Trypanosoma cruzi: Nifurtimox's effect on infectivity

Trypanosoma

cruzi:

Nifurtimox’s

Effect on Infectivity

RITA L. CARDONI, MAHIA TERESA RIMOLDI, AND MAWA M. E. DE BRACCO Instituto de Incestigaciones Mc(clica.~, Facultarl de Medicina, Uniuersidad Aires, Donato Alvarez 3000, Buenos Aires 1427, Argentina (Accepted

for publication

cle Buenos

2i June 1977)

CAIIIIONI, R. L., RIMOLDI, 31. T., AXD DE BRACCO, 11. hf. E. 1977. TrypanoPoma crrrzi: Nifurtimox’s effect on infectivity. Experimental ParasitoZogrJ 43, 370-375. hlice were protectctd from infection with Tryl~anosonla crud, Tulahlren strain, by Nifurtimo\[3-methyl4-( ~5’-nitrofnrfurylidene-nmi~~o)tetrahydro-4H-1,4-thiazine-l,l-dioxide] treatment with doses currently used for human therapy ( 10 mg/kg/day). of Nifrtrtimox Ivas Tl le effectiveness reflected by a significant decrease in the mortality of treated infected mice. However, cominocula of T. cruzi pretreated in uitro for 7 to 48 hr with the drug at concentrations parable to those reached in vitio in the peripheral blood of protected mice were as infective as controls, in spite of alterations in the growth of the parasites. In vitro incubation of the parasites up to 48 hr with plasma obtained from Nifurtimox-treated uninfected mice did not reduce their infectivity. These results suggest that the protective effect of Nifurtimox on experimental infection by T. crrrzi is not exerted solely by direct trypanocidal action. Whether the action depends on the long-lasting effect of the drug on parasite growth, on the modification of the host, or both remains to be established. INDEX DESCRIPTORS: Trypanosoma cruzi (Tulnhuen strain); Parasitic protozoa; Hemoflagellate; Chagas disease; Mouse protection; Nitrofurfurylidene; BAY 2502; 3-Methyl-4(5’-nitrofurfurylidene-amino) tetrahydro-4H-1,4-tbiazine-l,l-dioxide.

of the positive seroIogicn1 and parasitological tests has been demonstrated (Cerisola 1969; Cichero et al. 1969). In chronically infected adult patients, previous reports give controversial results, because no clear parameters of evaluation were developed; some workers reported reversal of the parasitological assay ( Wegner and Rohwedder 1972), but this was not proven by others (Cangado et al. 1969). Negative xenodiagnosis after Nifurtimos treatment was found only in hospitalized patients, and the failure to obtain similar results in the outpatient population had been ascribed to incorrect treatment. This is frequently the result of deleterious side effects by the drug, which only allow a maximum dose of 10 mg/kg/day for long-

INTRODUCTION

Nifurtimox [3-methyl-4-( S-nitrofurfurylidene-amino)tetrahydro-4H-1,4-thiazine-l,l-dioxide], a nitrofuran derivate, has been widely used for treatment of chronic and acute Chagas disease in Argentina (Bocca Tourres 1969; Levi and Neto 1971). Its en‘ectiveness for the treatment of Chagas, i.e., Trypanosoma cruzi, infection is currently evaluated by serological (complement fixation, hemagglutination, and immunofluorescence with specific antigens) and parasitological (xenodiagnosis) methods. It has been recommended for treatment of Chagas disease on the basis of the results obtained in children and adolescent patients during the acute and chronic phase of the disease. In both cases, reversal 370 Copyright All rights

@ 1977 by Academic of reproduction in any

Press, Inc. form reserved.

ISSN

0014 -4894

1'. CTUZi: NIFURTIMOX'S EFFECT ON INFECTIVITY

term treatment. Even with this protocol two-thirds of the patients develop serious complications and must interrupt treatment (Girardelli 1969; Lugones et al. 1969; Wegner and Rohwedder 1972). These controversial results cannot be accounted for by a differential sensitivity of the parasites, since both negative and positive xenodiagnoses after treatment have been found in patients from the same geographic area. However, strain variations in the drug protection from experimental Typanosoma cruzi infection in mice have been reported (Haberkorn and Gonnert 1972). The main purpose of this work was to investigate the effects of Nifurtimox on mice infected with Typanosoma cruzi. The study of the pharmacological action of this drug was approached by incubating the parasites in vitro with the drug at different concentrations and analyzing the infectivity of the pretreated parasites. In addition, the morphology of Nifurtimoxtreated parasites was observed after different times of incubation, When the drug is absorbed after oral ingestion, most of it is quickly metabolized and circulating metabolites are found in serum (Duhm et al. 1972). Accordingly we investigated the effects of these metabolic products in both in vivo and in vitro systems. MATERIALS AND METHODS

Trypanosoma cruzi, Tulahuen strain, maintained by mouse passage, were obtained from the Comision de Chagas, Catedra de Microbiologia y Parasitologia, Facultad de Medicina, Universidad de Buenos Aires. For in vivo studies groups of at least nine Rockland mice of either sex, 3-4 weeks old, were inoculated intraperitoneally with 2.5 x 10” bloodstream forms. Nifurtimox was given orally by gastric intubation at 10 mg/kg/day doses. For in vitro studies, bloodstream forms, obtained from mice infected 7 days before, were diluted in culture medium containing

371

2.8 gc/o of brain-heart infusion, 1 g’j/o of glucose, 10% of liver extract, 500 U/ml of penicillin, and 500 pg/ml of streptomycin, pH 7.4, at 28 C in a final concentration of 1 x 10” Trypanosoma cruzi/ml. T. cruzi counts were assessed as previously reported ( Kierszenbaum and Saavedra 1972). Handling of the parasites was performed under sterile conditions. Smears of parasites maintained in vitro for 7, 24, and 48 hr, with or without addition of Nifurtimox, were stained with May Griinwald-Giemsa. Trypomastigote, epimastigote, and dividing epimastigote forms were observed. The percentage of trypomastigote forms among the total number of parasites was determined. Nifurtimox ( Lampit ) without additions was provided by Bayer Laboratories, Argentina, and solubilized in 3% gelatin before use. Statistical comparisons between cumulative mortality curves were based on the Kolmogorov-Smirnov test. Differences were considered significant when P < 0.05 ( Siegel 1956). RESULTS

Efiect of Nifurtimox Treatment on Mice Infected with Trypanosoma cruzi Figure 1 shows the cumulative mortality curves from untreated mice inoculated with Trypanosoma crud and from infected mice treated daily with Nifurtimox according to different schedules. When mice were treated with the drug after infection (curve b) or before and after inoculation with T. cruzi (curve d) mortality was significantly lower than that of controls; however, the difference between both schedules of treatment was not significant. During treatment, smears of peripheral blood from infected mice stained with May Grunwald-Giemsa showed partially destroyed bloodstream forms of trypomastigotes. When Nifurtimox was given before infection (curve c) no differences from the control group (curve a) were observed. In this case, the last Nifurtimox dose was

CARDONI, c

RIMOLDI,

I--”

I

,_.....

~~----

b

,' : ;

'

.---------d / I'

i 10

__,

-.J

14

, 18

OAYS

AFTER

22

26

i0

INFECTION

1. Effect of Nifurtimox administration ( 10.0 m&kg/day) on the cumulative mortality of mice infected ip with 2.5 X 10’ Try~mmsomn cruzi. Curve a (-), untreated mice (n = 12); curve b (---), mice treated daily with Nifurtimox from 24 hr after the infec;ion until the end of the experiment (n = 9); curve c ( . . . . . . . ), mice treated daily with i\‘ifurtimox from 8 days before infection until the day of infection (n 1 9); and curve d (-.-.-), mice treated daily with Nifurtimox from 8 days before the infection until the end of the experiment ( n = 9).

given 4 hr before infection. At this time the highest concentration of drug was reached in peripheral blood of rats and dogs (Medenwald et al. 1972).

Effect of in Vitro Treatment with Nifurtimox on the Infectivity of Trypanosoma cruzi

Trypanosoma crusi, trypomastigote forms, at a final concentration of 1 X 10” cells/ ml were suspended in the culture medium described under Materials and Methods and pretreated in vitro with various concentrations of Nifurtimox before inoculation in mice. Data from the cumulative mortality curves from infected mice are shown in Table I. The infectivity of T. cruxi treated with the drug at 3.0 and 6.0 pg/ml for 7, 24, and 48 hr was similar to that of controls. When the drug was used at 45.0 pg/ml, the infectivity of the inoculum was significantly lower than that of the

AND

DE RRACCO

control, even when the inoculum was exposed to Nifurtimox for only 7 hr. Infectivity of the Nifurtimox-treated inocula was compared with that of a control inoculum kept under the same conditions without the drug because bloodstream forms show a decrease in infectivity when maintained under in vitro conditions. Morphological studies revealed that the percentage of trypomastigote forms decreased after in vitro culture in the absence of the drug, concomitantly to the appearance of epimastigote and dividing epimastigote forms of the parasite. This change was detected in the control after 24 hr of culture. As shown in Table II, the percentage of epimastigote and dividing epimastigote forms was reduced after addition of 3.0 and 6.0 pg/ml of Nifurtimox, and with 45.0 pg/ml of Nifurtimox only trypomastigote forms were observed.

Effect of Metubolic Products of Nifurtimox on the Infectivity of T. cruzi Uninfected mice were treated with Nifurtimos at 10.0 mg/kg/day for 8 days;

T. cruzi:

NIFURTIMOX’S

4 hr after the last dose the plasma was obtained from heparinized blood. This plasma was used as a source of the metabolic products of Nifurtimox. Also, unchanged drug is estimated to be present; this is supported by studies in treated dogs and rats (Duhm et al. 1972). Incubation of parasites with plasma containing Nifurtimox metabolic products failed to show any differences from controls even though the cells were exposed to these products for up to 48 hr (Table III). Control parasites pretreated with normal plasma showed no decrease in infectivity during the experiment. As in all tests, each sample was compared with a control kept under the same experimental conditions.

EFFECT

373

ON INFECTIVITY TABLE

III

Infectivity of Trypanosoma cruzi Pretreated in Vitro with Plasma from Nifurtimox-Treated Uninfected Mice PLY-

treatlrlentn

Plasma from mice

1~

(hr) 7

Untreated Treated

9 10

1.5.9 16.2

0 0

24

Untreated Treated

10 10

17.0 18.0

2:i 30

48

Cntreated Treated

10 10

17.0 22.0

20 50

L1Mice were infected ip with 2.5 X 105 T. with plasma from uninfected mice treated daily at 10.0 mg/kg for 8 days or with normal mice and 48 hr at 28 OC. * Median survival time = number of days at which 50% of the mice survived. c Percentage of mice surviving for 27 days

cruzi pretreated with Kifurtimox plasma for 7, 24, after

inoculation

after

inoculation.

DISCUSSION

The present work deals with the in vivo and in Vitro actions of Nifurtimox on infectivity of T~ypanosoma cruxi for mice. The experimental design followed a therapeutic protocol similar to that currently used in Chagas disease patients. When a single oral does of 15.0 mg/kg of Nifurtimox is given, the highest concentration of unchanged drug in human serum is 3.0 ,g/ml (0.5% of the total drug provided). With the current scheme of three daily treatments, a level of only 1.0 pg/ml in TABLE

II

Trypomastigote Forms of Trypanosoma cruzi in in Vitro Cultures Treated with Nifurtimox Nifurtimox, final concentration b&W

3.0 6.0 45.0

Trypomastigote

forms (‘%)*

v

24

48

100 100 100 100

19 2s 92 100

5 14 76 100

a Parasites, 1 X 10s/ml, were treated in vitro with 3, 6, and 45 /*g/ml of Nifurtimox for 7, 24, and 48 hr at 28 “C. b Determined as described under Materials and Methods; mean value of three determinations. c Hours of treatment.

human serum is reached. Moreover, repeated doses of Nifurtimox did not increase the serum levels of the unchanged drug because it is quickly eliminated (Medenwald et al. 1972). Previous reports (Gijnnert and Block 1972) indicate that the growth of intracellular parasites in HeLa cells may be impaired by Nifurtimox. Morphological alteration of intra- and extracellular forms was found after treatment with 1.0 pg/ml of Nifurtimox; however, even 7 days after treatment intact parasites persisted (Voight et al. 1972). In this work we also observed damage of bloodstream forms from treated mice, but we feel that morphological criteria are less reliable than infectivity for evaluating a drug to be used in humans. We found that in vitro incubation with 3.0 pg/ml of Nifurtimox had no significant effect on infectivity even after 2 days of treatment (Table I). On the other hand, 45.0 pg/ml of Nifurtimox decreases infectivity (Table I); this is in agreement with previous results using massive doses (1000 pg/ml) (Bock et aE. 1969). T. CTUZ~maintained in vitro for longer time periods (up to 48 hr) showed a time-related change in their morphology; the percentage of trypomastigote forms decreases, while epi-

374

CARDONI,

HIMOLDI,

mastigote and dividing epimastigote forms appeared. Nifurtimox interfered with this shift from trypomastigote to epimastigote forms of the parasite, and this effect was related to the dose of the drug. At 45.0 &ml, there was no transformation of the parasites. Most of the drug provided is absorbed and metabolized. Metabolic products remain in tissues and blood for a longer time than does unchanged drug. However, a progressive increase in the serum levels of the metabolites after repeated doses has not been experimentally proven (Duhm et al. 1972). To evaluate the possibility that the effect of Nifurtimox can be potentiated by its metabolic products present in plasma or by a plasmatic factor, we incubated the parasites in vitro with the drug plus metabolite-laden plasma for 2 days. A longer incubation period was not attempted since trypomastigote forms do not persist in our cultures without cells. We found a slight reduction in the infectivity of parasites exposed for 2 days to drug plus metaboliteladen plasma (Table III). It is noteworthy that trypomastigotes may not remain extracellular in the bloodstream for such a long period of time. At least under in vitro conditions, trypomastigotes penetrate culture lines of mammalian cells in less than 30 min (Dvorak and Hyde 1973). In this regard, drug pretreatment in viva did not alter the course of infection (Fig. 1, curve c) nor did it alter the effect of the postinfection treatment (Fig. 1, curve d). However, we demonstrated that in oivo postinfection treatment with Nifurtimox protected mice from Trypanosoma cruzi (Fig. 1, curve b). Other investigators (Bock et al. 1972) failed to show a protective effect with the same dosages used by us, e.g., when NMRI-Han mice infected with 10” parasites were treated for 4 days. We think that their results could be due to the number of drug doses as compared with the high number of parasites inoculated in the challenrine sten.

AND DE BRACCO

Previous reports (Bock et al. 1969) have shown that high doses of Nifurtimox given during a short time period were not protective, whereas long-lasting in viva treatment of infected mice with small doses seemed to yield the desired result. On the other hand, the protective effect of Nifurtimox could be due to additional mechanisms, such as drug-induced modifications in the immunologic reactivity of infected subjects. This has been shown in chronic Chagas patients undergoing Nifurtimox treatment (Lelchuk et nl. 1973). Whether these modifications in the host are directly related to the protective effects is currently under investigation in our laboratory. ACKNOWLEDGMENTS This work was supported by CONICET (Consejo National de Investigaciones Cientificas y T&micas) Grant 7617/76 Si.Sl.E. de B. is a career member, and R.L.C. and M.T.R. are research fellows from CONICET. We wish to express onr appreciation to Dr. J. A. hlanni for the helpful discussions he provided for undertaking this project and to A. M. Pizzi for her technical assistance. REFERENCES B~CCA TOU~RES, C. L. 1969. La enfermedad de Chagas en periodo agudo y su tratamiento con el BAY 2502. Boletin Chileno de Parasitologia

24, 24-27. Boce, hl., G~~XXERT, R., AND HABER~OIXN, A. 1969. Studies with BAY 2502 on animals. Boletin Chileno de Parusitologiu 24, 13-19. H., BOCK, M ., HABERGORN, A., HERLIXER, MAYER, K. H., ANU PETERSEN, S. 1972. The strncture-activity relationship of 4-( 5’-nitrofurfurylidene-amino) -tetrahydro-4H-1,4-thiazine-l,ldioxides against Trypanosonm cruzi. Arzneimittel-Forschng 22, 1564-1569. CANTADO, J. R., MARRA, U. D., LOPEZ, M., Moundo, O., FARIA, C. A. F., ALVARES, J. hl., AXII SALGADO, A. DE A. 1969. Toxicidnd y valor teraptutico de1 Bay 2502 en la enfermedad de Chagas cr6nica en tres esquemas posolbgicos.

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sitologia 24, 54-59. CICHERO, J. A., SECXJM, E., AND QUATROCHI, T. C. 1969. Evolucicin clinica. narusitolbeica. v

T.

CTUXi:

NIFURTIMOX’S

tolerancia a la droga de 33 niiios con infeccibn chag&ca crbnica tratados con Bay 2502. Boletin Chileno de Parasitologia 24, 59-62. DUIIA~ B., MAUL, W., MEDENWALD, II., PAN.%SCHKE, K., AND WEGNER, L. A. 1972. Investi-

gation on the pharmacokinetics of Nifurtimox3’S in the rat and dog. Arzneimittel-Forschung 22, 1617-1624. DVORAK, J. A., AND HYDE, T. P. 1973. Trypanosoma cruzi: Interaction with vertebrate cells in uitro. I. Individual interactions at cellular and subcellular levels. Experimental Parasitology 34, 268-283. GIRARDELLI, M. A. 1969. Evolucibn electroencefalogr6fica en nifios y adultos j6venes con infecci6n chag6sica crbnica tratados con BAY 2,502. Bobin ChiZeno de ParasitoZogia 24, 35-38. G~NNEIIT, R., AND BOCK, M. 1972. The effects of Nifurtimox on Trypanosomu crud in tissue cultures. ArzneimitteZ-Forschung 22, 1582-1586. HABERKORN, A., AND G~NNERT, R. 1972. Animal

experimental investigation into the activity of Nifurtimox against Trypanosoma cruzi. Arzneimittel-Forschung 22, 1570-1582. KIERSZENBAUM, F., AND SAAVEDRA, L. E. 1972. The effects of bacterial endotoxin on the infection of mice with Trypanosoma cruzi. Journal of ProtozooZogy 19, 655-657. LELCHUK, R., PATRUCCO, A., CHIALE, P., AND MANNI, J. A. 1973. Tripanosomiasis cr6nica

EFFECT

ON

INFECTIVITY

375

americana: Efecto de1 tratamiento sobre la respuesta celular inmune. Me&&a (Buenos Airen) 33, 656 (abst.). LEVI, G. C., AND NETO, V. A. 1971. Observacdes sBbre o tratamento de pacientes corn a forma crBnica da doen$a de Chagas mediante emp&go do compost0 nitrofurhnico “Bayer 2502” ou “Lampit.” Revista del Instituto de Medicina Tropical de SBo Paul0 13, 369-372. LUGONES, H. S., PERALTA, F., FEIJOO, D. C., AND DE MARTELETJR, A. E. A. 1969. Evoluci6n de la sintomatologia clinica y de la funci6n hepkica de la enfermedad de Chagas aguda tratada con Bay 2502. Boletin Chileno de ParasitoZogia 24, 19-24. MEIIENU’ALD,

II., BRANDAU, K., AND SCNOSSMANN, K. 1972. Quantitative determination of Nifurtimox in body fluids of rat, dog and man. Arzneimittel-Forschung 22, 1613-1617. SIEGEL, S. 1956. “Nonparametric Statistics,” pp. 127-136. McGraw-Hill, New York. VOIGHT, W. H., BOCK, M., AND GBNNERT, R. 1972. Ultrastructural observations on the activity of Nifurtimox on the causative organism of Chagas’ disease. I. Trypanosoma cruzi in tissue cultures. Arzneimittel-Forschung 22, 1586-1589. WEGNER, D. H. G., AND ROHWEDDER, R. W. 1972. Experience with Nifurtimox in chronic Chagas’ infection. Preliminary report. ArzneimittelForschung 22, 1635-1641.