Nitazoxanide, tizoxanide and a new analogue [4-nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide; NTB] inhibit the growth of kinetoplastid parasites (Trypanosoma cruzi and Leishmania mexicana) in vitro

Abstract

Sir, Parasitic diseases such as leishmaniasis and trypanosomiasis, both caused by protozoan parasites of the Kinetoplastida order, represent a serious problem to the health and the economy of many developing countries. The Leishmania species cause a variety of diseases, from self-healing cutaneous lesions to lifethreatening visceral infections. Clinical manifestations depend on the species of the infecting parasites. There are an estimated annual 1.5–2.0 million new cases of leishmaniasis, of which approximately 500000 belong to the visceral form, which is potentially fatal. American trypanosomiasis, or Chagas’ disease, is still one of the major causes of morbidity and mortality due to cardiovascular diseases in Latin America. Common chemotherapeutic agents currently used against both diseases are often inadequate since they require long courses of parenteral administration, may have toxic side effects or become less effective due to the emergence of resistant strains. Therefore, new, effective and inexpensive drugs that can be used to treat these diseases are urgently required. Nitazoxanide [2-(5-nitrothiazol-2-ylcarbamoyl)phenyl acetate; Alinia] is a broad-spectrum antiparasitic compound belonging to a nitroheterocyclic class named thiazolides. In humans, nitazoxanide is rapidly metabolized to tizoxanide, which is a compound that is as effective as the parent drug (Figure 1). Detailed in vitro and in vivo studies have been conducted regarding the efficacy of nitazoxanide and other nitroheterocyclic drugs against helminths, extracellular anaerobic protozoa and bacteria, intracellular parasites and viruses. – 6 Due to the limited information regarding the efficacy of nitazoxanide against kinetoplastid parasites such as Trypanosoma cruzi and Leishmania mexicana, the goal of this work was to show the potential of nitazoxanide and its major metabolite, tizoxanide, as antileishmanial and trypanocidal drugs. In this work, we evaluated the in vitro activity of nitazoxanide, tizoxanide and the newly synthesized analogue 4-nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide (NTB) (Figure 1) against T. cruzi and L. mexicana. The activity was compared with the activities of benznidazole and pentamidine, well-known drugs that act against T. cruzi and L. mexicana, respectively. Nitaxozanide was synthesized in our laboratory starting from the acylation of 2-amino-5-nitrothiazole with acetylsalicyloyl chloride and triethylamine in methylene chloride as solvent. An alkaline hydrolysis of nitazoxanide produced tizoxanide in good yields. NTB was synthesized by means of the same procedure, using 4-nitrobenzoyl chloride instead of acetylsalicyloyl chloride. All compounds were characterized by spectroscopic and spectrometric techniques and all data agreed with reported literature values. The growth inhibition test was performed on promastigotes of L. mexicana (MHOM/MX/84/ISETGS; clinical strain originally isolated from a patient with diffuse cutaneous leishmaniasis) and epimastigotes of T. cruzi (MHOM/MX/1994/Ninoa; clinical strain originally isolated from a patient with Chagas’ disease in the acute phase). Parasites were cultivated at 268C in Schneider’s Drosophila medium, supplemented with 10% fetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 mg/ mL). Biological assays were performed in 96-well plates and all compounds were evaluated in duplicate. Compounds were solubilized in dimethylsulphoxide and diluted in a liquid medium. Aliquots of 100 mL of compound solution and 100 mL of culture medium containing 10000 Leishmania promastigotes or 20000 T. cruzi epimastigotes were added to obtain concentrations of 10, 5, 2.5 and 1.25 mg/mL. Benznidazole (first-line antichagasic drug) and pentamidine (second-line antileishmanial drug) were used as positive controls. Cultures containing parasites without compound solution were also included. Plates were incubated at 268C for 72 h and the leishmanicidal activity of compounds was determined by direct count of parasites in a Neubauer chamber. The concentrations required to inhibit 50% of parasite growth (IC50) were calculated by probit analysis and are summarized in Table 1. It is clear from Table 1 that nitazoxanide and tizoxanide reduced the growth of the kinetoplastid parasites L. mexicana and T. cruzi in vitro with IC50s 2-fold lower than those of pentamidine and benznidazole, respectively. The newly synthesized analogue NTB was 2-fold more potent than nitazoxanide and tizoxanide and 6-fold more active