 series had been passaged 67 times and the NS series 57 times. The results are summar- ized in Table 2. At the time of examination of this material, the NK65 PS line passage material was able to tolerate the 1: 3 pyrimethamine/sulfadoxine mixture in daily doses of 25 mg/kg and the PSC line daily doses of 8 mg/kg of the mixture with 3 mg/kg chloroquine phosphate. The NS PS line could toler- ate 35 mg/kg of the mixture, and the PSC line 8 mg/kg mixture with 4 mg/kg chloroquine. PS WA PS _- -NK65 PSC -- _f/ 'N NS PSC N M A M J J TIME SCALE OF R.ES&AGS A S 0 N Fig. 1. Increasing exposure to antimalarial drugs in consecutive passages of P. berghei NS and NK65 strains. C = treatment with chloroquine alone; PS = treatment with a 1:3 pyrimethamine and sulfadoxine mixture; PSC = exposure to a 1 :3 pyrimethamine and sulfadoxine mixture plus increasing dosage of chloroquine. In NK65 and NS PSC lines the maximum dosage of chloroquine reached was 4 mg/kg per day. 380 PREVENTION OF DRUG RESISTANCE Table 1. A comparison of the responses to chloroquine, pyrimethamine, and sulfadoxine in the NK65 and NS series of P. berghei strains in the " 4-day suppressive test" 52nd passage 45th passage Test drug EDao a Resistance lao b EDso a Resistance lao b NK65 NK65 PS NK65 PSC NK65 C NS NS PS NS PSC NS C chloroquine 3.1 0.4 1.0 8.7 40 0.2 0.3 > 2(> LDloo) pyrimethamine 0.4 68 600 1.0 0.12 60 9.2 1.7 sulfadoxine 0.5 29 18 0.3 0.22 118 22 0.9 a mg/kg daily x 4 b 190 = 90 % effective dose for resistant line 90 % effective dose for parent strain (Note: NS parent line is maintained under chloroquine selection pressure) Table 2. Responses to pyrimethamine and sulfadoxine in the 67th passage of the NK65 series and 57th passage of the NS series of P. berghei strains in the " 4-day sup- pressive test" 67th passage 57th passage Test drug EDso Resistance Io EDso Resistance Io NK65 NK65 PS NK65 PSC NS NS PS NS PSC pyrimethamine 2.6 104 > 280 0.1 210 105 sulfadoxine 0.13 460 230 0.04 > 2500 1250 DISCUSSION These data may be interpreted as follows. Firstly, it is clear that resistance to chloroquine when used alone develops very rapidly in the NS line but more slowly in the NK65 strain of P. berghei. Never- theless, in both, a high level of resistance is attained rapidly. Resistance to pyrimethamine and to a sulfo- namide, when used alone, also develops rapidly. For comparison, this is illustrated in Fig. 2, which summarizes data from earlier work (1, 4). The rate at which resistance develops to a combination of pyrimethamine when given together with chloro- quine differs little from that at which resistance develops to pyrimethamine alone (P and CP in Fig. 2). In contrast, chloroquine slows the develop- ment of resistance to sulfonamide when the two are given together (00 and CO in Fig. 2), but resistance to the latter still reaches a high level. Pyrimethamine and sulfonamides only act additively with chloro- quine. However, pyrimethamine and sulfonamides potentiate each other's action. It was hoped that the exposure of P. berghei to the triple mixture of pyrimethamine, sulfadoxine, and chloroquine would slow down the rate at which the parasites became resistant to these three com- pounds. When we examine Fig. 1 and the data show- ing the daily doses of these compounds that could be tolerated by the various lines, it is apparent that this is the case, especially for chloroquine. However, while tolerance to the potentiating mixture of pyrime- thamine with sulfadoxine does increase, it does so more slowly than would resistance to the individual components if given alone. Reference to the data in Table 1 and Table 2 reveals a marked difference between the changes in response to pyrimethamine between the NK65 and the NS lines. Contrary to expectation, resistance developed to a much higher level in the NK65 PSC than in the NK65 PS line, whereas the reverse situation applied to the NS PSC and NS PS lines. In the latter case resistance devel- oped to a lower level in the NS PSC line exposed to the triple drug mixture. No such discrepancy is evident when we examine the data for sulfadoxine 381 W. PETERS 1000 £ C)C] E W 0 xJ a .2 o- .00 .CP .- %~~~~~~~~~J,JII-%J TIME SCALE OF PASSAGES (months) P/CP Fig. 2. Increasing exposure to antimalarial drugs in consecutive passages of P. berghei NK65. 00 = exposure to sulfaphenazole alone; CO = exposure to chloroquine plus sulfaphenazole (maximum dose of chloroquine reached was 5 mg/kg per day); P = exposure to pyrimethamine alone; CP = exposure to chloroquine plus pyrimethamine (maximum dose of chloroquine reached was 8 mg/kg per day). in Table 1, where the anticipated result was obtained, the triple mixture protecting the sulfonamide to some degree. However, continuing passage under drug pressure permitted a build-up of resistance to the sulfonamide to a high level in all four strains (Table 2), especially in those of the NS series. The relative resistance of PS and PSC lines was retained, but the overall level of resistance of the NS lines now exceeded that of the NK65 lines. (Some vari- ation is observed in the ED90 levels of the parent lines between Table 1 and Table 2. This is probably accounted for by unavoidable variations in the p-aminobenzoic acid and folic acid contents of the " standard " diet on which the test mice were fed on the two different occasions.) Outstanding in these observations is the manner in which P. berghei has retained its sensitivity to chloroquine in even the most recent passages of the NK65 PS, NK65 PSC, NS PS, and NS PSC lines. In the last two cases the parasites have actually regained some degree of sensitivity to chloroquine as compared with the parent NS line. It will be noted, too, that the NS line in both its 45th and 57th pas- sages is more sensitive than the NK65 line to pyri- methamine and to sulfadoxine. Moreover, the lines that were exposed to chloroquine alone and became highly resistant to this compound (i.e., NK65 C and NS C) retained their sensitivity to both pyrimeth- amine and sulfadoxine. On the basis of the experiments described here, it cannot be claimed that the long-term use of a mixture of pyrimethamine, sulfadoxine, and chloro- quine (PSC) does more to prevent P. berghei becom- ing resistant to the first two components than does the long-term use of a simple mixture of pyrimeth- amine with sulfadoxine, although chloroquine is " protected " from the development of resistance by the PSC mixture. In neither case is the development of resistance to pyrimethamine or sulfadoxine pre- vented, although the triple mixture does seem to be somewhat better in this respect, with the notable exception of the NK65 PSC line (noted above). We have shown previously (see Fig. 2) that the simple mixture of pyrimethamine with chloroquine also " protects " chloroquine to some degree (1), but that the mixture of chloroquine with a sulfonamide pro- vides some measure of protection to both com- ponents (4). Clearly no general rule can be laid down for the use of drug mixtures. Before recom- mending the administration of mixtures to " pro- 382 I PREVENTION OF DRUG RESISTANCE 383 tect" promising new antimalarials, such as the quinolinemethanols and phenanthrenemethanols that are currently undergoing clinical trial, it would be advisable to carry out further long-term experiments of this nature to see what happens, at least in a rodent malaria model, over some 50 or more pas- sages. It will also be necessary, of course, to investi- gate carefully whether new mixtures alter the levels of tolerability or toxicity of the various components to the host. ACKNOWLEDGEMENTS This study was supported in part by WHO and financial assistance was also received from the US Army Medical Research and Development Command through the European Research Office under Contract DAJA37-73-C-0576 and from CIBA-GEIGY Ltd., Basle, Switzerland. This paper is contribution No. 1291 from the US Army Research Program on Malaria. The author acknowledges the constant technical help in particular of Mrs J. H. Portus and Mr B. L. Robinson. RISUMI PREVENTION DE LA PHARMACORESISTANCE DANS LE PALUDISME DES RONGEURS PAR L'EMPLOI D'ASSOCIATIONS MEDICAMENTEUSES La presente recherche a 6t6 menee afin de comparer dans quelle mesure les stades asexues intraerythrocytaires du parasite du paludisme des rongeurs, Plasmodium berghei, acquierent une resistance A la pyriin6thamine, a la sulfadoxine et A la chloroquine administrees en combinaisons variables. L'information obtenue serait utile en vue de d6cider de l'emploi 6ventuel d'associations medicamenteuses de ce genre dans les campagnes anti- paludiques de masse. L'apparition de la resistance a la chloroquine dans le paludisme des rongeurs est inhib&e si l'on administre ce medicament en association avec de la pyrim6thamine et de la sulfadoxine, mais la pharmacor6sistance a I'egard de ces deux derniers composes continue a se d6velopper. II conviendrait d'etudier la valeur des associations m6di- camenteuses en tant que moyen de z proteger* la chloro- quine ou de nouveaux schizonticides utilisables dans la chimiotherapie de masse contre le paludisme humain. Aucune regle g6nerale ne pourra etre definie a cet egard tant qu'on n'aura pas essay6 diverses combinaisons de medicaments au cours de recherches de longue duree sur un modele experimental comme le paludisme des rongeurs. REFERENCES 1. PETERS, W. ET AL. Ann. trop. Med. Parasitol., 67: 143-154 (1973). 2. PETERS, W. ET AL. Ann. trop. Med. Parasitol., 64: 41-51 (1970). 3. PETERS, W. Exp. Parasitol., 17: 80-89 (1965). 4. PETERS, W. Nature, London, 223: 858-859 (1969).