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The primary in vitro evaluation of test compounds is performed against a broad panel of pathogens to allow proper evaluation of selectivity. This approach involves integrated and robotic logistics to attain a reasonably high throughput. Compound stock solutions are prepared in 100% DMSO at 20 mM or mg/ml. The compounds are serially pre-diluted (2-fold or 4-fold) in DMSO followed by a further (intermediate) dilution in demineralized water to assure a final in-test DMSO concentration of <1%. Test plates are identical for all screens and are produced as a single batch.
Trypanosoma cruzi, Tulahuen CL2, b galactosidase strain (nifurtimox-sensitive) is used. All cultures and assays are conducted at 37°C under an atmosphere of 5% C02. Assays are performed in sterile 96-well microtiter plates, each well containing 10 µl of the watery compound dilutions together with 190 µl of MRC-5 cell/parasite inoculum. Parasite growth is compared to untreated-infected controls (100% growth) and non-infected controls (0% growth) after 7 days incubation. Parasite burdens are assessed after adding the substrate CPRG (chlorophenolred ß-D-galactopyranoside). The change in color is measured spectrophotometrically at 540 nm after 4 hours incubation at 37 °C. The results are expressed as % reduction in parasite burdens compared to control wells and an IC50 is calculated. Nifurtimox is included as reference drug.
MRC-5 SV2 cells are cultured in Earl’s MEM + 5% FCSi. Other cell types (J774, L6, Vero, Hela, e.a.) may also be used for determination of cytotoxicity/selectivity. Assays are performed in 96-well microtiter plates, each well containing about 104cells/well. After 3 days incubation, cell viability is assessed fluorimetrically after addition of resazurin and fluorescence is measured (lex 550 nm, lem 590 nm). The results are expressed as % reduction in cell growth/viability compared to untreated control wells and a CC50 is determined. Cytotoxic reference compounds include tamoxifen.
Two Leishmania species (L.infantum MHOM/MA(BE)/67 and L.donovani MHOM/ET/67/L82) are used. The strains are maintained in the Golden Hamster and spleen amastigotes are collected for preparing infection inocula. Primary peritoneal mouse macrophages are used as host cell and are collected 2 days after peritoneal stimulation. Assays are performed in 96-well microtiter plates, each well containing 10 µl of the compound dilutions together with 190 µl of macrophage/parasite inoculum. After 5 days incubation, parasite burdens (mean number of amastigotes/macrophage) are microscopically assessed after Giemsa staining. The results are expressed as % reduction in parasite burden compared to untreated control wells and an IC50(50% inhibitory concentration) is calculated. Pentostam® and miltefosin are included as the reference drug.
Two strains of P.falciparum are used: 1/ GHA strain (P.falGHA), derived from a Ghanese patient and chloroquine sensitive and 2/ W-2 strain (P.falW2) from CDC/Indochina III and resistant to chloroquine, quinine, pyrimethamine, but susceptible to mefloquine. The strains are maintained in RPMI-1640 medium supplemented with hypoxanthine, Hepes, NaHC03, and O+ human serum together with 2-4% washed human O+ erythrocytes. All cultures and assays are conducted under an atmosphere of 4% C02, 3% 02 and 93% N2. Assays are performed in 96-well microtiter plates, each well containing 10 µl of the watery compound dilutions together with 190 µl of the malaria parasite inoculum (1% parasitaemia, 2% HCT). After 72h incubation, plates are frozen and stored at –20°C. After thawing, 20 µl of each well is transferred into another plate together with 100 µl Malstat reagent and 20 µl of a 1/1 mixture of PES (phenazine ethosulfaat, 0.1 mg/ml) and NBT (Nitro Blue Tetrazolium Grade III, 2 mg/ml). Change in colour is measured spectrophotometrically at 655 nm. The results are expressed as % reduction in parasitaemia compared to control wells. Artesunate and chloroquine are included as reference drugs.