Quantitative Assessment of Antimalarial Activity in Vitro by a Semiautomated Microdilution Technique ROBERT E

ANTIMICROBIAL AGENTS AND CHEmOTHEzRAY, Dec. 1979, p. 710-718 Vol. 16, No.6 0066-4804/79/12-0710/09$2.00/0 Quantitative Assessment of Antimalarial Activity In Vitro by a Semiautomated Microdilution Technique ROBERT E. DESJARDINS,t* CRAIG J. CANFIELD,' J. DAVID HAYNES,2 AND JEFFREY D. CHULAY2 Department ofPharmacology' and Departnent ofImmunology,2 Walter Reed Army Institute ofResearch, Washington, DC 20012 Received for publication 10 September 1979 A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microti- tration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed. The recent development of methods for the tem is partially automated for rapid analysis of continuous in vitro culture ofthe human malaria the data. parasite Plasmodium falciparum (4, 21) pro- vides a valuable resource for the investigation of MATERIALS AND METHODS new approaches to malaria chemotherapy. Di- Preparation of parasites. The parasite inocula rect measurement of the antimalarial activity of used in these experiments consisted of two isolates of potential new drugs against parasites grown in P. fakciparum. The African Uganda I strain was orig- vitro is now feasible. inally obtained by Martin and Arnold from a child in Kampala, Uganda, in 1965 (9). It has been widely used Resistance of P. fakciparum to chloroquine is in phase II clinical studies in human volunteers and an important consideration in the treatment and has retained its susceptibility to chloroquine, quinine, prevention ofmalaria in many parts ofthe world, and pyrimethamine. The Vietnam Smith strain, ob- including South and Central America (12), tained in 1969 from an American soldier at the Walter Southeast Asia (25), India (20), and the African Reed Army Medical Center, was first reported by continent (6). It is therefore essential to use well- Craig J. Canfield in 1971 (2). That patient, showing an characterized susceptible and resistant strains of R-I level of resistance to the combination of sulfalene the parasite in the evaluation of potential new and trimethoprim after treatment failures with other chemotherapeutic agents to detect cross-resist- antimalarial drugs, provided the parasite isolate des- standard anti- ignated Smith strain (2). This strain was later adapted ance with chloroquine and other to owl monkeys (24) and has also been used extensively malarial drugs. in human volunteer studies, where its resistance to The method described in this report can pro- chloroquine, quinine, and pyrimethamine has been vide quantitative measurements of the antima- amply documented (10). larial activity of large numbers of compounds, The two strains of parasites were grown continu- based on the inhibition of uptake of a radiola- ously in stock cultures by a modification of the meth- beled nucleic acid precursor by the parasite dur- ods of Trager and Jensen (21) and Haynes et al. (4). A ing short-term cultures in microtitration plates. 6% suspension of human type A+ erythrocytes was The parasites used are continually available prepared in culture medium which consisted of pow- The dered RMPI 1640 (GIBCO Laboratories, Grand Island, from long-tenn maintenance cultures. sys- N.Y.) diluted in sterile water with 25 mM HEPES (N- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; t Present address: Wellcome Research Laboratories, Re- Calbiochem, La Jolla, Calif.), 32 mM NaHCO3 search Triangle Park, NC 27709. (GIBCO), and 10% heat-inactivated (40 min at 56°C 710 VOL. 16, 1979 ASSESSMENT OF ANTIMALARIAL ACTIVITY IN VITRO 711 and then clarified by centrifugation) human type A+ i- fresh frozen plasma (in acid-citrate-dextrose antico- agulant). Stock cultures were maintained in 5.0 ml of the 6% erythrocyte suspension in 25-ml tissue culture CQOQO"O0KP""o flasks (Corning Glass Works, Corning, N.Y.). The o ~ 'OK ,>OqBQ1Q flasks were flushed with a gas mixture consisting of 5% 02, 5% C02, and 90% N2 (Air Products Corp., Allen- town, Pa.), sealed, and incubated at 370C. Best results were obtained in individual experiments when the F 88 growth rate in the stock cultures was high as indicated by a doubling of the parasitemia every 24 h. This was accomplished by daily changes of the culture medium and by dilution with fresh erythrocytes every 2 or 3 H QQQQQQQQQQQQ days so that less than 2% of the cells were infected at L any time. For each experiment, samples of the stock Li cultures were further diluted in culture medium con- FIG. 1. Representation of a microtiter plate, with taining sufficient noninfected type A+ human eryth- 96 wells arranged in 8 rows (A-H) and 12 columns rocytes to yield a final hematocrit of 1.5% and parasit- (1-12). When prepared as described in the text, wells emia of 0.25 to 0.5% in preparation for addition to the 1 through 8 of row A serve as a parasite control (no microtitration plates. drug present), and wells 9 through 12 of row A serve Preparation of drugs. Preparation of compounds as an erythrocyte control (no drug and no parasites). in a form suitable for addition to the microtitration Each compound ispresent in duplicate columns, over plate at a known concentration required consideration a 64-fold range, with the highest concentrations in of the aqueous solubility and sterility of each agent. row B and in twofold dilutions to the lowest concen- Many compounds were relatively insoluble in water trations in row H. and had to be dissolved initially in an organic solvent. These problems were overcome by using a combination of sterile water and ethanol as the solvent. Each A constant volume (200 id) of the parasitized eryth- compound was first dissolved in a measured volume of rocyte suspension described above was added to each either water or ethanol, which was then diluted with well of the microtiter plate except the last four wells its counterpart to yield a 70% ethanol-30% water mix- of row A, to each of which 200 A1 of an equivalent ture containing a known concentration of the drug. suspension of nonparasitized type A+ human eryth- This solution was allowed to stand at room tempera- rocytes in culture medium was added. The total vol- ture for 30 min. Further dilutions were then made by ume in every well was then 225 pl. The first eight wells addition of the compound in the 70% ethanol solution of row A, containing no drugs, served as a parasite to a measured volume of culture medium with con- control. The last four wells of row A, containing nei- stant mixing. Plasma proteins in the culture medium ther drugs nor parasites, served as a nonparasitized presumably provided binding sites for compounds erythrocyte controL The parasitized erythrocytes were which might otherwise have precipitated in an exposed to each compound in two columns, with the aqueous solution. The final dilution contained less highest concentration in row B and the lowest concen- than 0.1% ethanol, which had no measurable effect on tration in row H. Two plates were prepared for each the parasites in this system. experiment; the Uganda I strain of the parasite was Preparation of microtitration plates. Microti- added to one, and the Smith strain was added to the tration techniques were used to measure the activity other. of a large number of compounds efficiently. The mi- After preparation as above, the plates were placed crotiter plate (Cooke Laboratory Products, Alexan- in a humidified airtight box (Instrumentation Depart- dria, Va.) used consisted of 96 flat-bottom wells, ar- ment, Walter Reed Army Institute ofResearch, Wash- ranged in a matrix of eight rows (A through H) and 12 ington, D.C.), which was then flushed with a gas columns (1 through 12) (Fig. 1). The plates were mixture of 5% 02,5% C02, and 90% N2 and sealed. The prepared and the parasites were harvested by using box was then placed in an incubator at 37°C for 24 h. strict aseptic techniques inside a laminar flow hood in Preparation of isotope and labeling of para- the following sequence. An Eppendorf pipette or a sites. Uptake of [G-3H]hypoxanthine (Amersham/ Microdrop I (Cooke Laboratory Products) multiple- Searle Corp., Arlington Heights, Ill.) was used as an well filling pipette was used to place 25 id ofthe culture index of growth of the parasites. The isotope is sup- medium in each well of the microtiter plate. Twenty- plied as a lyophylate (1,000 mCi/mmol) in ampoules five microliters of the drug solution, prepared as de- containing 1.0 mCi. The contents of a single ampoule scribed above, was then added to each of two adjacent were dissolved in 2.0 ml of 50% ethanol to provide a wells in the second row (B) ofthe plate. Six compounds stock solution which was stored at -20°C. In prepa- were thereby accommodated by each plate. After the ration for addition to the microtiter plates, the ethanol drugs were added to the wells of row B, an Automatic was evaporated from a 0.2-ml sample of the stock Diluter (Cooke Laboratory Products) was used to solution, and 4.9 ml of culture medium was added to make serial twofold dilutions across the plate in each the remaining 0.1 ml of the isotope in water.

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