J. PROTOZOOL. 2 4 ( 4 ) , 544-547 (1977).

Growth-Inhibition Drug Test with Trypanosoma cruzi Culture Forms* B. R. SCHLEMPER, JR., E. CHIARI and Z. BRENER

Deportment

Parasitology, Z.C.B.,Univetsity of hJinas Gerais and Centro de Pesquisas X m i Rochoit, F I O C l l U Z , C.P. 17-13, Belo Horizonte, Minas Gerais, Brazil of

SYNOPSIS. A 48-hr drug screening test is described which evaluates inhibition of exponential growth of T . cruzi culture forms by electronic cell count. About 80% of drugs active in uico produced a > 50% growth inhibition, whereas among compounds inactive in vivo, only 19.6% induced such inhibition. Advantages of this test are low cost, rapid results, small amounts of drugs needed, and feasibility without animal facilities. Comparative studies showed that culture forms are not suitable for screening additives to prevent transmission of T. cruri by banked blood. Index Key Words:

N

Trypanosoma cruzi; culture forms: drug screening test.

0 drugs are known which surely cure Chagas Disease. Nevertheless, extensive screening tests against T. cruzi are not

Drug Activity in Experimentally Infected Mice.-Groups

of

5 male 18-20 g albino mice were inoculated intraperitoneally

apparently being performed and only a few drugs have recently reached clinical trial ( 6 ) . A practical in vztro screening test with T . cruzi culture forms which might be less costly than in vivo experiments has been insufficiently explored; studies of the effects of standard drugs in vitro and in the vertebrate are needed. The present paper describes a 48-hr screening test based on inhibition of exponential growth of T . cruzi culture forms, as evaluated by cell counter. The in citro results were compared with data from treating inoculated animals. This in uitro test as a model for screening drugs to be added to banked blood in endemic areas has also been investigated with T. cruzi bloodstream forms, using compounds active against culture forms. MATERIALS A K D M E T H O D S

Trypanosoma cruzi strain MR was isolated from a Triatoma infestans collected in Rio Grande do Sul, Brazil ( 7 ) . Cultivation.-The flagellates were grown in L I T (liver infusion-tryptose) broth (8). Only cultures in log phase, show100% increase in population in 24 hr were used. Cultures ing were transferred daily in Roux flasks to keep multiplication rates high. Counting Flagellates.-An electronic counter “Toa Medical Electronics,” Model CC-1002 B was used. T h e flagellates were diluted a t 1 :500 in Paul’s solution [NaCl, 7 g ; citric acid, 10.5 g; 40% (v/v) formalin, 1 ml; distilled water, 1 liter]; counting was carried out at a R? F“ threshold. In Vitro Screening Test.-Water-soluble drugs were diluted in distilled water, passed through a Millipore membrane (pore size 0.20 p m ) mounted in plastic filter holders. In some experiments, drugs poorly soluble in water were diluted in methanol, hot acetone or an emulsifying agent (Cremophor, Bayer) . T h e cultures were diluted with L I T medium to 12-18 x 106 flagellates/ml; 1.9 ml of this suspension was placed in 18 X 150 mm flatbottom screwcap tubes; 0.1 ml of the diluted drugs was added, to a final concentration of 10 and 100 pg/ml. Control tubes received 0.1 ml of the solvent. The tubes were incubated at 28 C for 24 and 48 hr and flagellates per ml counted. Two tubes were used for each drug dilution. Final results were the mean of 3 consecutive counts. Drug activity was estimated by calculating percent of growth inhibition in the treated tubes against controls. Examinations for contamination, morphologic changes, and viability of the flagellates were done in all experiments.

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with 100,000 T . cruzi bloodstream forms. Treatment started the day after inoculation and was extended for 7 consecutive days. Water-soluble drugs were given intraperitoneally, poorly soluble compounds orally, in doses equivalent to % to I/lo of the LD,,. Bloodstream trypomastigotes were determined in treated and control animals on the 5th and 7th days of infection ( 5 ) . Activity Against T. cruzi Bloodstream Forms in Vitro.-Acutely ill mice, with high parasitemia, were killed and the blood collected in an anticoagulant solution used in blood banks (citric acid, Na citrate, Na phosphate). Infected blood (0.1 m l ) having I0 to 50 parasites per 50 microscope fields ( X 400) and 0.01 ml of the drugs diluted with distilled water to 2.0 mg/ml were mixed and placed in wells of hemagglutination plates. The mixture was homogenized in an electric rotator for 10 min and incubated at 4 C for 24 hr. The blood was then examined for motile flagellates ( X 400). As controls, blood samples without drug and with crystal violet at 2.0 mg/ml (which kills T . cruzi bloodstream forms) were examined. Compounds Tested.-All drugs tested in vitro had been investigated in T . cruzi-infected mice. Accordingly, 2 groups of drugs have been studied: 17 drugs active and 51 inactive in vico. T h e following drugs active in vivo were used: L-5-morpho1inomethy I - 3 - ( -5 -nit rof urfurylideneamino ) - 2 -oxazolidinone HCI (NF-902), L-furaltadone and furazolidone (Eaton Laboratories) ; carbidium sulphate, a phenanthridinium (Wellcome Laboratories) ; diallylmalonyl-( -4-amino-2-methylquinolyl-6-amide) acetate ( “Cruzon” ) from Imperial Chemical Industries; 3-methyl-4-( 5’nitrofurfurylidene-amino) tetrahydro-4H-t thiazine-1,l-dioxide (nifurtimox (Bayer); 8-[6-N’- (3-hydroxybutyl)] piperazinohexylamino-6-methoxyquinoline, compound 349C59 (Wellcome Lab-1,3,4-thiaoratories) ; 2-amino-5- ( l-rnethyl-5-nitro-2-iniidazolyl) diazole (Lederle); 1-(5-nitro-2-furyl)-2-[5-amino-2-( 1,3,4-thiadiazolyl ) ]-ethylene (Hb- 126) (Boehringer ) ; 5-nitro-2-furaldehydesemicarbazone; 1-2 nitro- 1 -imidazolyl- 1,2-propanediol (Ro 59963), N-N-dimethyl-2-nitro-1-imidazole-butyramide (Ro 72430), I-2-nitro-l-imidazolyl ) -3-methoxy-2-propanol (Ro-0582) ; N-benzyl-2-nitro-1-imidazoleacetamide ( R o 7-105 1) ; two 2-nitroimidazol derivatives ( R o 7-0913 and Ro 1053) from Roche Laboratories. T h e drugs inactive in vitro had been selected from compounds with different chemical structures which had been submitted to routine testing in experimentally infected mice. RESULTS

This paper is one of a group published in honor of Seymour H. Hutner. * Supported by the National Research Council, Brazil.

Figure 1 compares growth inhibition of T . cruzi forms induced by drugs active or inactive in vivo. Among the drugs showing

544

DRUGTESTFOR T . cruzi

545

TABLE1. Percentage of growth inhibition of T. cruzi culture forms (after 24 and 48 h r ) induced b y a c h e drugs.

% growth

inhibition after 24 hr

Drum L-Furaltadone NF-902 1-I 2-Nitro-1-imidazolvl) 3-methoxy-2-propanol ' 3- (2-Nitro-1-imidazolyl) 1,2-Propanediol 2-Amino-5-( l-methyl-5nitro-2-imidazolyl) 1,3,4-thiadiazole Ni trofurazone N-Benzyl-2-nitro-1imidazoleacetamide 8-Aminoquinoline derivative (349C59) Carbidium sulphate N-N-Dimethyl-2-nitro1-imidazole-butyramide Furaltadone Furazolidone Nifurtimox HB-126 Emetine derivative* Isoquinoline derivative* Phenyl-ethyl-amine derivative* Phosphonium derivative* Cruzon

% growth

inhibition after 48 hr

10 m/ml

100 pdml

10 P.a/ml

100 pLg/ml

43 31

55 46

54 54

82 76

17

50

40

76

30

40

66

72

21 19

47 40

59 23

65 66

10

39

23

63

10 20

28 39

17 46

51 57

23 -~

23 13 6 11 11 43

57 44 42 27 33 43 49

41 21 23 9 16 22 69

80 70 47 33 40 73 72

12 33 13

50 47 13

24 52 13

80

A

"1 Drugs

in

inactive

vivo

72 19

Growth

inhibition (%)

*Drugs inactive i n vivo.

suppressive effects in animal infections, 79% of the compounds produced > 50% growth inhibition after 48 hr at 0.1 mg/ml, whereas among the drugs inactive in uiuo only 19.6% induced such a degree of inhibition, and most drugs inhibited only 1020% of culture growth. Among the 10 drugs inactive in uiuo which inhibited growth > 50%, three were members of chemical series already known to include compounds active against T . cruzi, e.g. emetine (14) and quinolines ( 1 1 ) ; one is a known in uitro inhibitor of nucleic acids synthesis (actinomycin D). On the other hand, from the 4 drugs active in uiuo showing 50% growth inhibition, one is a bisquinaldine (Cruzon) which is also inactive against T . cruzi in tissue culture (15) and 3 others are poorly soluble nitrofurans which, nevertheless, also clearly inhibited growth (Table 1 ) . The highest inhibition rates were detected with concentrations of 0.1 mg/ml and 48-hr incubation (Table l ) , suggesting these conditions are best for screening. T h e reproducibility of results with several active compounds is shown in Fig. 2. The soluble compounds active against culture forms were tested in vitro against T . cruzi bloodstream trypomastigotes. No correlation emerged from comparison of drug activity in culture forms, in the vertebrate, and in bloodstream forms in uitro (Table 2 ) .

Fig. 1. Percent of growth inhibition of T. cruti culture forms induced by drugs active and inactive in animals experimentally inoculated with this parasite. tiveness depends on the drug serum concentrations, whereas in T . cruzi it depends also on its action at the intracellular level. Many more types of drugs would have to be tested to prove or disprove this idea. Unfortunately, our knowledge of the drug sensitivities of lower trypanosomatids is too preliminary for surmises as to which lower trypanosomatids would serve as

Growth-inhibition drug test with Trypanosoma cruzi culture forms.

J. PROTOZOOL. 2 4 ( 4 ) , 544-547 (1977). Growth-Inhibition Drug Test with Trypanosoma cruzi Culture Forms* B. R. SCHLEMPER, JR., E. CHIARI and Z. BR...
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