Plasmodium falciparum: mefloquine resistance produced in vitro*

Bo Izati 6 ec4erc31 Bulletin of the World Health Organization, 62 (3): 433-438 ( 1984) Plasmodium falciparum: mefloquine resistance produced in vitro* C. LAMBROS' & J. D. NOTSCH2 Camp and Smith strains of the human malaria parasite Plasmodium falciparum became resistant to mefloquine after continuous cultivation in thepresence of the drug. The 50% inhibitory dose (ID50) valuesfor mefloquine, as assessed by [3H]hypoxanthine incorp- oration, were found to have increased 4-fold, from 3 1¶g/l to 12 Ag/l. The ID5o values obtained by morphological examination of the cultures increased 10-fold. Resistance was stable in both strains either when grown in a drug-free medium or when keptfrozen in liquid nitrogen. The mefloquine-resistant Camp strain remained sensitive to chloroquine and amodiaquine, and became slightly more resistant to quinine; there was increased sensitivity to pyrimethamine. The mefloquine-resistant Smith strain remained sensitive to amodia- quine and resistant to pyrimethamine; there was increased resistance to quinine, and an increase in sensitivity to chloroquine. Malaria continues to be one of the most widespread diseases in the world. Since 1974 there has been a re- surgence of malaria in areas such as Central and South America, and south and south-east Asia where the disease had seemed to be declining (1). A major problem in malaria control is the development of drug-resistant strains of Plasmodium falciparum. Chloroquine resistance exists now in Latin America and Asia (2-5) and is developing in Africa (6, 7). Resistance has also appeared against quinine (8) and the antifolates (9). Mefloquine, DL-erythro-2,8-bis(trifluoromethyl)- ca-(2-piperidyl)-4-quinolinemethanol hydrochloride (WR 142 490), is the only drug (at present available for experimental tests only) for the treatment and pro- phylaxis of multidrug-resistant falciparum malaria in man (10). Potential mefloquine resistance in fal- ciparum malaria has to be considered since resistance can be induced in strains of the rodent malaria para- * From the Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC 20307, USA. The opinions or assertions expressed in this article are the views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Requests for reprints should be addressed to Dr C. Lambros. Research Parasitologist. 2 Research Technician. site, P. berghei (11-12). Emergence of human strains resistant to mefloquine would therefore lead to seri- ous clinical and public health problems. Recently, a patient in Thailand with RII resistant falciparum malaria was identified (13). We examined the induction of resistance to meflo- quine in vitro in two strains of P.falciparum and determined their respective susceptibilities to other antimalarial drugs. MATERIALS AND METHODS Parasites The multidrug-resistant Viet Nam Smith and the Malayan Camp strains of P.falciparum (14) were used as test organisms. Stock cultures were main- tained as previously described (14). The culture medium consisted of RPMI 1640a supplemented with human A+ erythrocytes and 100o heat-inactivated human A+ plasma. Each culture flask contained a total volume of 5 ml and was flushed with a gas mix- ture of 5%o 02, 5Wo CO2 and 90%7o N2. Stock cultures " Gibco Laboratories, Grand Island, NY, USA. 4415 -433- 434 C. LAMBROS & J. D. NOTSCH were always diluted to parasitaemias of either 0. Wo (4-day growth) or 0.2% (for 3-day growth). Both strains attained final parasitaemias of 2.5-3.5%. Drugs Chloroquine diphosphate, mefloquine hydro- chloride, amodiaquine hydrochloride, quinine sul- fate, and pyrimethamine were obtained from the US Army Drug Development Program Inventory in powder form. Induction of mefloquine resistance Mefloquine was dissolved in 70% ethanol to a concentration of 1 g/l. Subsequent dilutions were then made with RPMI 1640 plus plasma (minus NaHCO3) to achieve stock solutions of 200 to 500 times the required experimental concentrations. The solutions were divided among polypropylene tubes and frozen at - 20 'C. To produce resistance, both strains were con- tinuously exposed to a starting mefloquine concen- tration of 5 yg/l. Culture medium containing the drug was changed daily and the parasites were sub- cultured with non-infected erythrocytes every 3-4 days. At the point where growth of the exposed tines attained the same level as those of the non-exposed parent lines, as determined by Giemsa-stained blood films, medium containing progressively higher con- centrations (10, 15 and 20 ig/l) of mefloquine was added to the cultures. The mefloquine-resistant (MR) strains were unaffected by the concentration of mef- loquine (13.5 yg/l) that is lethal to the parent strains. Drug susceptibility tests Two approaches were taken to determine drug sensitivities. One was the radioisotopic method of Desjardins et al. (14, 15), using incorporation of [G_3HJhypoxanthineb by the parasites as the index of parasite viability and antimalarial activity. Chloro- quine, mefloquine, amodiaquine, and quinine were dissolved in 70%7o ethanol to a concentration of 1 g/l; pyrimethamine was dissolved in a 50/50 (vol/vol) mixture of dimethyl sulfoxidec and ethanol. Sub- sequent dilutions were made with complete culture medium. Each drug was serially diluted 2-fold in the microtitration plates for a total of seven concen- trations over a 64-fold range. The final red blood cell suspension was 1 0o and the parasitaemia at the start of the experiment was 0.4-0.60o. In this test system the 50%o inhibitory dose (ID5o) is defined as the drug concentration corresponding to 50%lo inhibition of the uptake of radiolabelled hypoxanthine by the parasites New England Nuclear, Boston, MA, USA. ' igma Chemical Co., St. Louis, MO, USA. as analysed by nonlinear regression analysis (15). Because our preliminary studies indicated a lack of correlation between hypoxanthine uptake and para- sitaemia in the MR-strains versus mefloquine, a microscopic method to assess antimalarial activity was carried out. To the first two wells of a micro- titration plate were added 200 A1 of complete culture medium without drug; the remaining ten wells re- ceived 200 Al of culture medium containing a graded series of five drug concentrations (two wells for each drug concentration). Ten microlitres of a 50Wo sus- pension of parasitized erythrocytes were then added to all of the wells. Cultures that had been maintained in the presence of mefloquine were grown in drug-free medium for 48 h prior to introduction into the micro- titration wells. The final erythrocyte suspension was 2.38% and the parasitaemia at the start of the experi- ment was 0.4-0.60%o. After slight agitation to ensure uniform settling of the erythrocytes, the plates were placed in an airtight box, flushed with a gas mixture of 507o 02, 5%7o C02, and 90% N2, and placed in an incubator at 37 °C for a 48-h period with regener- ation of the gas phase after 24 h. The ID50 values for the antimalarial drugs tested were determined by nonlinear regression analysis after counting the number of parasites per 10 000 erythrocytes at the end of the 48-h growth period. The ID50 in this test system is defined as the drug concentration producing 50% reduction of parasite growth as compared with the un- treated controls. RESULTS The time course of development of resistance to different concentrations of mefloquine in the two strains is illustrated in Fig. 1. After one month of con- 100 80 MEFLJOUINE 80 ( s/l _j 40 CD CAMP o s 233,~~~~~~~~~~~~~~~~~~~~2 ~ 0 5 s-a2l20 5 404 X BCD X100 80 60- 40- SMITH20- 0 5 10 15 20 25 30 35 40 45 TIME (days) Fig. 1. Time course of resistance to mefloquine in two strains of P. falciparum in continuous culture. Parasit- aemias of both parent lines ranged from 2.5% to 3.5%. Percent control represents the ratio of the growth of the drug-exposed line to the original culture line. MEFLOQUINE RESISTANCE OF PLASMODIUM FALCIPARUM tinuous culture in the presence of 5 Ag/l of drug, com- parable growth was achieved between the experi- mental lines and the strains from which they had been derived. Increasing the mefloquine concentrations to 10, 15, and then 20 ftg/l produced a similar course of development of resistance at each successive concen- tration in both strains. Microscopic examination of the cultures revealed the presence of pigment. The in vitro response of the Camp and MR-Camp strains to several antimalarial compounds, assessed by both radiolabelled hypoxanthine incorporation and microscopic examination, is presented in Table 1. The MR-Camp strain retained its susceptibility to chloroquine and amodiaquine, and became slightly more resistant to quinine. Most surprising was the increased sensitivity to pyrimethamine. Differences in Table 1. Comparative susceptibilities of normal Camp strains and mefloquine-resistant Camp strains of P. falciparum against antimalarial drugs ID50 (tgll)' Ratio of IDso of MR-Camp Camp MR-Camp" to ID0 of parent Camp Drug 3H ME' 3H ME 3H ME Chloroquine 8.5 6.1 9.6 10.9 1.1 1.8(0.78)d (0.32) (0.88) 11.2) Mefloquine 4.9 3.4 12.0 34.0 2.4 10.0 (1.02) (0.32) (1.9) (1.5) Amodiaquine 8.1 3.7 7.4 5.2 0.91 1.4 (5.0) (0.14) (2.0) (0.94) Quinine 22.0 31.0 65.0 69.0 2.9 2.2 (3.2) (3.6) (4.1) (5.4) Pyrimethamine 505.0 325.0 28.0 13.0 0.05 0.04 (59.0) (49.0) (3.8) (2.6) a Mean of 4 determinations. b Parasites used were from those grown in 20 ug/l mefloquine. c 3H, radiolabeled hypoxanthine; ME, microscopic examination. d Numbers in parentheses are the standard error of the nonlinear regression analysis. Table 2. Comparative susceptibilities of normal Smith strains and mefloquine-resistant Smith strains of P. falciparum against antimalarial drugs ID50 (Ag1l)' Ratio of ID50 of MR-Smith Smith MR-Smithb to ID5o of parent Smith Drug333DH' ME' 3H ME 3H ME Chloroquine 68.0 58.0 24.0 23.0 0.35 0.39 (0.42) (2.4) (1.7) (2.5) Mefloquine 3.5 3.9 12.0 36.0 3.4 9.3 (0.16) (0.41) (1.2) (1.6) Amodiaquine 9.2 5.7 6.2 10.2 0.67 1.8 (4.8) (2.9) (2.0) (0.44) Quinine 55.0 48.0 193.0 234.0 3.5 4.8 (5.7) (3.2) (15.0) (12.0) Pyrimethamine > 1355 > 1000 > 1355 > 1000 a Mean of 4 determinations. b Parasites used were from those grown in 20 ag/l mefloquine. c 3H, radiolabeled hypoxanthine; ME, microscopic examination. d Numbers in parentheses are the standard error of the nonlinear regression analysis; minus sign indicates that the value is out of the test range. 435 C. LAMBROS & J. D. NOTSCH ID50 values for mefloquine versus MR-Camp ob- tained by the two methods were noted (Table 1): microscopic determinations gave higher IDso values (34 Ag/l) than did the radiolabelled hypoxanthine procedure (12 Ag/l). Table 2 presents the response of the Smith and MR-Smith strains to antimalarial compounds. The MR-Smith strain retained susceptibility to amodia- quine and resistance to pyrimethamine; resistance to quinine was increased while sensitivity to chloroquine was increased. Differences were noted in the ID50 values obtained by the two methods for mefloquine versus MR-Smith (Table 2): ID50 values obtained by morphological examination (ID50 = 36 ig/l) were greater than those obtained with hypoxanthine (ID50 = 12 Ag/l). When both strains were removed from drug pres- sure (mefloquine concentration = 20 Ag/l) and grown in drug-free medium, the resistance to mefloquine was stable for up to six months and their respective sensitivities to pyrimethamine and chloroquine were retained. Resistance was also stable in both strains after storage in liquid nitrogen for six months (data not shown). DISCUSSION Our approach of continuous exposure of parasites to mefloquine to produce resistant lines in vitro is similar to that used by Nguyen-Dinh & Trager (16) for inducing resistance to chloroquine but differs from that of Brockelman et al. (17), who used dis- continuous exposure to the drug. This study indicates that P.falciparum has the genetic capability of developing resistance to mefloquine. Furthermore, because of the changes in drug response demonstrated by our resistant strains (increased susceptibility to either chloroquine or pyrimethamine), it is possible that compounds whose efficacies have decreased over the years owing to development of drug resistance may again be of value. Peters et al. (11) have shown that P. berghei can easily develop resistance to mefloquine, especially if the parasites were already resistant to chloroquine. In our studies with P.falciparum, mefloquine resistance came about more quickly in the chloroquine-sensitive strain than in the chloroquine-resistant one (Fig. 1). Drug susceptibility studies by Kazim et al. (12) and Merkli et al. (18) with mefloquine-resistant P. berghei (derived from a chloroquine-sensitive parent line) showed a continued sensitivity to chloroquine, amodiaquine, and pyrimethamine but an increased resistance to quinine. On the other hand, Ager (personal communication) has observed that chloro- quine-resistant P. berghei was cross-resistant to mef- loquine and mefloquine-resistant parasites exhibited cross-resistance to chloroquine. In none of the P. ber- ghei studies was there evidence for collateral sus- ceptibility, i.e., increased susceptibility to one drug concomitant with development of resistance to another. Some evidence for this was shown by our MR-strains. MR-Camp became more sensitive to pyrimethamine than was the parent strain, and the MR-Smith strain became more sensitive to chloro- quine; both showed increased resistance to quinine in varying degrees. Collateral susceptibility has been observed in trypanosomatids (19) where isomet- amidium and oxophenarsine had greater activity against clones of a Leptomonas resistant to acriflav- inium, quinapyramine, diminazene, stilbamidine, and pentamidine than the parent strain. The different results between the two methods for the determinations of ID5o for mefloquine versus MR-Camp and MR-Smith strains suggest that the change that occurred in these strains may be of a bio- chemical nature that limits the use of radiolabelled hypoxanthine as an index of parasite viability. On the other hand, the erythrocyte suspension used in the morphological assay was 2.38 times that of the radio- isotopic assay which may indicate that much more mefloquine might be needed to exert parasite inhi- bition though the ID50 values of the other anti- malarials tested were generally in agreement between the two methods. In summary, we have demonstrated that strains of P.falciparum developed resistance to mefloquine in vitro and that the resistance appears to be stable in the absence of drug pressure. It is now possible to per- form chemotherapeutic analysis and to investigate the mechanisms of drug resistance in malaria by com- parisons of resistant strains and the sensitive strains from which they were derived. ACKNOWLEDGEMENTS The authors wish to thank Dr G. J. McCormick, Dr W. A. Reid, Dr C. J. Canfield and Dr J. D. Haynes for suggestions made during the preparation of the manuscript. They also thank the staff of the Walter Reed Blood Donor Center for providing plasma. This paper has been designated as contribution no. 1678 to the Army Research Program on Antiparasitic Drugs. 436 MEFLOQUINE RESISTANCE OF PLASMODIUM FALCIPARUM 437 RESUME PLASMODIUM FALCIPARUM: RESISTANCE INDUITE IN VITRO A LA MEFLOQUINE Le paludisme reste une des maladies les plus repandues dans le monde. Un des problmes cruciaux de la lutte anti- paludique est l'apparition de souches de Plasmodium falci- parum resistantes aux medicaments. La mefloquine (WR 142 490) est le seul medicament recent dont on dispose actuelle pour le traitement et la pro- phylaxie du paludisme humain A falciparum polypharma- coresistant. Une resistance potentielle de ce parasite A la mefloquine doit etre envisagee vu qu'une resistance induite est possible chez des souches de P. berghei, agent du palu- disme des rongeurs. L'induction d'une mefloquino-resis- tance a et etudiee in vitro chez deux souches de plasmodies et leur sensibilite respective A d'autres antipaludeens a et mesuree. On a employe comme organismes d'epreuve des souches multiresistantes, la souche Camp de Malaisie et la souche Smith du Viet Nam. Le milieu de culture etait du RPMI 1640 supplemente d'erythrocytes humains A+ et de 10%o de plasma humain A+ inactive par la chaleur. Chaque flacon de culture contenait au total 5 ml et l'on y a fait circuler un courant gazeux constitue de 5%7o de 02, 5%o de CO2 et 90%7o de N2. Pour l'etude de la resistance, la mefloquine a d'abord et mise en solution dans l'ethanol A 700%o puis diluee avec le milieu de culture (sans NaHCO3) de facon A obtenir des solutions-meres 200 A 500 fois plus concentrees que la solu- tion necessaire aux experiences. La solution finale a e re- partie en tubes de polypropylene et congelee A -20 'C. Pour induire la resistance chez les deux souches exposees en permanence A une concentration de mefloquine, on les a d'abord fixees A 5 Lg/l. Quand les lignees exposees at- teignaient un niveau de developpement semblable A celui de la lignee parentale non exposee, les concentrations de medi- cament dans le milieu etaient augmentees progressivement (10, 15 et 20,ug/1). Deux methodes ont et employees pour determiner la pharmacosensibilite. La premiere, la methode radioisoto- pique, utilise comme indicateur de 1'activite antipaludique l'incorporation d'hypoxanthine tritiee [G-3H] par le para- site. La seconde, la methode microscopique, consiste a compter le nombre de parasites pour 10 000 erythrocytes apres 48 heures de culture. Les medicaments soumis a l'epreuve sont la chloroquine (diphosphate), la mefloquine (chlorhydrate), l'amodiaquine (chlorhydrate), la quinine et la pyrimethamine (sulfate). Dans les deux methodes, la dose inhibitrice mediane (DI5o) de chaque medicament a et calculee par regression non lineaire. Apres un mois de culture ininterrompue en presence de 5 Ag/l de mefloquine, on a obtenu un developpement comparable des lignees experimentales et des souches dont elles etaient issues. L'augmentation de la concentration a 10, 15 puis 20 yg/l de mefloquine a provoque l'accroisse- ment de la resistance chez les deux souches, dans des temps comparables. On a constate pour les deux souches que la D15o mesur6e au moyen de l'hypoxanthine marquee avait et multipli6e par 4, alors qu'elle avait decuple d'apres l'examen morpho- logique. 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