Effects of clomipramine on Trypanosoma cruzi infection in mice

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(2001)95,529-533

Effects of clomipramine

on Trypanosoma

cruzi infection

in mice

H. W. Rivarola’, A. R. Fernzindez*, J. E. Enders’, R. Fretes3, S. Gea4 and P. Paglini-Oliva’ ‘Chtedru de Fz’sica Biokdica, Facultad de Ciencias Midicas, Universidad National de Cdrdoba, Santa Rosa 1085, 5000 Cdrdoba, Argentina; 2Consejo National de Investigaciones Cientifcas y Tknicas de la Repziblica Argentina; jC&edra de Histologia, Embriologia y Genitica II, Facultad de Ciencias Mbdicas, and 4Departamento de Inmunologia, Facultad de Ciencias Quimicas, Universidad National de Cdrdoba, 5016 Chdoba, Argentina Abstract Trypanosoma cruzi, widely distributed in Latin American countries, provokes Chagas disease, characterized by cardiomyopathy and mega-viscera. The drugs used currently for treatment of acute Chagas disease are highly toxic; the side-effects are undesirable and patients may abandon treatment. We have previously demonstrated that clomipramine (CLO) exerts trypanocidal effects upon epimastigotes and trypomastigotes in vitro with anticalmodulin activity. The present study analyses the effectiveness of CL0 treatment in mice infected with a low number of T. cruzi, an animal model that reproduces acute, indeterminate and chronic phases of this trypanosomiasis. In this work, our results demonstrated that CL0 5 mg/kg daily for 30 days, or 2 doses of CL0 40 mg/kg given intraperitoneally at 1 h and 7 days after infection, was not toxic for the host, but was effective against the parasite in that parasitaemias became negative and only mild heart structural and electrocardiographic alterations were detected in the chronic phase in the group treated with CL0 5 mg/kg. In mice treated with CL0 40 mg/kg, none of these alterations was detected. Cardiac p receptor density and affinity returned to normal in the chronic stage in both experimental groups. T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets. It has been reported that both can be inhibited by CLO, a tricyclic drug used in clinical therapeutics. We have shown that CL0 strongly decreased the mortality rate and electrocardiographic alterations; in addition cardiac fi receptor density and heart histology returned to, or close to, normality 135 days post infection. These results clearly demonstrated that CL0 treatment modified significantly the natural evolution of T. cruzi infection. Keywords: Chagas disease, Trypanosomacruzi, animal models, mice, chemotherapy, clomipramine, histology, heart, electrocardiography

Introduction One of the most important factors that affect health and produce sudden death in Latin American countries is &e parasite, Trypanosoma cruzi, which is widely distributed there. T. cntzi causes Chagas disease. which is characterized by cardiomyopathy &d mega-Viscera. About 24 million people are infected out of which 2 million live in Argentina (WHO, 1991; SEGURA et al., 1999). Studies of Ca*+ homeostasis in this parasite are important because they have offered potential targets fo; chemotherapy (DO&MPO, 1993; DOCAMPO et-al., 1993, ~~~~;DOCAMPO&MORENO. 1996:Z~AN~etul.. 1998). In addition, both parasite’and Gost-cell Ca2’ homeostasis seem to be important factors in the hostparasite relationship (MORRIS et al., 1991, 1998; DOCAMPO & MORENO, 1996). It has also been reported that T. cruzi has a complex profile of calcium-dependent calmodulin-binding proteins (DOCAMPO 8.1VERCESI, 1989; VERCESI et al., 199 l), and that their concentrations are modified in the different parasite life-stages (ORR et al., 1992). These proteins represent potential drug targets (STOPPANI, 1993) because of their relationship with Ca2+ homeostasis. Phenothiazines and some related compounds are tricyclic drugs used in psychiatric treatment, and as antihistaminics and antiemetics. Among their different biological activities (MOLNAR et al., 1993a, b), they have marked anticalmodulin properties (PROZIALECK & WEISS,~~~~;BONDY,~~~~;STOPPANI,~~~~,~~~~). The drugs used in current treatment of acute Chagas disease (nifurtimox and benznidazole) have high toxicity and undesirable side-effects which may result in the patient giving up treatment (CASTRO & DIAZ DE TORANZO, 1988; MORELLO, 1988; GORLA et al., 1989). Also, the genetic structure of the parasite (BLANCO, 1993) leads to the frequent appearance of strains more

Address for correspondence: Patricia Paglini-Oliva, D. Rapela 3258, Residential V. Sirsfield, 5016 Cbrdoba, Argentina; fax +54 351 4617150, e-mail [email protected]

resistant to treatment. Therefore there is a real need for investigation into other drugs effective for Chagas disease. Clomipramine is a tricyclic drug that exerts trypanotidal effects upon epimastigotes and trypomasti&tes in vitro (BARIOGLIO et al., 19871 with anticalmodulin activity. In this study we analyseh the effect of clomipramine upon T. cruzi infection in mice to determine its probable therapeutic effect. Treatment effectiveness was assessed by studying parasitaemia, survival, serology, electrocardiography, density and affinity of the cardiac receptors and heart histopathology. Materials and Methods Experimental infection Blood collected into heparinized tubes from mice infected with the Tulahuen strain of T. cruzi was used as the inoculum for another IO0 mice (Swiss albino males, each weighing 30 & 1 g). The parasite concentration in this blood was estimated in a Neubauer haemocytometer, so that each mouse could be inoculated, intraperitoneally, with blood containing about 55 bloodstream trypomastigotes. The mice were then divided into the following groups: (i) control: treated daily with isotonic saline -intraperit&eally (n = 20); (ii) treated intraneritoneallv with clominramine (CL01 KGrna Chemical Co., 8t Louis, MI,- USA) 5 mg/kg dailifor 30 days (CL0 5) (n = 40); (iii) treated with CL0 40 mg/kg at 60 min post-infection (pi) and 7 days pi (CL0 40) (n = 401. These CL0 doses were selected Because a pievious’study (BAIUOGLIO et al., 1987) had shown them to be effective in the acute phase of T. cruzi infection. A Neubauer haemocytometer and tail-vein blood samnles collected twice a week from dav 7 nest-infection were used to follow parasitaemias in each infected mouse. Survival was monitored every day, at 13:O0. Serology Blood samples were collected 35, 75 and 135 days post-infection, representing the acute, intermediate and chronic phases of the infection, respectively. These

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samples were then assayed for antibodies to ‘cruzipain’ antigen (see below), by ELISA (GEA et al., 1992).

At least 30 areas from each slice were examined with a X40 objective.

Cruzipain antigen Epimastigotes of the Tulahuen strain were grown at 26°C in neutralized, liver-digest medium (Oxoid, Basingstoke, Hampshire, UK) supplemented with 0.5% t@ptose, 10% fetal calf serum, 200 mg/mL haemin and 100 U/mL streutomvcin. Thev were harvested while in exbonential grow&, centri&ged (5000 g, 10 min), and washed with phosphate-buffered saline. A cysteine-protease inhibitor (1 mM N-a-ptosyl-L-lysine chloromethyl ketone) was added before the pelleted parasites were lysed by 8 cycles of freezing and thawing. The parasite homogenate was centrifuged (105 000 g) and cruzipain was purified from the supernatant solution by affinity chromatography (LABRIOLA et al., 1993). The absence of enzyme activity in the (non-reduced, unboiled) antigen preparation was checked by polyacrylamide gel electrophoresis at pH 5.7, in gels containing 0.1% gelatin (Sigma); after incubation to allow any protease to hydrolyse the gelatin, the gels were stained with Coomassie Blue R250.

Statistical analysis Data were compared by analysis ofvariance, Student’s t test and multiple comparison by Tukey tests; significance level was set at 0.05.

Electroca;dic$$ams were obtained with an electrocardiographic apparatus (Model FD-16, Fukuda Denshi, Tokyo, Japan), under Ketalar HCl (Parke Davis) 10 mg/kg anaesthesia, for non-infected, infected, and infected and clomipramine-treated groups at 3575 and 135 days pi. The electrocardiographic tracings were obtained with 6 standard leads (dipolar leads Dl, D2, D3 and unipolar leads aVR, aVL, Ave). The tracings were recorded at a paper speed of 50 mm/s and a calibration amplitude of 1 mV = 10 mm. Adrenergic /3' receptors The l3’ adrenereic receutor binding was nerformed in right aid left venthcles of&e experimental groups under study at 30,75 and 135 days pi. A pool of 2 ventricles was homogenized in 10 volumes of ice-cold homogenization buffer (250 mMsucrose. 1 mMM&l?. and 20 mMTrisHCl, p-H-7.4). Homogehates werece&ifuged at 2000 g for 10 min. Pellets were homogenized again and centrifuged at 40 000 g for 30 min and twice with KC1 0.6 M in homogenization buffer only. The final pellet was suspended in incubation buffer (mM composition: 125 MgC&; 1.5 EDTA; 75 Tri-HCl; pH 7.65) in a volume of 1 mL/g of wet tissue. [3H]Dihydroalprenolol (r3H]DHA, specific activity 3.5 15 X lOI Bq/mol from NEN, Boston, USA) was used as radioligand in (3-adrenergic receptor binding assays. The experiments were carried out in triplicate with 100 pL of membrane suspension (480 mg protein) and [‘HIDHA (2.4-l I.5 nM) incubated at 37°C for 10 min in a final volume of 1 mL. The incubation was concluded by adding 1 mL of cold incubation buffer to each tube and rapidly filtering the contents under reduced pressure through Whatman GF/B filters. The filters were dried and transferred to vials to count radioactivity in Aquasol Universal LSC cocktail (NEN). Specific binding was defined as the difference in radioactivity bound in the absence or presence of propanolo1 1 @. Dissociation constant (&) and maximum [3H]DHA binding (B,,,) were determined by a saturation curve and Scatchard analysis using GraFit (Erithacus Software, Staines, UK). Histopathology Assessments were made 30, 75 and 135 days pi. The animals were killed by ether anaesthesia and the hearts dissected. The organ was fured in buffered (pH 7.0) 10% formalin and embedded in paraffin. The tissue was cross sliced from the apex to the auricles. The slices (5 pm thick) were stained with the haematoxylin-eosin technique. A total of 50 slices from each group were analysed.

Results Parasitaemias Figure 1 shows the parasitaemia evolution observed in infected mice with and without treatment. It can be seen that untreated mice had a peak of parasitaemia by day 35 post inoculation, decreasing anh becoming negative from dav 45. whereas mice treated with CL0 5 rng/kg daily for-30 days or with 40 mg/kg in only 2 doses shc&ei significantly lower parasitaemias remaining in values close to zero along the evolution of the infection. Electrocardiography The electrocardiographic studies (Table) showed that untreated infected animals had increased alterations with T. cruzi infection evolution. Treated animals presented lower conduction disturbances from 75 days pi to the end of the experiment. No electrocardiographic alterations were detected in mice from the CL0 5 treated group at 135 days, the chronic phase. Cardiac p adrenergic receptor affinity and density The Table shows the analysis of cardiac fi adrenergic receptors on myocardium membranes obtained from uniifected, infe&ed (in the acute, indeterminate and chronic phase) and infected and CL0 5 and CL0 40treated mice at the same periods. At 35 days pi the affinity in the CL0 5 group was diminished (P < 0.01). A marked decreaseof &e receptor density can be obse’rved also in the CL0 5 mice (P < 0.01) at the indeterminate stage. Both CL0 treatments resulted in density and affinity returning to normal values in the chronic phase. Serology The anti-cruzipain IgG evolution along the 3 stages of the experimental T. cruzi infection (0.36 & 0.1 optical density) showed that the antibody levels in the CL0 5 and CL0 40-treated groups were lower than in untreated mice. However their titres remained higher than observed in uninfected animals, until the end of the experiment (35 days pi: 0.8 * 0.1; 75 days pi: 0.17 * 0.1; 135 days pi 0.15 f 0.1). Antibody optical density in the uninfected group (n = 10) were 0.04 z!=0.01. Antibody titres were similar for both treatments (CL0 5 and CL0 40) during the 3 stages of the disease. Survival The survival of the treated animals at the end of the experiment (135 days) was 75% in the CL0 5 group and

0

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100 60 80 Days Post-infection

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Fig. 1. Parasitaemia evolution in T. cruzi-infected mice that were untreated (0; n = 20) or treated with clomipramine 5 mg/kg daily for 30 days (A; n = 40) or with clomipramine 40 mg/kg at 1 h and 7 days post infection (U; n = 40). Results are given as mean f SE.

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70% in the CL0 40 group. Two years later all these surviving animals were still alive. Only 30% of the untreated animals were alive 135 days pi (P < 0.01) (Fig. 2). Two years after infection none of the untreated mice had survived. His tology The hearts from infected and untreated mice presented inflammatory infiltrates with mononuclear cells and amastigote nests, which are histopathological alterations typical of acute chagasic myocarditis (Fig. 3a). Hearts from CL0 5 and CL0 40 mice, sacrificed 35 days pi, showed focalized inflammatory infiltrates without amastigote nests. Another study carried out 75 days pi showed a mild infiltrate located in the intraventricular septum. At the end of the experiment, 135 days pi in the chronic phase, CL0 5-treated mice showed a few inflammatory infiltrates in the right ventricle, but no structural alterations were detected in hearts from mice treated with CL0 40 (Fig. 3b). Discussion There is an indisputable and urgent need for new, safe and effective drugs for the treatment of Chagas disease (FAIRLAMB, 1999). Although vector control has been done reasonably well, vector transmission of the disease remains and alternative transmission modes such as blood transfusion, transplantation or transplacentary are still of clinical concern. Thus, new drugs are required to replace the currently used drugs, nifurtimox and benznidazole, which are often poorly tolerated and frequently ineffective (FKARDI & BRENER, 1987; FAIRLAMB, 1999). Clomipramine is a tricyclic antidepressive drug of similar structure and biological action as phenothiazines that has shown trypanocidal activity against epimastigotes and trypomastigotes in vitro, probably through anti-calmodulin action (BAEIOGLIO et al., 1987). In the present study we investigated the probable efficiency of CL0 in the treatment of T. crux infection, in mice inoculated with a low number of parasites, in order to analyse the different disease stages. Two treatment schedules were used: CL0 5 mg/kg daily for 1 month or 2 doses of CL0 40 mg/kg each, 1 h after T. cmzi infection and 7 days later. The 2 schedules gave similar effective results upon parasitaemia levels, which remained close to zero from the beginning. Survival of treated groups was 70-75% in the chronic stage (135 days pi) whereas untreated mice at this stage presented a survival of only 30%. Two years later survival 100

80

S 60 "m .2 E 2 40

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80

100

120

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Days Post-infection Fig. 2. Survival of T. cmzi-infected mice that were untreated (m), or treated with clomipramine 5 mg/kg daily for 30 days or with clomipramine 40 mg/kg at 1 hand 7 days post infection 0. Survival of these animals was monitored until 1 year post infection and percentages did not change over this time.

Fig. 3. (a) Myocardium from T. cruzi infected and untreated mice with inflammatory infiltrates. (b) Hearts from 7’. cm&infected mice that were treated with clomipramine 40 mg/kg at 1 h and 7 days post infection (pi), sacrificed 135 days pi, showed no structural alterations. (Haematoxylin and eosin.)

of treated animals remained as above, whereas none of the untreated mice had survived. Antibody titres were significantly lower with both CL0 treatments, but they did not reduce to the values in control (uninfected) mice. This is not surprising because a low parasitaemia was verified during the acute phase of infection despite the treatment, and the time elapsing (135 days) may not be enough for serum negativity. The values in treated mice are in agreement with previous work from our laboratory (RIVAROLA et al., 1999).

The electrocardiographic studies showed that treated animals presented lower conduction disturbances during the chronic stage, and no electrocardiographic alterations were detected in mice from the CL0 5 group at 135 days. Small CL0 doses daily for 30 days proved to be more effective, at least to prevent electric conduction disturbances. These results show that it is necessary to maintain CL0 at low blood concentrations constantly during the first month of T. cruziinfection; this is the time of parasite maximum reproduction in target organs. These results agree with the cardiac histology in that 135 days pi the myocardium from treated animals showed few inflammatory infiltrates, absence of fibrosis or normal cardiac structure (CL0 40). Cardiac p receptor affinity and density are modified in a particular manner in each T. cruzi infection phase (FERNANDEZ et al., 1994, 1995; ENDER~ et al., 1995), indicating different degrees in cardiac function alteration. For this reason, CL0 treatment efficiencv was also evaluated through 6 receptor behaviour. The results indicated that 6 receptor activity was similar to that in uninfected mice except for the CL0 5 group, which had affinity changes in the acute stageand decreasing density in the indeterminate stageofthe experimental disease.In the chronic stage, mice given either of the treatments showed afXnity and density of cardiac /3receptors similar to those of uninfected mice. Knowledge of the parasite biochemical pathways is likely to facilitate the search for effective drugs. Some essential T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets (BORGES et al., 1995; HUNTER, 1995; FAIRLAMB, 1999; STOPPANI, 1999) because it has been reported that both can be inhibited by phenothiazines or tricyclic-related compounds (STOPPANI 1993, 1999; BONDY, 1995; DOUGLAS, 1995). CL0

belongs to a tricyclic group of drugs that have been reported to be selective inhibitors of trypanothione reductase (KRWTH-SIEGEL & COOMSS, 1999). The therapeutic effect verified in the CLO-treated animals could be related to the ability of this drug to selectively inhibit trypanothione reductase and calmodulin, bearing in mind that Ca2+ homeostasis is essential not only for the parasite but also for its relationship with the host (DOCAMPO, 1997; ZHANG et al., 1998) and that

CL0 was toxic for T. cruzi, but did not induce damage in the host. Our findings show that CLO, which is generally used in clinical psychiatry, was also effective in treatment of experimental Chagas disease, since it proved to modify significantly the natural evolution of the infection. These results demonstrate that calmodulin and trypanothione reductase inhibitors may become new drug candidates for acute, indeterminate and chronic Chagas disease. Acknowledgements

This work was supported by grants from the Council for Sciences and Technology of C6rdoba (CONICOR) and the National University of C6rdoba (SECYT). References

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(199 1). Modulation of host cell metabolism by Trypanosoma cruzi. Parasitology To&y, I, 82-87. Morris, S. A., Tanowitz, H. B., Hatcher, V., Bilzikian, J. I’. & Wittner, M. (1998). Alterations in intracellular calcium following infection of human endothelial cells with Trypanosoma cruzi. Molecular and Biochemical Parasitology, 29, 213-221. On, G. A., Tanowitz, H. B. & Wittner, M. (1992). Trypanosoma cruzi: stage expression of calmodulin binding proteins. Experimental Parasilology, 74, 127-133. Prozialeck. W. C. & Weiss. B. f 1982). Inhibition of calmodulin by phegothiazines and ielated drugs: structure-activity relationships. .~oumal of Pharmacolo~-. and Experimental Therspy, 222;509-516. Rivarola, H. W., Fernindez, A. R., Enders, J. E., Fretes, R., Gea, S., Suligoy, M., Palma, J. A. & Paglini-Oliva, A. (1999). Thioridazine treatment modifies the evolution of Typanosoma cruzi infection in mice. Annals of Tropical Medicine and Parasitolo~. 93. 695-702. Segura, E. ‘i, S&a Estani, S., Esquivel, M., G6mez, A. & Salombn, 0. D. (1999). Control of the transmission of Typanosoma cruziin Argentina. Medicina, 59, 91-96. Stoppani, A. 0. M. (1993). The chemotherapy of Chagas’ disease. Medicina (Buenos Aires), 53, 3. Stoppani, A. 0. M. (1999). The chemotherapy of Chagas’ disease. Medicina (Buenos Aires), 59, 147- 165. Vercesi, A. E., Hoffman, M. E., Bemades, C. F. & Docampo, R. (199 1). Regulation of intracellular calcium homeostasis in Typanosoma cruzi. Effects of calmidazolium and trifluoperazine. Cell Calcium, 12, 361-369. WHO (1991). Chagas’ Disease: Tropical Disease Progress in Research 1989- 1990. Geneva, Switzerland: World Health Organization, Technical Report Series, no. 8 11. Zhang, H. S., McDonald, T., Tanowitz, H. B. Wittner, M. Weiss, L. M., Bilezikian, J. P. & Morris, S. A. (1998). Intracellular Ca*+ homeostasis in Typanosoma cruzi. Journal of Eukariotic Microbiology, 45, 80-86. Received accepted

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OF TROPICAL MEDICINE Garnham Fellowships

2000; revised 14 February 28 Februa y 2001

2001;

AND HYGIENE

Professor Cyril Garnham was one of the UK’s leading parasitologists in the 20th century and his work was characterized by outstanding achievement as both laboratory scientist and field worker in the tropics. The special place that Garnham occupies among his colleagues is recognized by the Fund set up in his memory to establish research fellowships for young scientists.

The aim of the Garnham Fellowship is to encourage young scientists to carry out short-term field projects. Suitable applicants are invited to apply to the Fund, which is administered by the Royal Society of Tropical Medicine and Hygiene. There are no restrictions by nationality or age, and fellowship of the Royal Society of Tropical Medicine and Hygiene is not a requirement. Applications from non-Fellows should be supported by a Fellow who can attest to the value of the project and to the competence of the applicant to carry out the work. One Garnham Fellowship of up to A2000 will be awarded annually The Garnham Fellowship is to be used for short-term field projects of up to 2 years’ duration l Preference will be given to topics in parasitology or medical entomology and to applicants with less than 5 years’ postdoctoral experience l Applicants are required to submit a detailed project, with costing of the work proposed, and a supporting statement from their head of department or supervisor, at least 6 months before the date of commencement l A short report should be submitted within 3 months of completion of the study l l

Application forms may be obtained from the Administrator, Royal Society of Tropical Medicine and Hygiene, Manson House, 26 Portland Place, London, WlB lEY, UK; fax +44 (0)20 7436 1389, e-mail [email protected] The closing date for receipt of applications is 15 September annually.