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JAC Advance Access originally published online on July 19, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):610-614; doi:10.1093/jac/dkl259
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Trypanocidal activity of the phenyl-substituted analogue of furamidine DB569 against Trypanosoma cruzi infection in vivo

Elen M. de Souza1, Gabriel M. Oliveira1, David W. Boykin2, Arvind Kumar2, Qiyue Hu2 and Maria De Nazaré C. Soeiro1,*

1 Lab. Biologia Celular, DUBC, Instituto Oswaldo Cruz FIOCRUZ, Rio de Janeiro, RJ, Brasil 2 Department of Chemistry, Georgia State University Atlanta, USA

*Corresponding author. Tel: +55-21-2598-4331; Fax: +55-21-2260-4434; E-mail: soeiro{at}ioc.fiocruz.br

Received 16 February 2006; returned 10 April 2006; revised 26 May 2006; accepted 29 May 2006


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Objectives: Aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. One such compound is furamidine (DB75) and we recently reported that one of its analogues, an N-phenyl analogue (DB569), exhibits higher trypanocidal dose and time-dependent effects against different forms of Trypanosoma cruzi as compared with DB75. Our present aim was to investigate the efficacy of DB569 in a T. cruzi mouse model.

Methods: The trypanocidal activity of the compound was evaluated by clinical, parasitological, histopathological and biochemical investigations.

Results: Treatment with DB569 significantly reduced cardiac parasitism, partially increased the survival rates of mice and lowered the levels of alanine aminotransferase and creatinine indicating a protective role against renal and hepatic lesions caused by the parasite infection.

Conclusions: Altogether, the data support the potential effect of this class of compounds against T. cruzi and motivate the screening of new diamidines for efficacy against Chagas' disease.

Keywords: aromatic diamidines , therapy , infected mice , Chagas' disease


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Chagas' disease, caused by the protozoan Trypanosoma cruzi, affects over 16–18 million people in endemic areas of Latin America1 and triggers an important myocardial pathology, which is poorly understood. Despite the fact that T. cruzi infection is an important cause of mortality and morbidity, no vaccines or safe chemotherapeutic agents are available. Recent reviews clearly point to the need for finding more efficient and less toxic drugs.2,3 In this respect, aromatic diamidines represent a promising class of DNA-targeted antiparasitic agents.4 Furamidine, a bis-amidine, displays potent activities against microorganisms such as Pneumocystis jiroveci, Plasmodium falciparum and Trypanosoma rhodesiense. Furthermore, a methamidoxime prodrug of furamidine is currently undergoing Phase III clinical trials against human African trypanosomiasis.5

We recently reported the in vitro antiparasitic activity of furamidine (DB75) and its N-phenyl-substituted analogue (DB569).6 Both DB75 and DB569 showed similar DNA minor groove binding at AT-rich DNA sequences; however, they presented distinctly different uptake profiles in cancer cells.5 Addition of phenyl groups to the amidines significantly improved the trypanocidal and leishmanicidal activity.6 DB569 presented higher in vitro activity than DB75 against two T. cruzi stocks corresponding to biodemes I (sylvatic cycle) and II (domestic cycle). However, Dm 28c parasites, representative of biodeme I, were less sensitive than Y parasites, representative of biodeme II. Transmission electron microscopy showed that the morphological changes were more severe in the nucleus and kinetoplast of DB569-treated parasites, resulting in mitochondrial enlargement and fragmentation of the kinetoplast into condensed bodies.6 These results prompted us to investigate the efficacy of DB569 in a T. cruzi mouse model.


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Parasites

Bloodstream trypomastigotes of the Y strain were used and harvested by heart puncture from T. cruzi-infected Swiss mice at the parasitaemia peak, as described previously.6

In vivo infection

Swiss male mice (25–30 g) were obtained from the animal facilities of CECAL (FIOCRUZ, Rio de Janeiro, Brazil). Mice were housed 8 per cage and kept in a conventional room at 20–25°C under a 12/12 h light/dark cycle. The animals were allowed to equilibrate for 7–15 days before starting the experiments and were provided sterilized water and chow ad libitum. Infection was performed by intraperitoneal (ip) injection of 104 bloodstream trypomastigotes. Age-matched non-infected mice were maintained under identical conditions.

All assays were run 3–5 times and procedures were carried out in accordance with the guidelines established by the FIOCRUZ Committee of Ethics for the Use of Animals (CEUA 0099/01).

Experimental groups

The animals were divided into the following groups: non-infected (G1), infected and non-treated (G2), infected and treated with 20 mg/kg DB569 (G3), infected and treated with 50 mg/kg DB569 (G4), non-infected and treated with 50 mg/kg DB569 (toxicity) (G5) and infected and treated with 100 mg/kg benznidazole (G6). At least eight mice from each group were used for analysis at each different day post-infection (dpi) and five independent experiments were performed.

Drugs and treatment schedules

The synthesis of DB569 has been reported previously.6 Stock solution (5 mM) of diamidine was prepared in DMSO and mice received 0.1 mL ip daily injection of 20 mg/kg/mouse or 50 mg/kg/mouse DB569, using two different schedules: (i) from 8 (parasitaemia peak) to 14 dpi and (ii) from 3 to 12 dpi. Benznidazole (N-benzyl-2-nitroimidazoyl acetamide, Rochagan, Roche, Rio de Janeiro, Brasil) was administered at 100 mg/kg by oral route (gavage) starting at 3 dpi for 10 consecutive days.

Parasitaemia and mortality

Mortality was checked daily until 21 dpi and expressed as the percentage of cumulative mortality (%CM).7 Parasitaemia was individually checked by direct microscopic counting of parasites in 5 µL of blood, as described previously.7 At 8, 15 and 21 dpi, body weight was evaluated, blood was collected from the tip of the tail of mice of all experimental groups for the determination of plasma levels of creatine kinase cardiac isoenzyme, alanine aminotransferase and creatinine, and the mice were sacrificed and the hearts were quickly removed and processed for histopathological analysis. The plasmas were immediately analysed as described previously.7

Biochemistry

  1. Creatine kinase (E.C. 2.7.3.2 [EC] ) cardiac isoenzyme (CK-MB): For the analysis of cardiac injury, CK-MB activity was determined using a commercial kit (Granutest kit, Merck Darmstadt, Germany) as recommended by the manufacturer. The results were expressed in mean variation of absorbance obtained in each of seven sequential readings at 1 min intervals ({Delta}A340/min) using a 340 nm filter. The variance (ANOVA) analysis was used to compare non-treated and treated groups and results were considered statistically significant with P ≤ 0.05.
  2. Alanine aminotransferase (ALT) and creatinine: To evaluate hepatic dysfunction, the activity of ALT was analysed in all groups of mice at the end of drug administration (at 13 dpi) following manufacturer recommendations (LabTest Diagnostica SA, Minas Gerais, Brazil) and the results expressed as enzyme concentration (IU/L). For renal dysfunction, creatinine levels were measured as indicated (LabTest Diagnostica SA) and were expressed as mg/dL. In all assays, non-treated and treated groups were compared using analysis of variance (ANOVA) and results were considered statistically significant with P ≤ 0.01.

Histopathological analysis

At 8, 15 and 21 dpi, hearts were removed, cut longitudinally, rinsed in ice-cold phosphate-buffered saline (PBS) (10 mM sodium phosphate, 0.015 M NaCl, pH 7.4) and fixed in Millonig-Rosman solution (10% formaldehyde in PBS). The fixed tissues were dehydrated and embedded in paraffin. Sections (3 µm) stained by routine haematoxylin–eosin (HE) were analysed by light microscopy. The extent of fibrosis and the number of amastigotes' nests and of inflammatory infiltrates (more than 10 mononuclear cells) were determined in at least 200 fields (total magnification, x63) for each slide. The mean number of amastigotes' nests or inflammatory infiltrates per field was obtained from at least three mice per group (8, 15 and 21 dpi), with three sections from each mouse. Fibrosis was further studied by Masson trichrome. Student's t-test was applied to ascertain the significance of the observed differences and results were considered statistically significant with P ≤ 0.01.


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DB569 (Figure 1) shows significant in vitro activity against extracellular and intracellular forms of T. cruzi6 and we report here its efficacy in a mice model. Figure 2 shows the results of the treatment performed from 8 to 14 dpi using 20 and 50 mg/kg DB569. Both treatments led to lower mortality levels as compared with non-treated mice (G2), with, respectively, 25% and 12.5% decrease in the cumulative mortality (Figure 2a). Treatment of non-infected mice with 50 mg/kg DB569 (G5) did not result in death of the mice, showing no overt toxicity at the higher dosage of the diamidine. As expected, the infected and non-treated group (G2) presented 100% of mortality at 20 dpi. Both doses of DB569 partially reduced the number of circulating parasites as compared with non-treated mice (Figure 2b). As expected, the infection by T. cruzi induced a loss of body weight when compared with non-infected animals and the treated mice (20 and 50 mg/kg) regained, only slightly, body weight at 15 dpi (data not shown).


Figure 1
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  		Figure 1. Structure of DB569.

 

Figure 2
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  		Figure 2. Treatment of T. cruzi-infected mice (104 Y strain/mice) with DB569 from 8 to 14 dpi: (a) percentage of cumulative mortality; (b) kinetics of parasitaemia; and (c) CK-MB activity at 15 dpi. Symbols in (a) and (b): open circles, non-infected (G1); filled diamonds, infected and non-treated (G2); filled squares, infected and treated with 20 mg/kg DB569 (G3); filled triangles, infected and treated with 50 mg/kg DB569 (G4); and open triangles, non-infected and treated with 50 mg/kg DB569 (toxicity) (G5). Bars in (c): G1, non-infected; G2, infected and non-treated; G3, infected and treated with 20 mg/kg DB569; G4, infected and treated with 50 mg/kg DB569; and G5, non-infected and treated with 50 mg/kg DB569 (toxicity). Asterisks indicate significant differences in relation to the non-treated group, P ≤ 0.05.

 
CK-MB, a well-known marker of cardiac damage induced by T. cruzi infection,8 was significantly higher in the non-treated group (G2) as compared with the mice treated with 20 mg/kg DB569 (P ≤ 0.05) (Figure 2c). However, administration of 50 mg/kg DB569 to infected mice did not reduce enzyme activity compared with the non-treated group. The non-infected and treated mice (toxicity group) did not present increased levels of CK-MB suggesting that DB569 did not cause cardiotoxicity (Figure 2c).

Since the treatment with 20 and 50 mg/kg DB569 led to similar results (body weight and parasitaemia levels), and as the higher dose induced higher cardiac lesions and mortality rates, subsequent experiments were performed administering 20 mg/kg DB569 from 3 to 12 dpi. As a positive control, infected mice were treated orally with 100 mg/kg benznidazole.9

Non-treated mice presented high parasitaemia levels peaking at 8 dpi, and DB569 treatment resulted only in a slight decrease in parasitaemia whereas benznidazole induced a remarkable reduction in the number of circulating trypomastigotes (Figure 3a). Although DB569 led to a 40% reduction of the mortality compared with non-treated mice, all the benznidazole-treated and the non-infected mice survived (data not shown).


Figure 3
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  		Figure 3. Treatment of T. cruzi-infected mice (104 Y strain/mice) with 20 mg/kg DB569 from 3 to 12 dpi. Kinetics of (a) parasitaemia and (d) CK-MB activity of open circles, non-infected (G1); filled diamonds, infected and non-treated (G2); filled squares, infected and treated with 20 mg/kg DB569 (G3); and filled circles, infected and treated with 100 mg/kg benznidazole (G6). (b) Amastigotes' nests and (c) inflammatory infiltration in the heart of: white bars, infected and non-treated (G2); black bars, infected and treated with 20 mg/kg DB569 (G3); and grey bars, infected and treated with 100 mg/kg benznidazole (G6). Asterisks indicate significant differences in relation to the non-treated group, P ≤ 0.01.

 
The analysis of heart parasitism showed that DB569 dramatically reduced (P ≤ 0.01) the parasite load at 15 dpi approaching that of benznidazole therapy, which completely abolished the cardiac parasitism (Figure 3b). Benznidazole-treated mice did not present considerable inflammatory infiltration; however, DB569-treated ones showed cardiac inflammation similar to that in non-treated animals both at 15 and 21 dpi (Figure 3c). At 21 dpi, heart fibrosis was lower in the treated mice than in non-treated ones (data not shown). Determination of CK-MB plasma activity showed higher cardiac lesions in the infected and non-treated group compared with the non-infected one (Figure 3d). Benznidazole significantly decreased enzyme levels, indicating a reduction of cardiac lesions, while such an effect was not seen in the DB569-treated group (Figure 3d).

The measurement of ALT and creatinine showed that infection by T. cruzi induces important hepatic and renal lesions; for the DB569-treated group lower levels of ALT (P = 0.004) and creatinine (P = 0.0001) were found (data not shown). Although presenting a high antiparasitic effect, benznidazole did not prevent renal damage (data not shown).


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Chagas' disease is an incurable pathology and despite the fact that other tissues can also be affected heart myofibres represent important targets for the parasite. Although an effective inflammatory response is needed for controlling parasite growth, excessive or uncontrolled inflammation can contribute to the cardiac damage.8 Previously reported data suggest that tissue damage is direct or indirectly induced by T. cruzi itself.9

Although initiatives of both the Pan American Health Organization (PAHO) and World Health Organization (WHO) have led to vector control in the Southern Cone countries and consequently to a decrease in the incidence of new infections, Chagas' disease remains an unsolved problem due to (i) the failure to treat chronic cases, (ii) the high level of acute cases in certain endemic areas and (iii) the occurrence of new resistant parasite strains.2 Consequently new drugs are urgently needed.

Aromatic diamidines represent an important class of DNA minor groove binders of high therapeutic interest as antiparasitic agents, being effective against protozoa parasites.5 Since we previously showed the higher antiparasitic activity against bloodstream trypomastigotes of DB569 compared with DB75, this N-phenyl analogue was selected for in vivo analysis.

Starting at 8 dpi treatment with DB569 doses of 20 and 50 mg/kg showed that (i) both doses partially reduced the mortality and parasitaemia levels and (ii) the dose of 20 mg/kg caused less cardiac lesions compared with the non-treated group. These results led us to treat the mice with 20 mg/kg from 3 to 12 dpi. In the present study we found that although DB569 did not reduce the number of circulating bloodstream trypomastigotes, it was able to decrease the mouse mortality. Both tissue parasitism and fibrosis were reduced similar to that observed with benznidazole. Since histopathological analysis showed lower cardiac parasitism in DB569-treated mice, the cardiac damage detected by CK-MB activity in this group was probably due to cardiac inflammation and not due to the tissue parasitism itself. The parasitaemia levels were similar in DB569-treated and untreated groups; however, a marked reduction in heart parasitism was noted for the treated group. Consequently, the origin of the circulating parasites may be other affected tissues and/or that DB569 reduced heart parasitism by acting in synergism with cardiac inflammatory cells and/or even exacerbating their microbicidal ability. In fact, our previous in vitro data corroborate this latter hypothesis, since parasite elimination was more effective after treatment of infected phagocytic cells as compared with cardiac ones.6 This effect on the microbicidal activity of phagocytes is now under investigation.

DB569 diminished both renal and hepatic damage caused by the parasite infection, which may be due to reduced tissue parasite load as we noted in the cardiac samples. The reduced renal and hepatic damage as well as reduced heart parasitism possibly contributed to the increased survival rates found in the DB569-treated infected mice group.

Currently, there is no efficient therapy for Chagas' disease,10 pointing to the urgent need for less toxic and more effective new drugs. In this context, aromatic diamidines have been explored against a wide range of microorganisms and represent potential compounds to be assayed against T. cruzi.6 Currently, a novel prodrug of furamidine (DB75), 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), is in Phase III clinical trials for first-stage African trypanosomiasis and P. jiroveci.5 The high activity of this class of compounds justifies the present study and further in vitro and in vivo investigations with other analogues.


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None to declare.


   Acknowledgements
 
MNCS and EMDS would like to thank Dr Solange Lisboa de Castro for manuscript review and Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and PAPES IV/FIOCRUZ for financial support. The funding to D. W. B. by the Bill and Melinda Gates Foundation is gratefully acknowledged.


   References
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1 World Health Organization. (1999) Chagas Disease. Tropical Disease Research, 18th Program Report, UNDP/WB/TDR (WHO, Geneva).

2 Coura JR and de Castro SL. (2002) A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz 97:3–24.[Medline]

3 Paulino M, Iribarne F, Dubin M, et al. (2005) The chemotherapy of Chagas' disease: an overview. Mini Rev Med Chem 5:499–519.[CrossRef][Web of Science][Medline]

4 Wilson WD, Nguyen B, Tanious FA, et al. (2005) Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity. Curr Med Chem AntiCancer Agents 5:389–408.

5 Soeiro MNC, De Souza EM, Stephens CE, et al. (2005) Aromatic diamidines as antiparasitic agents. Expert Opin Investig Drugs 14:957–72.[CrossRef][Web of Science][Medline]

6 De Souza EM, Lansiaux A, Bailly C, et al. (2004) Phenyl substitution of furamidine markedly potentiates its antiparasitic activity against Trypanosoma cruzi and Leishmania amazonensis. Biochem Pharmacol 68:593–600.[CrossRef][Web of Science][Medline]

7 De Souza AP, Olivieri BP, de Castro SL, et al. (2000) Enzymatic markers of heart lesion in mice infected with Trypanosoma cruzi and submitted to benznidazole chemotherapy. Parasitol Res 86:800–8.[CrossRef][Web of Science][Medline]

8 Teixeira MM, Gazzinelli RT, Silva JS. (2002) Chemokines, inflammation and Trypanosoma cruzi infection. Trends Parasitol 18:262–5.[CrossRef][Web of Science][Medline]

9 Higuchi ML, De Brito T, Reis MM, et al. (1993) Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: light microscopy and immunohistochemical findings. Cardiovasc Pathol 2:101.

10 Croft SL, Barret MP, Urbina JA. (2005) Chemotherapy of trypanosomiases and leishmaniasis. Trends Parasitol 21:508–12.[CrossRef][Web of Science][Medline]


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