Effect of tripanosomicide benznidazole (Rochagan®) on the biodistribution of sodium pertechnetate (Na99mTcO4) in Wistar rats

DRUG INTERACTION AND SURGERY, AND THE BIODISTRIBUTION OF RADIOPHARMACEUTICAL

Effect of tripanosomicide benznidazole (Rochagan®) on the biodistribution of sodium pertechnetate (Na^99mTcO₄) in Wistar rats

Vanessa Santos de Arruda Barbosa^I,*; Cecília Maria de Carvalho Xavier Holanda^I,II; Roseane Pereira da Silva^I; Daniel Pereira de Oliveira^II; Maurício Ferreira da Silva Júnior^II; Elias Herculano de Oliveira^II; Maria Helena Constantino Spyrides^III; Aldo Cunha Medeiros^I,IV

^ICentro de Ciências da Saúde; Universidade Federal do Rio Grande do Norte; Av. Nilo Peçanha, s/n; [e-mail]; 59012300; Natal - RN - Brasil

^IIDepartamento de Microbiologia e Parasitologia; Centro de Biociências; Universidade Federal do Rio Grande do Norte; Av. Salgado Filho, 3000; 59078-970; Natal - RN - Brasil

^IIIDepartamento de Estatística; Universidade Federal do Rio Grande do Norte; Av. Salgado Filho, 3000; 59078970; Natal - RN - Brasil

^IVDepartamento de Cirurgia; Universidade Federal do Rio Grande do Norte; Av. Nilo Peçanha, s/n; 59012300; Natal - RN - Brasil

ABSTRACT

Benznidazole, a drug with specific anti-Trypanosoma cruzi activity, is used in the treatment of Chagas' disease. The radiopharmaceutical sodium pertechnetate (Na^99mTcO4) is used to obtain diagnostic images of the stomach, thyroid, parathyroids, salivary glands, brain and in the study of esophageal reflux and blood flow. This study aimed at evaluating in vivo the influence of benznidazole treatment on the sodium pertechnetate biodistribution in Wistar rats. The percentage of radioactivity per gram (%ATI/g) of various organs (brain, heart, esophagus, stomach, small intestine, large intestine, spleen, liver, muscle and blood) was determined. Comparing the treated rats with the controls, we observed that sodium pertechnetate biodistribution did not change when administered to rats treated for thirty days with benznidazole.

Keywords:Antiparasite, Radiopharmaceutical, Technetium-99m, Benznidazole, Biodistribution, Trypanosoma cruzi

RESUMO

O benzonidazol é um quimioterápico com atividade específica anti-T. cruzi utilizado por milhares de pessoas para tratamento da doença de Chagas. O radiofármaco pertecnetato de sódio (Na^99mTcO4) é utilizado na obtenção de imagens diagnósticas do estômago, tireóide, paratireóides, glândulas salivares, plexo coróide, cérebro e de estudos de refluxo esofágico e de fluxo sanguíneo. Esse trabalho objetivou avaliar in vivo a influência do tratamento crônico com o benzonidazol na biodistribuição do radiofármaco pertecnetato de sódio em ratos Wistar. O percentual de radioatividade por grama (%ATI/g) de vários órgãos (cérebro, coração, esôfago, estômago, intestino delgado, intestino grosso, baço, fígado, músculo e sangue) foi determinado. Comparando-se o grupo controle e o tratado observou-se que o pertecnetato de sódio não possui sua biodistribuição alterada quando administrado em ratos tratados por trinta dias com a droga benzonidazol, mostrando não prejudicar a interpretação de diagnósticos por imagem.

INTRODUCTION

Chagas' disease is an endemic disorder caused by the flagellate protozoan Trypanosoma cruzi. It is a serious medical and social problem in Brazil and several Latin American countries, affecting 18 million individuals, with 300 thousand new cases every year (WHO, 2002). T. cruzi can invade multiple host cells, generating megacolon, megaesophagus and chagasic heart disease (Santos et al., 2005; Texeira et al., 2006). Benznidazole (Bz), commercially known as Rochagan®, is a drug with specific anti-T. cruzi activity and in vitro, available in Brazil since the 1970s (Coura and Castro, 2002). It contains N-benzyl-2-nitro-1-imidazolacetamida, which acts directly on the macromolecule synthesis by a covalent link with cellular components. It has demonstrated low efficiency and high toxicity, especially in the chronic phase of the disease and its treatment can last for up to 60 days, depending on clinical manifestations and host tolerance (Cançado, 2005).

The most widely used treatment scheme is 10 mg/kg/day in adults and less than 5 mg/kg/day in children (Castro et al., 2000; Cançado, 2002; Urbina and Docampo, 2003; Dias, 2004). Following oral administration, Bz is absorbed by the intestine, and is bound to plasma proteins and red blood cells to be distributed throughout the body. Maximum plasma concentrations are achieved in two to four hours. The half-life of plasma elimination is approximately twelve hours (Morilla et al., 2005).

Among the many diagnostic tools that can be used in tropical diseases, scintigraphy is widely used in the anatomic and functional analyses of organs and systems (Braga, 2002). Technetium-99m (^99mTc), in the form of sodium pertechnetate (Na^99mTcO4), is a radionuclide that connects to a wide variety of molecules and cells (Thrall and Ziessman, 2003; Saha, 2004). When injected intravenously, it is distributed through the veins and interstitium and is used to obtain diagnostic images of the stomach, thyroid, parathyroids, salivary glands, brain and in the study of esophageal reflux and blood flow (Saha, 2004; Owunwanne et al., 1995; Thrall and Ziessman, 2003).

Several drugs can interfere with the biological behavior of radiopharmaceuticals used in scyntigraphic examinations. They can change the biological effect of the radiopharmaceutical and their interaction can lead to hypo or hyper uptake of radiopharmaceuticals in a particular organ, causing incorrect diagnosis or misinterpretation of results. Repeated scintigraphy may result in unnecessary radiation for patients (Bernardo-Filho et al., 2005; Gomes et al., 2002). Although Bz is the drug currently recommended by the National Foundation of Health, Brazil, and is used by thousands of people, little is known about its action mechanism, its effects on host cells or its toxicity. Thus, it is important to study the effect of Bz on the biodistribution of the radiopharmaceutical sodium pertechnetate in laboratory animals subjected to chronic treatment with this drug. The aim of this work was to assess in vivo the influence of Bz on the biodistribution of sodium pertechnetate in rats.

MATERIALS AND METHODS

Twelve male Wistar rats weighing 200-250g from the Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte (UFRN), Natal- RN, Brazil were used. The protocol was conducted in accordance with Brazilian College of Animal Experimentation guidelines and was approved by the Research Ethics Committee of Onofre Lopes Hospital-UFRN (08/2007). The animals had free access to water and standard food for rodents (Labina Purina ®) and were randomly allocated to 2 groups: control and treated. The treated group (n=6) received 5 mg/kg/day of Bz diluted with sorbitol, by gavage. The control rats (n=6) received only sorbitol. The animals were treated for 30 days. On the last day of treatment the rats received 0.1 mL of Na^99mTcO4 (3.7 MBq) via orbital plexus.

The Na^99mTcO4 was eluted in a ⁹⁹Mo/^99mTc generator (Instituto de Pesquisas Energéticas e Nucleares, São Paulo, Brazil). After 60 minutes, all animals were quickly killed under anesthesia with xylazine (20 mg/kg) and ketamine (50 mg/kg), by intraperitoneal via. Samples were harvested from the brain, heart, esophagus, stomach, small intestine, large intestine, spleen, liver, muscle and blood. The tissue samples were washed in 0.9% saline, weighed on a precision scale (Mark 160®, Bel equipment, Italy) and the percentage of radioactivity per gram of tissue (%ATI/g) was determined in an automatic gamma counter (Wizard 1470, Perkin-Elmer, Finland). The efficiency of the gamma counter was 86%, as specified by the manufacturer. The biochemical and hematological dosages were performed in automated equipment TermoKonelab 60i, Abbot and Cell-Dyn 3500R, Abbot, respectively. Data were presented as mean ± standard deviation. The percentage of radioactivity per gram (%ATI/g) was determined by dividing the percentage of total radioactivity of each sample by its weight in grams. The ATI%/g was compared using the non-parametric Mann-Whitney test and the biochemical and hematological parameters by Student's t-test, considering the level of statistical significance at p<0.05 in both tests. Statistica 6.0 software was used.

RESULTS

Table 1 shows the relationship between the controls and the Bz-treated rats. No statistically significant difference (p>0.05) in biodistribution of sodium pertechnetate was observed in any of the organs.

Table 2 shows significant differences in alanine aminotransferase (ALT) dosage and the percentage of neutrophils and lymphocytes, when comparing the treated and control rats. All the other parameters such as iron, total protein, aminotransferase aspartate (AST), creatinine, glucose, red blood cells, hemoglobin, hematocrit, white blood cells and platelets showed no significant differences, when the two groups were compared (p>0.05).

DISCUSSION

The biological behavior of radiopharmaceuticals used for diagnosis in nuclear medicine is well established in the scientific literature. The interaction between a drug and a radiopharmaceutical may alter its biodistribution and result in an unexpected effect (Gomes et al., 2002, Bernardo-Filho et al., 2005).

It is of critical importance to know which drugs can interfere with the normal biodistribution of radiopharmaceuticals, to avoid the misinterpretation of scintigraphic images. Several authors have shown that radiopharmaceutical biodistribution can be altered by natural and synthetic products, radiotherapy, surgery and diet (Gomes et al., 2002; Xavier-Holanda et al., 2002; Passos et al, 2002; Bernardo et al., 2004; Moreno et al., 2005; Santos-Filho and Bernardo-Filho., 2005; Holanda et al., 2006; Araújo-Filho et al., 2007).

Glucantime, an anti-Leishmania drug, increased the uptake of the radiopharmaceutical methylene disphosphonic acid, labeled with technetium-99m (^99mTc-MDP) in the spleen, kidney, testicles, heart and liver of rats (Xavier-Holanda et al., 2002). The biodistribution of ^99mTc-MDP was also changed in various organs of rats treated with the antimalarials artemisinin and mefloquine (Holanda et al., 2006). Araujo-Filho et al. (2007) reported changes in sodium pertechnetate biodistribution in the thyroid, lung, pancreas, spleen and muscle after biliopancreatic bypass surgery with duodenal switch in rats. Moreno et al. (2005) showed that Ginkgo biloba extract can change the sodium pertechnetate biodistribution in the kidneys, liver and duodenum. Santos-Filho and Bernardo-Filho. (2005) showed that Hypericum perforatum extract reduced the uptake of the radiopharmaceutical in bone, muscle and thyroid.

Some studies show that Bz can have adverse effects on adrenal cortex, esophagus and colon cells in rats and that it exerts a mutagenic and carcinogenic effect (Diaz, 2000, Castro et al., 2006, de Castro et al., 2003). Because of its antigenic and toxic effect, Bz can cause several undesirable reactions, mainly in the nervous system and gastrointestinal tract (Cançado, 2002).

During benznidazole treatment (30 to 60 consecutive days), skin reactions, gastrointestinal disorders, nausea, paresthesia, or symptoms of peripheral polyneuritis may occur, especially after prolonged treatment or with excessive doses of Bz. The depression of the bone marrow with neutropenia, thrombocytopenic purple and hepatotoxicity are serious effects of Bz that demand caution when they are being used (Cançado, 2005). In our study, neutropenia was found in Bz-treated rats, but no hepatic and intestinal mucous membrane damage was observed. Although Bz can cause these several undesirable reactions, our results were shown positive in relation to interaction Bz-radiopharmaceutical. Both Bz and sodium pertechnetate are bound to plasma proteins, but it seems that there is no competition between them for the same connection sites (Coura and Castro, 2002; Owunwanne et al., 1995). Bz also, probably, does not interfere in the intestinal mobility and does not alter blood flow. The alteration of these factors could modify the biodistribution of the radiopharmaceutical (Owunwanne et al., 1995). Data from biochemical and hematological parameters also lead us to believe that Bz, administered in equivalent dose to treat Chagas' disease in humans, had no toxic effect on the organs studied, nor did it cause enough tissue damage to promote a change in the uptake of Na^99mTcO₄.

In this study we demonstrated that the administration of benznidazole didn't alter sodium pertechnetate biodistribution to important target organs, what demonstrate that this result is satisfactory for its use in patients with Chagas' disease. In spite of, this experimental were performed in Wistar rats.

ACKNOWLEDGEMENTS

The authors thank the Liga Norteriograndense against Cancer for the support, Michael Germain from Canada, for the revision of English language and Italo Medeiros Azevedo for his help with the experiments.

Received: August 12, 2008;

Revised: September 02, 2008;

Accepted: September 04, 2008

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*

Author for correspondence

Publication Dates

• Publication in this collection
  20 Feb 2009
• Date of issue
  Dec 2008

History

• Accepted
  04 Sept 2008
• Reviewed
  02 Sept 2008
• Received
  12 Aug 2008