a Departamento de Parasitologia, ICB, UFMG, Avenida Antônio Carlos, 6627, Belo Horizonte, MG and b Laboratório de Doença de Chagas, Centro de Pesquisas René Rachou, FIOCRUZ, Avenida Augusto de Lima, 1715, Caixa Postal 1743, CEP 30190-002, Belo Horizonte, MG, Brazil
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The control of Chagas' disease in Brazil for the past 10 years has been focused on intervention directed against vectors.4 Despite the success of vector control, with possible eradication of the vectorial transmission, there are still millions of infected people who will depend on medical care for at least the next 30 years.
In spite of the large number of infected patients, there are no commercially available drugs with high efficacy that can be used for mass and/or individual treatment. Specific chemotherapy with the two drugs available, benznidazole and nifurtimox, has been indicated for the treatment of the brief acute phase, with a cure rate of 5070%.5 Prolonged follow-up studies with chronically infected patients, however, show a very low cure rate (820%) in patients treated for more than 10 years.57 Furthermore, both drugs can cause several side-effects including hypersensitivity reactions (dermatitis), peripheral neuritis, weight loss, gastrointestinal disturbances and peripheral polyneuropathy.5 In addition, differences in the susceptibility and natural resistance of a large number of T. cruzi isolates to nitro-derivatives has also been suggested as an important factor in the low rates of cure in treated chagasic patients.810 The improvement of currently available chemotherapy is thus a research priority in those countries where vectorial transmission has been controlled.
The existence of T. cruzi isolates that are naturally resistant to benznidazole and nifurtimox has stimulated the search for alternative drugs suitable for the treatment of T. cruzi infection.1117 Among the several compounds tested in vitro and in vivo against T. cruzi, it has been demonstrated that ketoconazole, an imidazole derivative highly effective in the treatment of topical and systemic fungal infection, is able to protect mice against fatal T. cruzi infection.1315 Ketoconazole, however, failed to cure chronic phase human clinical and experimental Chagas' disease.15
Some studies have also been performed to evaluate the effects of various drugs in vitro, with axenic as well as infected tissue culture, demonstrating that ofloxacin, a bacterial topoisomerase II inhibitor, is able to inhibit both proliferation and differentiation of T. cruzi in axenic cultures in vitro.16,17 No evidence, however, of in vivo activity of this drug has so far been provided.
Synergy between drugs can be a valuable way to improve treatment efficacy in several diseases.1820 Using this approach, Urbina et al.21 demonstrated that the combination of ketoconazole with lovastatin has an anti-proliferative effect in vitro on T. cruzi epimastigote growth in LIT media and on amastigotes in tissue cultures. Unfortunately, these results were not observed in vivo when the same combination was used to treat infected mice.15 A synergic effect of ketoconazole plus terbinafine and lovastatin, inhibitors of ergosterol biosynthesis, has also been demonstrated both in vitro and in a murine model Chagas' disease.21
In view of the trypanosomicidal effect of ketoconazole and ofloxacin described previously, and the fact that these drugs are commercially available for human chemotherapy, we investigated in this study whether their combination with benznidazole could have a synergic effect and improve the effectiveness of treatment of Chagas' disease.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benznidazole (2-nitro-N-(phenylmethyl)-1H-imidazole-1-acetamide) is synthesized by F. HoffmanLa Roche, and commercially available in Brazil as Rochagan (Produtos Roche Químicos e Farmacêuticos, Rio de Janeiro, Brazil). It has been used as a standard drug for human Chagas' disease treatment in Brazil. Ketoconazole is a derived imidazole produced by Janssen Pharmaceutics and commercially available in Brazil as Nizoral (Janssen-Cilag Farmacêutica, São José dos Campos, São Paulo, Brazil). Ofloxacin is a bacterial topoisomerase II inhibitor, synthetic quinolone synthesized by Janssen Pharmaceutics and commercially available in Brazil as Ofloxan (Janssen Farmacêutica, São José dos Campos, São Paulo, Brazil).
Trypanosoma cruzi isolates
Three isolates of T. cruzi previously characterized by Filardi & Brener10 according to their susceptibility to benznidazole and nifurtimox in vivo were used; susceptible CL,22 moderately resistant Y23 and naturally resistant Colombiana.24 All isolates of T. cruzi were obtained as infected blood samples frozen in liquid nitrogen (as described by Filardi25) from the T. cruzi cryobank at Laboratório de Doença de Chagas, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, MG, Brazil. All isolates were maintained in the laboratory by passages in SwissWebster female mice.
Infection and treatment schedules
SwissWebster female mice, 2024 g, were obtained from the animal facility at Centro de Pesquisas René Rachou (FIOCRUZ). Animals were inoculated intraperitoneally with 1 x 104 blood forms of T. cruzi. The inoculated animals were divided into 13 groups of 1014 animals and received the various drug combinations. All compounds were suspended in distilled water using 4% gum arabic and each animal received 0.25 mL of drug suspension daily by gavage. Drug combinations consisted of benznidazole plus either ketoconazole or ofloxacin. Benznidazole was administrated in daily doses of 25, 50 or 100 mg/kg/day. One hour after benznidazole treatment, the second drug was administered at a dose of 120 mg/kg/day or 100 mg/kg/day for ketoconazole or ofloxacin, respectively. Treatment started immediately after the detection of patent parasitaemia (810 days after infection for Y isolate and 1215 days after infection for CL and Colombiana isolates) and was administered for 20 consecutive days, for all drug combinations. One group of 11 animals received ketoconazole (120 mg/kg/day) starting 24 h after infection with Y isolate. Control groups consisted of untreated animals and those given each of the compounds alone.
Parasitaemia evaluation
Blood examination was performed daily to assess levels of parasitaemia, as described by Brener.26 Briefly, 5 µL of blood from the tail were transferred to a microscope slide, covered with a coverslip and examined for living flagellates by direct optical microscopy. Results were expressed as number of trypomastigotes/5 µL blood. After treatment, parasitaemia was followed for different periods of time, depending on the previously described peak of parasitaemia for different isolates of T. cruzi (6 days for Y and 20 days for CL and Colombiana).
Test for cure assessment
Cure was assessed by using parasitological (haemoculture and xenodiagnosis) and serological (anti-live trypomastigote antibodies) methods. Haemoculture was performed as described by Filardi & Brener10 and xenodiagnosis as described by Brener.26 Analysis of anti-live trypomastigote antibody was performed as described by Martins-Filho et al.27 Results were compiled as animals cured and those not cured. Animals were considered cured only when all tests showed negative results.
Statistical analysis
The 2 analysis or Fisher's Exact test was used to compare differences in cure rate between the groups receiving benznidazole, ketoconazole or ofloxacin alone with those receiving combinations of drugs. Differences were considered statistically significant at P < 0.05. All statistical analyses were carried out using Version 6 of Epi-Info software from the CDC.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine whether the time between the infection and the beginning of treatment had any influence on the cure rate, we performed a parallel study in which animals infected with the Y isolate were submitted to different treatment schedules with ketoconazole. Infected animals received ketoconazole (120 mg/kg/day for 20 days) starting 24 h and 9 days after infection. We observed a 100% cure (11/11) for the animals treated 24 h after infection, but none of the animals (0/11) treated 9 days after infection were cured. These data demonstrated that the time span between the infection and the beginning of chemotherapy with ketoconazole is of fundamental importance in the effectiveness of treatment with ketoconazole.
Combination of benznidazole and ketoconazole
As ketoconazole was ineffective in animals infected with the Y isolate when therapy started 9 days after infection, the effect of ketoconazole combined with benznidazole was determined in continuing infections. For this purpose, animals infected with the CL, Y and Colombiana isolates of T. cruzi were treated immediately after the detection of patent parasitaemia, for all different drug combinations.
The cure rate observed after treatment with benznidazole, ketoconazole and their combination are presented in Table I. The results demonstrate that in mice infected with the CL isolate a combination of benznidazole and ketoconazole resulted in a synergic effect, increasing the cure rate (P < 0.05). This effect can be clearly noted when comparing the percentage of cured animals after treatment with benznidazole (25 and 50 mg/kg/day) or ketoconazole (120 mg/kg/day) alone with those receiving the combination of benznidazole plus ketoconazole. Treatment with ketoconazole (120 mg/kg/day) alone or benznidazole (50 mg/kg/day) alone cured 0 or 9.1% of animals, respectively. In contrast, treatment with ketoconazole (120 mg/ kg/day) plus benznidazole (25 or 50 mg/kg/day) cured 71.4 or 100%, respectively.
|
Although the combination of benznidazole (100 mg/kg/ day) and ketoconazole (120 mg/kg/day) produced 30.8% cure in animals infected with the Colombiana isolate of T. cruzi in comparison with the treatment using the drugs alone, this difference was not statistically significant (P > 0.05).
Combination of benznidazole and ofloxacin
The cure rate observed after treatment with benznidazole and ofloxacin alone and combined is presented in Table II. No cure was observed when animals infected with CL, Y or Colombiana isolate were treated with ofloxacin alone at 100 mg/kg/day. We did not find any differences in the cure rate in animals treated with benznidazole plus ofloxacin in comparison with those treated with benznidazole alone for any of the isolates.
|
The level of parasitaemia was investigated in animals infected with T. cruzi after chemotherapy with ofloxacin. The Figure shows the levels of parasitaemia in animals infected with CL, Y and Colombiana isolates of T. cruzi after treatment with ofloxacin. Parasitaemia was followed for different periods of time, depending on the peak of parasitaemia described previously for the T. cruzi isolates (6 days for Y, and 20 days for CL and Colombiana). By using this approach, we ensured that any in vivo trypanosomicidal effect of ofloxacin could be identified as a reduction in the high levels of parasitaemia observed in untreated animals. As observed in the Figure
, despite its reported in vitro activity,16 ofloxacin did not have any trypanosomicidal effect in vivo. It is interesting to observe that in animals infected with Y isolate at day 2 after treatment, the level of parasitaemia was increased in the ofloxacin group in comparison with the untreated group. This result emphasizes the total lack of effect of ofloxacin as a trypanosomicidal drug for in vivo treatment.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper we investigated a possible synergic effect of benznidazole with ketoconazole or ofloxacin in mice inoculated with isolates of T. cruzi naturally resistant and susceptible to benznidazole and nifurtimox. Our data demonstrated that a combination of benznidazole with ketoconazole induces a synergic effect in mice infected with the CL and Y isolates of T. cruzi. The results with isolate Y were particularly impressive as this isolate is partially resistant to benznidazole. No differences were observed, however, in animals infected with the Colombiana isolate, indicating that synergic effects with benznidazole and ketoconazole are influenced by the isolate of parasite and that this may be important in further studies searching for drug combinations.
It is interesting to note that our data show that ketoconazole alone was not enough to cure animals infected with the Y isolate of T. cruzi, when treatment was started 9 days after infection; however, when dosing started 24 h after infection, animals were cured. These data are in agreement with those of McCabe et al.,14 who demonstrated that although mice infected with four different isolates of T. cruzi can be markedly protected from death by early treatment with ketoconazole, the therapeutic effectiveness was dependent on the delay between the infection and the beginning of treatment. They showed that the results can range from 100 to 0% survival from day 5 to day 9 after infection. These results and ours emphasize the necessity of early treatment of Chagas' disease.
We have also investigated here a possible synergic effect produced by a combination of benznidazole and ofloxacin. Unfortunately, our data did not demonstrate any positive effect with such a combination, despite the results of Gonzales-Perdomo et al.,16 who showed that an inhibitor of bacterial topoisomerase II inhibits the proliferation and differentiation of T. cruzi in axenic cultures in vitro, as well as in infected tissue culture. Moreover, we found that treatment with ofloxacin increased the parasitaemia in animals infected with the Y isolate. A similar result in animals infected with the Y isolate has been reported by Brener et al.,15 who evaluated the effect of lovastatin combination with ketoconazole.
It is interesting to observe that the Colombiana isolate, which is naturally resistant to benznidazole, was not susceptible to any of the drug combinations tested here. Despite several studies investigating the mechanisms of drug resistance in different T. cruzi isolates, the mode of resistance of the Colombiana isolate is still unknown.
In conclusion, our results confirmed the importance of identifying among compounds already marketed those with synergic effects that are able to enhance the cure of Chagas' disease. This approach may help avoid the high costs and time-consuming research on toxicity and bioavailability of drugs for human consumption, the ultimate stage of the drug development process. Moreover, considering that several publications have shown the inefficacy of benznidazole and nifurtimox in treatment of Chagas' disease, our results support the use of combinations of benznidazole and ketoconazole in clinical trials, mainly in those cases of Chagas' disease when diagnosis is confirmed shortly after infection. The use of benznidazole and ketoconazole in humans could contribute to an increase in therapeutic efficacy, even though their use in isolation does not give the expected results.13 Finally, as suggested previously by Andrade et al.,9 the fact that particular isolates of T. cruzi each have their own level of susceptibility to certain drugs further supports the strategy of using different drug combinations to improve chemotherapy success in different geographical regions.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Higuchi, M. L. (1995). Doença de Chagas. Importância do parasita na patogenia da forma crônica cardíaca. Arquivos Brasileiros de Cardiologia 64, 2514.[Medline]
3 . Rezende, J. M. (1993). Manifestações digestivas da Doença de Chagas. In Gastroenterologia Clínica, (Dani, R. & Castro, L. P., Eds), pp. 172955. Guanabara Koogan, Rio de Janeiro.
4 . Schmunis, G. A., Zicker, F. & Moncayo, A. (1996). Interruption of Chagas' disease transmission through vector elimination. Lancet 348, 1171.[ISI][Medline]
5 . Cançado, J. R. (1985). Tratamento específico. In Cardiopatia Chagásica, (Cançado, J. R. & Chuster, M., Eds), pp. 32755. Fundação Carlos Chagas, Belo Horizonte.
6 . Ferreira, H. O. (1990). Tratamento da forma indeterminada da doença de Chagas com nifurtimox e benzonidazol. Revista da Sociedade Brasileira de Medicina Tropical 23, 20911.[Medline]
7 . Galvão, L. M., Nunes, R. M., Cançado, J. R., Brener, Z. & Krettli, A. U. (1993). Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 2203.[ISI][Medline]
8 . Brener, Z., Costa, C. A. & Chiari, C. (1976). Differences in the susceptibility of Trypanosoma cruzi strain to active chemotherapeutic agents. Revista do Instituto de Medicina tropical de São Paulo 18, 4505.
9 . Andrade, S. G., Magalhães, J. B. & Pontes, A. L. (1985). Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types. Bulletin of the World Health Organization 63, 7216.[ISI][Medline]
10 . Filardi, L. S. & Brener, Z. (1987). Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 7559.[ISI][Medline]
11 . De Castro, S. L. (1993). The challenge of Chagas' disease chemotherapy: an update of drugs assayed against Trypanosoma cruzi. Acta Tropica 53, 8398.[ISI][Medline]
12 . Urbina, J. A., Payares, G., Molina, J., Sanoja, C., Liendo, A., Lazardi, K. et al. (1996). Cure of short- and long-term experimental Chagas' disease using DO870. Science 273, 96971.[Abstract]
13 . McCabe, R. E., Araújo, F. G. & Remington, J. S. (1983). Ketoconazole protects against infection with Trypanosoma cruzi in a murine model. American Journal of Tropical Medicine and Hygiene 32, 9602.[ISI][Medline]
14 . McCabe, R. E., Remington, J. S. & Araújo, F. G. (1984). Ketoconazole inhibition of intracellular multiplication of Trypanosoma cruzi and protection of mice against lethal infection with the organism. Journal of Infectious Diseases 150, 594601.[ISI][Medline]
15 . Brener, Z., Cancado, J. R., Galvao, L. M., da Luz, Z. M., Filardi, L. de S., Pereira, M. E. et al. (1993). An experimental and clinical assay with ketoconazole in the treatment of Chagas' disease. Memórias do Instituto Oswaldo Cruz 88, 14953.
16 . Gonzales-Perdomo, M., De Castro, S. L., Meirelles, M. N. & Goldenberg, S. (1990). Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors. Antimicrobial Agents and Chemotherapy 34, 170714.[ISI][Medline]
17 . Pate, P. G., Wolfson, J. S., McHugh, G. L., Pan, S. C. & Swartz, M. N. (1986). Novobiocin antagonism of amastigotes of Trypanosoma cruzi growing in cell-free medium. Antimicrobial Agents and Chemotherapy 29, 42631.[ISI][Medline]
18 . Chunge, C. N., Gachihi, G., Muigai, R., Wasunna, K., Rashida, J. R., Chulay, J. D. et al. (1985). Visceral leishmaniasis unresponsive to antimonial drugs. III. Successful treatment using a combination of sodium stibogluconate plus allopurinol. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 7158.[ISI][Medline]
19 . Rosenthal, C. (1995). Resistência e uso combinado de drogas anti-HIV. Revista da Sociedade Brasileira de Medicina Tropical 28, Suppl. 2, 918.[Medline]
20 . Radloff, P. D., Philipps, J., Nkeyi, M., Hutchinson, D. & Kremsner, P. G. (1996). Atovaquone and proguanil for Plasmodium falciparum malaria. Lancet 347, 15114.[ISI][Medline]
21 . Urbina, J. A., Maldonado, R. A., Payares, G., Marchan, E., Lazardi, K., Aguirre, T. et al. (1991). In vitro and in vivo synergism of binary and ternary combinations of ergosterol biosynthesis inhibitors as antiproliferative agents against Trypanosoma cruzi: therapeutic implications. Memórias do Instituto Oswaldo Cruz 86, Suppl. I, 335.
22 . Brener, Z. & Chiari, E. (1963). Variações morfológicas observadas em diferentes amostras de Trypanosoma cruzi. Revista do Instituto de Medicina tropical de São Paulo 5, 2204.
23 . Pereira da Silva, L. H. & Nussenzweig, V. (1953). Sobre uma cepa de Trypanosoma cruzi altamente virulenta para camundongo branco. Folia Clinica et Biologica 20, 191208.
24 . Federici, E. E., Abelmann, W. H. & Neva, F. A. (1964). Chronic and progressive myocarditis in C3H mice infected with Trypanosoma cruzi. American Journal of Tropical Medicine and Hygiene 13, 27280.
25 . Filardi, L. S. (1975). Cryopreservation of Trypanosoma cruzi bloodstream forms. Journal of Protozoology 22, 398401.[ISI][Medline]
26 . Brener, Z. (1962). Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi. Revista do Instituto de Medicina tropical de São Paulo 4, 38996.
27 . Martins-Filho, O. A., Pereira, M. E., Carvalho, J. F. & Brener, Z. (1995). Flow cytometry, a new approach to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' Disease. Clinical and Diagnostic Laboratory Immunology 2, 56973.[Abstract]
Received 2 September 1999; returned 4 December 1999; revised 6 January 2000; accepted 25 January 2000