Plasmodium falciparum: Induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro

EXPERIMENTALPARASITOLOGY

67,354-360(1988)

Plasmodium falciparum: Induction of Resistance to Mefloquine Cloned Strains by Continuous Drug Exposure in Vitro AYOADE

W. K. MILHOUS, *Al N. F. WEATHERLY,* AND R. E. DESJARDINS~~

M. J. ODUOLA,*“,~

in

J. H. BowDRE,t

*Department of Parasitology and Laboratory Practice, School of Public Health, and TDepartment of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, 27514, U.S.A., and SWellcome Research Laboratories, Research Triangle Park, North Carolina, 27709, U.S.A.

Accepted for publication 18 August 1988 ODUOLA,A.M.J.,MILHOUS, JARDINS, R. E. 1988.

W.K.,

WEATHERLY,

N.F.,BowDRE,J.H.,ANDDEs-

Falciparum: Induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro. Experimental Parasitology 67,354-360. A genetically homogeneous population of Plasmodium fulciparum prepared by a single erythrocyte micromanipulation technique was used to produce lines of P. falciparum resistant to mefloquine hydrochloride in vitro. Parasites were maintained in a culture medium Plasmodium

containing gradually increased concentrations of mefloquine hydrochloride (CMP-men starting with 2 @ml. One of the mefloquine-resistant culture lines (Wt-mef) was obtained after 96 weeks of continuous culture in CMP-mef, the last 4 weeks in medium containing 40 r&ml of mefloquine hydrochloride. The W2-mef was four to six times more resistant to mefloquine than was the parent clone W2. Means of multiple determinations of 50% inhibitory concentrations (IC-50) of mefloquine hydrochloride against WZ-mef and clone W2 were 20.39 + 5.08 rig/ml and 4.50 2 1.94 r&ml, respectively. o 1988 Academic press, hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Plasmodium falciparum; Protozoa, parasitic; Malaria; Parent clone W2; Mefloquine-resistant culture line (WZ-mef); Culture medium with plasma (CMP); Culture medium containing mefloquine hydrochloride (CMP-meQ; Lactate dehydrogenase (LDH); Glucose phosphate isomerase (GPI); Exponential growth rate (R); 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes); Fresh Frozen Human Plasma (FFP). INTRODUCTION

The rapid spread of drug-resistant populations of Plasmodium faiciparum raises important questions concerning development of new drugs and the genetic basis of drug resistance in human malaria. Chloroquine resistance in rodent models appears to follow a classic Mendelian type of inheritance (Padua 1981; Rosario 1976). How’ Present address to which correspondence and reprint requests should be addressed: Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20307. * To whom correspondence and reprint requests should be addressed. 3 Present address: American Cyanamid Corp., Lederle Laboratories, Pearl River, NY 10965.

ever, little is known about the genetic basis of P. fulciparum resistance to either chloroquine or mefloquine, the latter being the newest antimalarial drug for treating chloroquine-resistant parasites. The use of a mefloquine-resistant strain produced from a genetically homogeneous sensitive strain may provide a model for studying the mechanism and evolution of mefloquine resistance in P. falciparum. In the present report a genetically homogeneous population of P. falciparum prepared by a single erythrocyte micromanipulation technique (Oduola et al. 1988) was used to produce lines of P. falciparum resistant to mefloquine hydrochloride in vifro. The use of a cloned strain undergoing continuous drug exposure permitted an initial induction of resistance in the parasite, 354

0014-4894/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

MEFLOQUINE-RESISTANT

P.fulciparum

and subsequent selection of a population with stable induced resistance. Sensitivities of the resistant parasite to mefloquine, halofantrine, empiroline, other experimental antimalarial compounds, and some standard antimalarial drugs were compared with those of the parent clone. MATERIALSANDMETHODS Culture medium. Standard culture medium containing powdered RPM1 1640 supplemented with 32 mM Hepes and 10% (v/v) FFP (CMP) was prepared every 3 to 4 days and stored at 4 C. CMP-mef was prepared by adding predetermined concentrations of a stock solution of mefloquine to the CMP. A sufficient amount of powdered mefloquine hydrochloride was dissolved in 70% ethanol to prepare a stock solution and stored at 4 C. Stock solutions of mefloquine were prepared every 3 weeks and evaluated against reference parasites before being used and at the end of the 3-week period. Parasites and culture conditions. Two cloned strains of Plasmodium falciparum prepared by single erythrocyte micromanipulation were used in the study. Clone W2 and clone Ml were prepared from the same isolates and their characteristics have been described elsewhere (Oduola et al. 1988). Their sensitivities to antimalarial drugs are identical. The parasites were maintained in continuous culture by standard techniques (Trager and Jensen 1976; Haynes et al. 1976). Briefly, an inoculum of the stock culture of each clone was prepared in a 6% suspension of type A+ erythrocytes in CMP and maintained in 50 ml sealed culture flasks (Coming Glass Works, Coming, NY, U.S.A.) as described previously (Oduola et al. 1985). The culture medium was changed daily with CMP, or with culture medium containing predetermined concentrations of mefloquine hydrochloride (CMP-mef). To maintain parasitemias between 0.5 and 2%, each culture was diluted every 2 to 4 days with culture medium containing a suspension of noninfected erythrocytes. Thin blood smears were prepared from each culture prior to dilution, and daily from the “drugexposed” cultures to assess parasite growth rates and microscopic appearance. The exponential growth rates (R) (Oduola et al. 1985) for each culture line were determined every other day as follows: R=

PRODUCED

355

VitrO

sion of human type A + erythrocytes. Continuous exposure of the parasites to mefloquine was initiated by replacing the culture medium in one culture line (drugexposed culture) with CMP-mef containing 2 @ml of mefloquine, a concentration equivalent to the 10% inhibitory concentration (IC-10) of the drug against clone W2. Parallel cultures were maintained in CMP without drug as controls. The concentration of mefloquine in the CMP-mef was increased stepwise as the growth rate and microscopic appearance of the “drugexposed” culture became comparable with the control culture. Appearance of morphological degeneration or decreased R in the parasites was promptly followed by reduction of drug concentration until the growth rate became comparable with that of the control and the microscopic appearance was normal. Samples of each culture line were assayed against mefloquine and standard antimalarial drugs every 2 to 4 weeks, and prior to increasing the concentration of mefloquine in the drug-exposed cultures. Subcultures were prepared from the drug-exposed cultures whenever there was a significant decrease in the parasite’s sensitivity to mefloquine. Aliquots of the parasites growing at that drug concentration were frozen and stored at - 70 C or in liquid nitrogen. Isoenzyme patterns GPI and LDH for the parasite population were determined using a cellulose acetate electrophoresis technique (Cuoto et al. 1983) every 8 to 10 weeks, and subsequent to changes in the parasite’s susceptibility to mefloquine. The concentration of mefloquine in CMP-mef at the conclusion of the experiment was 40 &ml. Drug susceptibility testing. In preparation for antimalarial drug susceptibility testing, an inoculum of the drug-exposed culture was diluted with a suspension of noninfected erythrocytes and maintained in culture medium without drug for 36 to 48 hr. The susceptibility of each culture line to standard antimalarial drugs, mefloquine hydrochloride, and the experimental antimalarial drugs (WR 172435; WR 122455; WR 184306; WR 194965; WR 180409, and SW-58C) shown in Fig. 1 was determined using a semiautomated microdilution technique and incorporation of [3H]hypoxanthine (Desjardins et al. 1979). The 50% inhibitory concentrations (K-50) of each compound against the drugexposed culture and the control cultures were analyzed by a nonlinear regression technique (Desjardins et al. 1979; Oduola et al. 1986) and compared.

1nAZ - InA, tz-t,

A,,A, = number of parasites per milliliter at times t, and t,, respectively. Variations in R values represent influence of culture conditions on parasites multiplication and reinvasion of erythrocytes in culture. Induction of resistance. Inocula from continuous stock cultures were prepared in 5 ml of a 6% suspen-

in

RESULTS

The growth rates (R) for clone W2 maintained

in standard

culture

medium

ranged

from 1 to 2 during 2 years of continuous culture. An R value of 2 represents optimal multiplication and reinvasion of erythro-

356

ODUOLA

ET AL. H

H -0 00 oT;1

w

CF3

CF3 EFLOQUINE

- HOCIL$H2S03H UR 122.455

WOFANTRINE

-H3P04

UR 184.806

ENPIROLINE

-CH3SO*OH

UR 172.435

1. Structural configurations of mefloquine, halofantrine, enpiroline (WR 180409), WR 122455, WR 184806, and WR 172435. FIG.

cytes by clone W2 in culture medium supplemented with 10% (v/v) human plasma. Lower R values indicate decrease in parasite multiplication, reinvasion, or both under specified culture conditions. Changes in the growth rates for the drug-exposed culture (clone W2) during the induction experiments are shown in Fig. 2. The parasites grew at a rate similar to that of the control culture (R = 1 to 2.5) during 6 weeks of continuous culture in CMP-mef containing 3 to 9 r&ml of mefloquine hydrochloride. The growth rate decreased gradually to 0.8 when the concentration of mefloquine hydrochloride was increased above 10 &ml. Further decreases were observed with stepwise increases of drug concentration from 10 to 20 rig/ml, but improved as the parasites adapted to grow in CMP-mef containing 20 rig/ml of mefloquine. Growth rates of the parasite fluctuated between 0.9 and 1.8 during the following 32 weeks of continuous culture as the concentration of mefloquine was increased from 20 to 34 rig/ml.

Changes in microscopic appearance of the parasites were first observed when the concentration of mefloquine hydrochloride in CMP-mef was increased from 8.2 to 10 rig/ml, and at subsequent higher drug concentrations. Changes included presence of small vesicles and pigment in parasite cytoplasm of the ring stages. The trophozoites, which were small and compact, also had a diffuse staining cytoplasm. The microscopic appearance of the parasites improved with temporary reduction in drug concentration and after a period of adaptation at the higher drug concentrations. The isoenzyme patterns for the clones exposed to drug and those of subsequent subcultures maintained in medium without drug were identical to those of the control clones that had been maintained in standard culture medium. The control culture and the drug-exposed culture sublines exhibited GPI type I, and LDH type I activities. Clone W2 is resistant to chloroquine, quinine, and pyrimethamine, but sensitive to

P. fUkipUrum

MEFLOQUINE-RESISTANT

619

7121

913

11117 +-I982

115

1128

PRODUCED

in vitro

357

519

1983-t

FIG. 2. Changes in growth rates (H) of a culture line of clone W2, maintained in culture medium containing increasing concentrations (0) of mefloquine hydrochloride over % weeks. Decrease in growth rates and subsequent removal of drug pressure between December 1982 and January 1983 resulted from lack of normal manipulation of parasites due to unforeseen events. Changes in parasites susceptibility to mefloquine hydrochloride were noted on two occasions (*).

mefloquine and halofantrine. The initial responses of the clone to mefloquine hydrochloride, chloroquine diphosphate, quinine sulphate, and halofantrine before the induction experiments were 3.0 ? 0.3 rig/ml, 122.5 ? 5.9 &ml, 133.8 2 9.3 rig/ml, and 1.1 + 0.1 &ml, respectively. Changes in susceptibility to mefloquine in the drugexposed culture occurred in two phases. The clone became significantly less susceptible to mefloquine after 20 weeks of continuous culture in CMP-mef. During this period, the concentration of mefloquine in the culture medium was gradually increased from 2 to 12 rig/ml. At that time when changes in susceptibility occurred, the parasites had been growing for 4 weeks at the higher drug concentration. The mean K-50 for mefloquine against a subculture (WMC-II) prepared at this level of drug exposure was 12.3 + 0.6 &ml. The concentration of mefloquine in the drug-exposed culture line was gradually increased during the following 6 weeks from 12 to 22.5 r&ml without any significant change in the parasite’s susceptibility to mefloquine. Subsequent attempts to increase the drug concen-

tration from 22.5 to 24 rig/ml resulted in a decreased growth rate (R = 0.3, Fig. 2), and degenerated microscopic appearance of the parasites. Consequently, the drug was eliminated and the parasites were maintained in medium without drug for 27 days. During that period, the growth rates and microscopic appearance of the parasites improved (R = 0.9 to 1.2, Fig. 2) and the parasites adapted to grow in medium containing concentrations of mefloquine gradually increased from 15 to 24 r&ml. Although subsequent attempts to increased the drug concentration first to 26 rig/ml and then from 28 to 30 rig/ml resulted in a decreased growth rate, brief reductions in drug pressure were usually followed by successful growth at the higher drug concentration. A second shift in the susceptibility of the drug-exposed culture to mefloquine occurred after the parasites had been growing in medium containing 30 rig/ml of mefloquine for 6 weeks (96 weeks of drug pressure). The IC-50 for mefloquine against a subculture (W2-met) prepared at this level of drug exposure was 26.4 + 1.8 &ml. The

358

ODUOLA

stock drug-exposed parasites continued to grow in medium containing 40 &ml of mefloquine for 4 weeks. Means of multiple determinations of IC50 for mefloquine hydrochloride, halofantrine hydrochloride, WR 172435, WR 122455, WR 184806, WR 194965, and WR 180409 against WZmef determined during 18 months of continuous culturein medium without drug are shown in Table I. The cross-resistance patterns of WZmef to mefloquine and experimental antimalarial compounds are shown in Fig. 3. W2-mef was four to six times more resistant to mefloquine than was the parent clone W2. The K-SOS for halofantrine, WR 122455, WR 172435, WR 180409, WR 184806, and WR 194965 against WZmef were two to three times higher than were those of the control parasites. WZmef was more sensitive to chloroquine (IC-50 = 88.6 + 13.9 rig/ml) than the control clone, but remained resistant to quinine (IC-50 = 186.7 + 36.9 ng/ ml). There were no statistically significant differences in the sensitivities of either culture line to BW-58C (mean IC-50s: clone W2 = 0.88 2 0.4 &ml, WZmef = 1.18 r 0.5 @ml). Clones prepared from the mefloquine resistant line (W2-mef) after 12 months of continuous culture in medium without drug were three to five times more resistant to mefloquine than was clone W2. The mean IC-50s for mefloquine against clone 11801, TABLE I Comparative in Vitro Susceptibilities of Clone W2 and a Mefloquine-Resistant Line (W2-mef) to Mefloquine and Experimental Antimalarial Compounds K-50 Clone Mefloquine Halofantrine Enpiroline (WR 180409) WR 172435 WR 122455 WR 184806 *n=5.

W2

*4.50 + 1.94 1.20 f 0.50 3.28 3.30 3.55 3.13

2 + * f

1.87 1.44 1.69 1.64

+ ISD*

(&ml) W2-mef 20.39 2.85

2 5.08 + 0.85

10.14 6.10 6.78 8.65

f + 2 +

5.33 0.88 0.90 5.62

ET AL.

0

25

250 -

x t

.D .O

150-

loo-

0 . 0 .

: . . ; 8

A8 e 'E '5 a

FIG. 3. Susceptibilities and cross-resistance patterns of (0) clone W2 and (0) a mefloquine resistant line (WZmef) to mefloquine hydrochloride; quinine sulphate; chloroquine diphosphate; halofantrine hydrochloride; WR 180,409; and WR 194,965.

clone 11802, and clone 11804 were 21.38 * 1.56 &ml, 18.87 f 0.6 rig/ml, and 19.9 & 0.9 rig/ml, respectively. After 3 years of continuous culture in medium without drug pressure, WZmef and the clones prepared from the population 2 years ago have remained three to five times more resistant to mefloquine than the unexposed parent culture . DISCUSSION

The results of these experiments indicate that stable resistance to mefloquine can be generated by continuous culture of a previously sensitive clone of Plasmodium fulciparum in culture medium containing increasing concentrations of drug. Identical results were obtained in similar experiments with clone Ml. One of the mefloquine-resistant culture lines (W2-mef) obtained at the conclusion of the induction experiments was four to six times more resistant to mefloquine than was the parent clone. Development of resistance to mefloquine in these parasites differs from other reports in that the use of genetically homogeneous cloned strains of a sensitive clone of P. fulcipurum precludes the possibility of a mixed population from which an initial

MEFLOQUINE-RESISTANT

P.

fUki~UW?l

selection of a resistant line could have occurred. Previous reports on production of parasites with reduced sensitivities to antimalarial drugs in vitro (Lambros and Notsch 1984; Nguyen-Dinh and Trager 1978; Brockelman et al. 1981) have been in heterogeneous parasite populations with components having varied degree of resistance to the drugs used. The use of heterogeneous parasite populations in those reports may well have resulted in a selection of parasite populations with innate resistance to the drug. Although the selected natural drug-resistant populations may represent a product of one mode of drug resistance in nature, they do not provide a reasonable model for determining and evaluating the genetic or biochemical changes that may accompany acquisition of resistance in the parasites. Such parameters are of utmost importance in drug development, especially with regard to structure and activity of new compounds and potential for cross-resistance with currently used antimalarial drugs. To illustrate this point, Trager et al. (1981) selected a chloroquineresistant clone from an isolate of P. fulciparum (FCR-3) which was thought to be sensitive and that had been used earlier to produce a chloroquine-resistant line (Nguyen-Dinh and Trager 1978). Accidental infection of a laboratory worker with the same apparently sensitive isolate after 4 years of continuous culture in vitro also failed standard chloroquine treatment (Jensen et al. 1981). Studies demonstrated that a R-I level of resistance was present and the patient was successfully treated with a combination of quinine sulfate (650 mg three times daily for 10 days), pyrimethamine (Daraprim 25 mg twice daily for 3 days), and sulfadiazine (2000 mg four times daily for 5 days) (Jensen et al. 1981). Similarly, a Viet Nam Smith isolate which resulted in a mefloquine-resistant infection in a volunteer study (Cosgriff et al. 1985) was previously used by Lambros and Notsch (1984) in producing a mefloquine-resistant line by continuous drug exposure in vitro.

PRODUCED

in Vitro

359

Only one previous report on induction of drug resistance in vitro involved the use of a cloned line of P. fulciparum (Banyal and Inselburg 1986) and, in that study, mutagenic agents were used to facilitate induction of resistance to pyrimethamine. Although the mechanism(s) involved in the development of resistance to mefloquine in these clones has not been delineated, the assumption is that genetic changes in the parasites are involved. It is evident that stable resistance was not obtained until after an extended period of time, but changes in the population of the parasites may have occurred during the early period. It is possible that changes in the chromosomes conferring mefloquine resistance occurred in the parasite population during the initial drug exposure and a population with the induced resistance was subsequently selected by the continued drug pressure. The hallmark of changes in sensitivity of the parasites to mefloquine strongly suggest that genetic changes preceded final selection in the process. The ability of the parasite population at certain points during the experiments to grow in the presence of high levels of drug with minimal changes in the parasite’s sensitivity to mefloquine supports this observation. The high level of resistance to mefloquine in the parasites was accompanied by cross-resistance of varying degrees to some experimental antimalarial compounds (Fig. 3 and Table I). There appears to be a consistent decrease in susceptibility of the parasites to halofantrine and to a group of other experimental antimalarial compounds developed at the Walter Reed Army Institute of Research. The increased resistance to halofantrine, however, may not be of any clinical significance since its IC-50 against the parasites is only slightly increased and may still be within a therapeutic level. The parasites which were originally resistant to chloroquine and quinine showed statistically significant increase in sensitivity to chloroquine, but remained resistant to quinine after acquisition of resistance to

360

ODUOLA

mefloquine. Although the WZmef line is more sensitive to chloroquine, its K-50 remained within a range for parasites with a known clinical chloroquine resistance profile. Susceptibility of the W2-mef line to chloroquine is also increased by simultaneous exposure to chloroquine and verapamil, a phenomenon so far only observed in chloroquine-resistant parasites (Martin et al. 1987). The availability of this mefloquineresistant line and the parent sensitive clone provides an important resource for antimalarial drug testing, and for evaluating the mechanisms of action of mefloquine. In addition, it will be valuable for studying the genetic processes involved in acquisition of resistance, and for studies of crossresistance among candidate antimalarial compounds and mefloquine . ACKNOWLEDGMENTS This investigation received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. We are grateful to Dr. Jim Lovelace and Dr. Dennis Kyle for review of the manuscript, and to Mr. Bruce Alexander for technical assistance. We thank Mrs. Julia Hardy for her help in the preparation of the manuscript. REFERENCES BANYAL, H. S., AND INSELBURG, J. 1986. Plasmodium falciparum: Induction, selection, and characterization of pyrimethamine-resistant mutants. Experimental Parasitology 62, 61-70. BROCKELMAN, C. R., MONKOLKEHA, S., AND TANARIYA, P. 1981. Decrease in susceptibility of Plasmodium falciparum to mefloquine in continuous culture. Bulletin of the World Health Organization 59, 249-252. COSGRIFF, T. M., PAMPLIN, C. L., CANFIELD, C. J., AND WILLET, G. P. 1985. Mefloquine failure in a case of falciparum malaria induced with a multidrugresistant isolate in a non-immune subject. American Journal of Tropical Medicine and Hygiene 34, 692693. CUOTO, A., ROSARIO, V. E., AND WALKER, D. 1983. Analise enzimatica de 56 amostrad de Plasmodium falciparum de Bacia Amazonia (Brazil). Malarial. D. Trop. 35, 11-19. DESIARDINS, R. E., CANFIELD, C. J., HAYNES, J. D., AND CHULAY, J. D. 1979. Quantitative assessment

ET AL.

of antimalarial activity in vitro by semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710-718. HAYNES, J. D., DIGGS, C. L., HINES, F. A., AND DESJARDINS, R. E. 1976. Culture of human malaria parasites Plasmodium falciparum. Nature (London) 263,767-769. JENSEN, J. B., THOMAS, C. C., AND CARLIN, J. M. 1981. Clinical drug-resistant falciparum malaria acquired from cultured parasites. American Journal of Tropical Medicine and Hygiene 30, 523-525. LAMBROS, C., AND NOTSCH, J. D. 1984. Plasmodium falciparum: Mefloquine resistance produced in vitro. Bulletin of the World Health Organization 62, 433-438. MARTIN, S. K., ODUOLA, A. M. J., AND MILHOUS, W. K. 1987. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235, 899-901. NGUYEN-DINH, P., AND TRAGER, W. 1978. Chloroquine resistance produced in vitro in an African strain of human malaria. Science 200, 1397-1398. ODUOLA, A. M. J., ALEXANDER, B. M., WEATHERLY, N. F., BOWDRE, J. H., AND DESIARDINS, R. E. 1985. Use of non-human plasma substitute in cultivation and antimalarial drug susceptibility studies with Plasmodium falciparum in vitro. American Journal of Tropical Medicine and Hygiene 34, 20% 215. ODUOLA, A. M. J., MILLER, R., AND MILHOUS, W. K. 1986. Standardized in vitro cultivation and drug susceptibility testing of Plasmodium falciparum in epidemiology and malaria control. In “Proceedings, Ninth International Congress of Infectious and Parasitic Diseases, Munich, W. Germany.” ODUOLA, A. M. J., WEATHERLY, N. F., BOWDRE, J. H., AND DESIARDINS, R. E. 1988. Plasmodium falciparum: Cloning by single-erythrocyte micromanipulation and heterogeneity in vitro. Experimental Parasitology 66, 86-95. PADUA, R. A, 1981. Plasmodium chabaudi: Genetics of resistance to chloroquine. Experimental Parasitology 52, 419-426. ROSARIO, V. E. 1976. Genetics of chloroquineresistance in malaria parasites. Nature (London) 261, 585-586. TRAGER, W., AND JENSEN, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 674675. TRAGER, W., TERSHAKOVEC, L., LYANDVER, T. L., STANLEY, H., LANNERS, N., AND GUGERT, E. 1981. Clones of the malaria parasite Plasmodium falciparum obtained by microscopic selection: Their characterization with regard to knobs, chloroquine sensitivity and formation of gametocytes. Proceedings of the National Academy of Sciences, U.S.A. 78, 65274530.