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Modifications in Chagas´Disease Evolution
Using Tricyclic Drugs

Rivarola, Hector; Fernández A., Ruth;
Enders, Julio; Paglini Oliva, Patricia

Cátedra de Física Biomédica, Facultad de Ciencias Médicas,
Universidad Nacional de Córdoba, Córdoba, Argentina

SUMMARY
Chagas´disease is caused by the protozoan Trypanosome cruzi. About 24 million people suffer from this disease in developing countries of South and Central America. The two currently drugs use to trest the infection, nifurtimox and benznidazole, frequently produce adverse reactions and in some cases are mutagenic. The search of more effective and less toxic drugs are here presented. Some T. cruzi enzymes as trypanothione reductase and calmodulin represent targets for phenothiazines and related compounds that are able to inhibit both enzymes, that play an essential role in the parasite life. Clomipramine (5mg/Kd/day for 1 month) CLO and thioridazine (80 mg/Kg/day for 3 days) THI, belong to these drugs and have been found to be trypanocidal in "in vitro" studies. They were used as treatment for mice infected with 50 tripomastigotes of T. cruzi Tulahuen strain (control). The effectiveness was detected studding parasitemias, survival, electrocardiography histopathology and affinity and density of cardiac beta receptors that were significantly affected in Chagas´disease.
Parasitemias were lower in treated mice (control 255±20, CLO 85±5, THI 98±3 parasites/µl, p< 0.01 respectively) and became negative by day 30 using either of the drugs. By day 135 (chronic phase) 80 % of untreated mice were dead, but 80% of treated survived at this time. Two years later this mice were alive. Low frequency of mild electrocardiographic abnormalities (prolonged PQ segment and QRS segment) were detected in treated groups , p< 0.001 than infected. Hearts from treated mice showed minor inflammatory infiltrates in the acute phase, but none structural abnormalities were detected in the chronic phase (135 days post infection). At this time, treated animals had significantly lower cardiac beta receptors affinity [Kd (nM):CLO 4,56±0.98, THI 5.48±0.21] and higher density [Bmax (fmol/mg protein): CLO 65.01±4.88, THI 72.34±1.06] when compared with untreated infected mice Kd (nM) 11.21± 0.26, Bmax (fmol/mg protein) 53.33±0.71, p<0.01. Values from treated animals were not different from those of uninfected animals (Kd: 3.61±0.05, Bmax 71.97±0.36) except for THI treated that the affinity remained higher.
Both tricyclic drugs are in clinical use, for this the present results can be the basis for new agents for the treatment or prevention of Chagas´disease.

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INTRODUCTION
   Chagas´disease is caused by the protozoan Trypanosome cruzi. About 24 millions people are infected in Latin America out of which 2 millions live in Argentina (WHO 1991; Segura et al. 1999).

   The current drugs used in acute Chagas´disease treatment, (Nifurtimox and Benznidazole) have high toxicity so their side effects are not desirable and patient gives up the treatment (Castro & Diaz de Toranzo 1998; Morello 1988) for this chemotherapy of Chagas'disease is still an open field and remains as an unsolved problem.

   T. cruzi enzymes such as trypanothione reductase and calmodulin, represent potential drugs targets because they play an essential role in the life of this organism (Engels and McKerrow 1995), so drugs as phenothiazines and related compounds that inhibit these enzymes (Bondy 1995, Paglini-Oliva et al. 1998, Stoppani 1999 ) are potential chemotherapic compounds.

   Phenothiazines and some related compounds are tricyclic drugs used in psychiatric treatments, and as antihistaminic and antihemetic. Clomipramine and thioridazine are drugs that belong to that series, and exert trypanocidal effects upon epimastigotes and tripomastigotes in vitro (Barioglio et al. 1987, Paglini et al. 1998).

   The purpose of this study was to analyze the effect of Clomipramine and Thioridazine upon T. cruzi infection in mice to determine its probable therapeutic effect . Treatment effectiveness was assessed by studying parasitemia, survival, electrocardiography , density and affinity of the cardiac receptors and histopathology of heart.

MATERIAL AND METHODS
   Thioridazine hidrochloride (THI) (Sandoz) 80 mg/Kg/day for 3 days, oral way and Clomipramine (CLO) (Sigma Chemical Co.) 5 mg/Kg/day for 30 days intraperitoneally were used.
Albino Swiss male mice were challenged with 55 tripomastigote forms of T. cruzi, Tulahuen strain, by an intraperitoneal injection.Then they were divided into the following groups: a) uninfected (n:20) b)control: treated daily with isotonic saline intraperitoneally (n:20), c) treated with THI (n:10) d) treated intraperitoneally with CLO (n:10). Parasitemias were determined in Neubawer hemocytometer from blood samples obtained from the tail 2 times a week . Electrocardiograms were obtained with an electrocardiographic apparatus (Fukuda), under Ketalar HCl (Parke Davis) anesthesia 10 mg/kg, at 35, 75 and 135 days post infection . The six standard leads(dipolar leads D1, D2, D3 and unipolar leads aVR, aVL, aVF) were studied recorded at 50 mm/s.

   b adrenergic receptors binding was performed in microsomal fraction of right and left ventricles from the 4 groups under study 30, 75 and 135 days post-infection. 3H/dihydroalprenolol ( NEN, USA) was used as radioligand and the experiments were carried out as previously (Fernández et al 1995).Dissociation constant (Kd) and maximum 3H/DHA binding (B max) were determined by a saturation curve and Scatchard analysis using GraFit (Erithacus Software Limited).

   Histopathologic studies were made 30, 75 and 135 days post-infection fixing the organs and staining them with hematoxylin-eosin technique. A total of 50 slices from each group were analyzed. At least 30 areas from each slice were examined with a 40 x objective.

Statistical analysis
Data were compared by analysis of variance, Student´s t test and multiple comparison by Tuckey Test and Chi square for categoric variables; significance level was set at 0.05.

RESULTS
   Parasitemias were lower in treated mice (control 255±20, CLO 85±5, THI 98±3 parasites/ml, p< 0.01 respectively) and became negative by day 30 using either of the drugs. By day 135 (chronic phase) 80 % of untreated mice were dead, but 80% of treated survived at this time. Two years later this mice were alive.

   The electrocardiographic abnormalities observed (pulse rate, auricle-ventricle conduction, prolonged PQ segment, and intraventricular conduction, prolonged QRS complex) were significantly more frequent in the untreated mice. See Table 1A, Table 1B and Table 1C.


Tabla 1 A


Tabla 1 B

Tabla 1 C

Table 1A, Table 1B and Table 1C: Results of the electrocardiographic and ß-receptor studies of uninfected mice and mice infected with Trypanosoma cruzi and left untreated or treated with thioridazine (THI) and Clomipramine (CLO).

   Hearts from untreated mice showed inflammatory infiltrates, with mononuclear cells, and nests of amastigotes, the histopathological alteration typical of acute chagasic myocarditis. Hearts from treated with either drugs showed minor inflammatory infiltrates by day 35 and no detectable abnormalities by day 135(chronic phase).At this time,treated animals had significantly lower cardiac beta receptors affinity [Kd (nM): CLO 4,56±0.98, THI 5.48±0.21] and higher density [Bmax (fmol/mg protein): CLO 65.01±4.88, THI 72.34±1.06] when compared with untreated infected mice (Kd (nM) 11.21± 0.26, Bmax (fmol/mg protein) 53.33±0.71, p<0.01.Values from treated animals were not different from those of uninfected animals (Kd: 3.61±0.05, Bmax 71.97±0.36) except for THI treated that the affinity remained higher. Values of affinity and density during acute and indeterminate phase can be observed in Table 1A, Table 1B and Table 1C.

DISCUSSION
   There is an indisputable and urgent need for new, safe and effective drugs for the treatment of Chagas'disease (Fairlamb 1999). In the present we studied T. cruzi infected mice treated with the tricyclic drugs THI or CLO, ,that negativized their parasitemias by day 30 post infection (pi) and 80% of them survived until two year that was the moment that we ended our experiments, whereas the non treated ones only 20% were alive 24 months pi. Cardiac abnormalities seen in hearts of infected and treated mice were minor and the examined 135 days pi appeared to have normal cardiac histology , significantly less electrocardiographic alterations or none in CLO treated ones. At this time, treated animals had significantly lower cardiac beta receptors affinity and higher density when compared with untreated infected mice Values from treated animals were not different from those of uninfected animals except for THI treated that the affinity remained higher.

   It has been reported that some tricyclic compound ,as the used in the present work, bind selectively to T. cruzi enzymes such as trypanothione reductase and calmodulin (Bondy 1995; Douglas 1995; Stoppani 1999). These effect could be taking place and could help to explain the therapeutic effect verified in the THI and CLO treated animals, because the drugs were toxic for the parasite but almost induced no damage in the host.

CONCLUSION
   Our findings show that CLO and THI which are generally used in clinical psychiatry were also effective in treatment of experimental Chagas' disease, since it proved to modify significantly the natural evolution of the infection. These results demonstrated that calmodulin and trypanothine reductase inhibitors may form the basis for new agents for the treatment of Chagas disease.

REFERENCES

1. -Segura, E.L.; Sosa Estani, S.; Esquivel, M.; Gómez, A.& Salomón, O.D.(1999). Control of the transmission of Trypanosoma cruzi in Argentina. Medicina, 59, 91-96.

2. World Health Organization (WHO). (1991) Chagas´disease tropical disease progress in research 1989-1990. Tenth Program Report of the World Health Organization. Geneve.

3. Morello, A. (1988) The biochemestry of the mode of action of drugs and the detoxication mechanism in Trypanosoma cruzi. Comparative Biochem and Physiol, 90, 1-12.

4. Castro, J. A., Diaz de Toranzo, E. G. (1988). Toxic effects of nifurtimox and benznidazole, two drugs used against American Trypanosomiasis (Chagas´disease) Biom and Envir Sci, 1, 19-33.

5. Engel, J. C., and McKerrow, J.H. (1995). Physiological and ultrastructural changes induced by cysteine proteinase inhibitors in Trypanosoma cruzi . Mem. Inst. Oswaldo Cruz 90, 43-44.

6. Bondy, B. (1995). Resents trends in the field of calmodulin inhibitors. In Biological and Chemical Aspects of Thiazines and Analogs, Vol.1, eds Barbé, J., Keyser, H. & Soyfer, J.C. pp. 433- 434. San Gabriel, CA: English Associated.

7. Paglini-Oliva, P., Fernandez A. R., Fretes, R., Pelsman, A. (1998)Structural, ultrastructural studies and evolution of Trypanosoma cruzi infected mice treated with thioridazine. Exp. Mol. Pathol. , 65: 78-86.

8. Stoppani,A.O.M. (1999). The chemotherapy of Chagas´disease. Medicina, 59, 147-165.

9. Barioglio, S.R., Lacuara, J. L., Paglini?Oliva, P. (1987). Effects of clomipramine upon motility of Trypanosoma Cruzi. J Parasitol, 73, 451?452.

10. Fernández, A.R.; Enders, J.E.; Rivarola, W.; Paglini, P.; Palma, J.A. (1995). Acute Chagas' disease: study of beta receptors in two experimental models. Mem Inst Oswaldo Cruz ,90, 79.

11. Fairlamb, A.H. (1999). Future prospects for the chemotherapy of Chagas´disease. Medicina, 59, 179-187.

12. Douglas, K. T. (1995) Phenothiazines as trypanothione reductase inhibitors. In Biological and Chemical Aspects of Thiazines and Analogs, Vol.1, eds Barbé, J., Keyser, H. & Soyfer, J.C. pp. 317- 328. San Gabriel, CA: English Associated.

 

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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
Steering Committee
President
Dr. Raúl Bretal
Scientific Committee
President
Dr. Armando Pacher
Technical Committee - CETIFAC
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