Melt crystallization and thermal degradation profile of the antichagasic drug nifurtimox.

Despite nifurtimox (NFX) being a traditional drug for treating Chagas disease, some of its physicochemical properties are still unknown, especially its thermal behavior, which brings important outcomes regarding stability and compatibility. In this work, a comprehensive study of NFX's thermal properties was conducted to assist incremental innovations that can improve the efficacy of this drug in novel pharmaceutical products. For this purpose, thermal analyses associated with spectroscopy and spectrometry techniques were used. DSC analyses revealed that the melt crystallization of the NFX led to its amorphous form with the possible formation of a minor fraction of a different crystalline phase. Coats-Redfern method using TGA results indicated the activation energy of NFX non-isothermal degradation as 348.8 ± 8.2 kJ mol-1, which coincides with the C-NO2 bond dissociation energy of the 2-nitrofuran. Investigation of the isothermal degradation kinetics using FTIR 2D COS showed the possible detachment of radical NO2 and ethylene from the NFX structure, which could affect its mechanism of action. A preliminary mechanism for the thermal degradation of this drug was also proposed. The results enhanced the understanding of NFX's thermal properties, providing valuable insights, especially for developing NFX-based pharmaceutical products that involve thermal processing.

Trypanosoma cruzi Mitochondrial Peroxiredoxin Promotes Infectivity in Macrophages and Attenuates Nifurtimox Toxicity.

Trypanosoma cruzi is the causative agent of Chagas disease which is currently treated by nifurtimox (NFX) and benznidazole (BZ). Nevertheless, the mechanism of action of NFX is not completely established. Herein, we show the protective effects of T. cruzi mitochondrial peroxiredoxin (MPX) in macrophage infections and in response to NFX toxicity. After a 3-day treatment of epimastigotes with NFX, MPX content increased (2.5-fold) with respect to control, and interestingly, an MPX-overexpressing strain was more resistant to the drug. The generation of mitochondrial reactive species and the redox status of the low molecular weight thiols of the parasite were not affected by NFX treatment indicating the absence of oxidative stress in this condition. Since MPX was shown to be protective and overexpressed in drug-challenged parasites, non-classical peroxiredoxin activity was studied. We found that recombinant MPX exhibits holdase activity independently of its redox state and that its overexpression was also observed in temperature-challenged parasites. Moreover, increased holdase activity (2-fold) together with an augmented protease activity (proteasome-related) and an enhancement in ubiquitinylated proteins was found in NFX-treated parasites. These results suggest a protective role of MPX holdase activity toward NFX toxicity. Trypanosoma cruzi has a complex life cycle, part of which involves the invasion of mammalian cells, where parasite replication inside the host occurs. In the early stages of the infection, macrophages recognize and engulf T. cruzi with the generation of reactive oxygen and nitrogen species toward the internalized parasite. Parasites overexpressing MPX produced higher macrophage infection yield compared with wild-type parasites. The relevance of peroxidase vs. holdase activity of MPX during macrophage infections was assessed using conoidin A (CA), a covalent, cell-permeable inhibitor of peroxiredoxin peroxidase activity. Covalent adducts of MPX were detected in CA-treated parasites, which proves its action in vivo. The pretreatment of parasites with CA led to a reduced infection index in macrophages revealing that the peroxidase activity of peroxiredoxin is crucial during this infection process. Our results confirm the importance of peroxidase activity during macrophage infection and provide insights for the relevance of MPX holdase activity in NFX resistance.

Benznidazole-resistance in Trypanosoma cruzi: evidence that distinct mechanisms can act in concert.

Benznidazole is the main drug used to treat Trypanosoma cruzi infections. However, frequent instances of treatment failure have been reported. To better understand potential resistance mechanisms, we analysed three clones isolated from a single parasite population that had undergone benznidazole-selection. These clones exhibited differing levels of benznidazole-resistance (varying between 9 and 26-fold), and displayed cross-resistance to nifurtimox (2 to 4-fold). Each clone had acquired a stop-codon-generating mutation in the gene which encodes the nitroreductase (TcNTR) that is responsible for activating nitroheterocyclic pro-drugs. In addition, one clone had lost a copy of the chromosome containing TcNTR. However, these processes alone are insufficient to account for the extent and diversity of benznidazole-resistance. It is implicit from our results that additional mechanisms must also operate and that T. cruzi has an intrinsic ability to develop drug-resistance by independent sequential steps, even within a single population. This has important implications for drug development strategies.

Untargeted metabolomics reveals a lack of synergy between nifurtimox and eflornithine against Trypanosoma brucei.

A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.

Metabolization of nifurtimox and benznidazole in cellular fractions of rat mammary tissue.

Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.

Nifurtimox nitroreductase activity in different cellular fractions from male rat pancreas. Biochemical and ultrastructural alterations.

Nifurtimox (Nfx) is a nitroheterocyclic drug used in the treatment of Chagas' disease. It has serious side effects which frequently force to interrupt the treatment. Nfx toxicity has been linked to its nitroreduction to a nitroanion radical with a subsequent redox cycling which generate reactive oxygen species. We analyzed the ability of Sprague Dawley male rat pancreas to nitroreduce Nfx and whether this drug may cause deleterious effects in this organ. The microsomal fraction exhibited Nfx nitroreductase activity in the presence of NADPH under anaerobic atmosphere, which was fully inhibited under air but not altered when N2 was replaced by pure CO. The cytosol nitroreduced Nfx in the presence of hypoxanthine under N2; it was inhibited by allopurinol and negligible in aerobiosis. Nfx reached pancreatic tissue at 1, 3 or 6 h after intragastric administration (100 mg/kg). Six hours after drug administration, a significant increase in t-buthylhydroperoxide promoted chemiluminiscence was detected. Pancreatic protein sulfhydryl content significantly decreased at either 1, 3 or 6 h after Nfx administration. No changes in either protein carbonyl or in lipid hydroperoxides were observable. Ultrastructural alterations were observed in the endoplasmic reticulum and nuclei from acinar cells and in the insulin-containing granules from the pancreas. However, the seric amylase levels were not changed, but the blood glucose levels were slightly but significantly increased 24 h after Nfx administration. These studies might suggest that Nfx treatment could impose an increased risk to patients exposed to other insults provoking oxidative stress or having preexisting pathologies in the pancreas.

Reactions of nifurtimox with critical sulfhydryl-containing biomolecules: their potential toxicological relevance.

Nifurtimox (Nfx) is a drug used in the treatment of Chagas' disease, an endemic parasitic disease from Latin American countries. It produces undesirable side-effects in patients, frequently forcing the treatment to be stopped. Its toxic mechanism is not fully understood. In this work we describe purely chemical reactions of Nfx with relevant cellular sulfhydryl (SH) compounds. The compounds tested were glutathione (GSH), cysteine (RSH), lipoic acid (LA) and coenzyme A (CoA). All reacted with Nfx to give nitrite (NO(-) (2)). The relative reaction rates were CoA>LA>GSH>RSH. In studies with GSH and RSH the formation of nitrite was accompanied by decreases in Nfx concentration and increases in the formation of a reaction product revealed by HPLC. We failed to show the presence of liver cytosolic GST (GSH transferase activity)-mediated formation of NO2- from Nfx. These NO(-) (2)-releasing processes occurred under in vivo conditions in Nfx-treated Sprague-Dawley male rats (240-260 g body weight) at a dose of 100 mg Nfx kg(-1) p.o. In urine samples NO(-) (2) excretion was accompanied by unchanged drug and two unidentified more polar metabolites detectable by HPLC. The Nfx reactions with critical SH from molecules such as GSH, RSH, LA and CoA, and potentially others containing SH residues (e.g. enzymes or structural proteins), might have toxicological relevance not only for the Nfx side-effects but also for the chemotherapeutic effects on Trypanosoma cruzi. In addition, Nfx reactions with GSH might be crucial in Nfx detoxification.

[Ultrastructural alterations in colonic mucosa of nifurtimox treated rats]. / Alteraciones ultraestructurales en la mucosa del colon de ratas tratadas con nifurtimox.

Nifurtimox (Nfx) is a chemotherapeutic agent used in the treatment of acute Chagas' disease. Clinical and experimental studies with this nitroheterocyclic compound evidenced serious undesirable side effects. These were correlated with Nfx nitroreduction to a nitroanion radical followed by superoxide anion generation through a redox cycling process. The aim of this study was to verify whether the oral administration of Nfx to Sprague Dawley male rats (100 mg.kg-1, p.o.) produced any observable ultrastructural alteration in the cells of the colonic mucosa. Results showed that 24 h after Nfx administration there were observable alterations in this type of cells. They essentially consisted of moderate dilatation of their endoplasmic reticulum and intense dilatation of their Golgi complex. Already 1 and 3 h after Nfx administration, the original compound reached a concentration of 9.7 +/- 2.9 and 7.0 +/- 1.7 nmol.g-1 respectively in the colonic tissue. Studies on Nfx nitroreductase activity of colonic mucosa as determined spectrophotometrically and by HPLC methods showed that the microsomal fraction (from 0.72 +/- 0.29 to 0.26 +/- 0.04 nmol Nfx.min-1.mg-1 protein) but not the cytosol had the ability to nitroreduce Nfx. The results obtained show a correlation between the ultrastructural localization of injury and that of nitroreductase activity. The intense deleterious effects of Nfx in the Golgi apparatus suggest the potential occurrence of alterations in the synthesis/storage of secretory products of the colonic mucosa.

Alteraciones ultraestructurales en la mucosa del colon de ratas tratadas con nifurtimox / Ultrastructural alterations in colonic mucosa of nifurtimox treated rats

El nifurtimox (Nfx) es un fármaco empleado en el tratamiento del Mal de Chagas agudo que ha evidenciado en su uso clínico y en estudios experimentales efectos colasterales tóxicos que comprometen su empleo. Estos efectos fueron correlacionados con la nitrorreducción del Nfx a radical nitroanión y la generación de aniones superóxidos a través de un ciclo redox. El objetivo de este trabajo fue verificar si después de la administración oral de Nfx ( 100g/ kg-1) a rata macho Sprague Dawley se observan alteraciones ultraestructurales en el colon. Los resultados mostraron que 24 horas después de administrar el Nfx se observan alteraciones consistentes en una dilatación moderada del retículo endoplasmático y en una dilatación intensa del Complejo de Golgi en las células epiteliales colónicas. El Nfx está presente en el tejido colónico 1 y 3 horas después de su administración oral, en concentraciones de 9.7 + o - 2.9 y 7.0 + o - nmol/g-1 respectivamente. Los estudios de actividad nitrorreductásica del Nfx, espectrofotómetricos y por HPLC, en fracciones subcelulares, permiten establecer su presencia en la fracción microsomal, con valores de 0.72 + o - 0.29 y 0.26 + o - 0.04 nmol Nfx/min-1/mg-1 proteína , pero no en el citosol. Los resultados una correlación entre la localización del año observado y la fracción celular donde ocurre la nitrorreducción. El daño intenso del complejo de Golgi producido por el Nfx sugiere potenciales alteraciones en las funciones de síntesis y/o almacenamiento de productos de secreción de la mucosa colónica.

Action of new organometallic complexes against Leishmania donovani.

The action of 16 newly synthesized metal complexes having the general structure cis-Pt-(II)-Xn-Ln have been tested in vitro against the promastigote forms of Leishmania donovani. The metal complexes at 24 h and maximum dosages inhibited growth from 0%, e.g. in cis-Pt-nifurtimox, to 100%, e.g. in cis-Pt-(2,3,4,5,6-pentafluoroaniline)2Br2 or cis-Pt-pentamidine-I2. A study of the cytotoxicty of these latter complexes on the phagocytic cell line J-774 showed neither high cytotoxicity nor cytolysis. At the maximum dosage after 24 h of permanent contact with the cells (extreme, non-physiological conditions), cytolysis did not exceed 30%. For most of the compounds, cytolysis ranged from 0%, for cis-Pt-oxamniquine-Cl2 to 27.7%, for cis-Pt-pentamidine-I2. The compound cis-Pt-(2,3,4,5,6-pentafluoroaniline)2-Br2 caused up to 1.4% cytolysis under the above conditions. Parasites exposed to cis-Pt-pentamidine-I2 showed notably reduced DNA, RNA and protein synthesis, unlike those exposed to other compounds. Parasites examined by electron microscopy showed effects mainly on the nucleus, though in some cases the mitochondria were affected, altering the internal membranes of the cytoplasmic organelles. The in-vivo activity of the complex cis-Pt-guanethidine-Cl2 was evaluated in parasitized Wistar rats, in which the number of amastigotes per gram of spleen was reduced by 75% compared with controls.

Reduction of nitrofuran compounds by heart lipoamide dehydrogenase: role of flavin and the reactive disulfide groups.

In order to elucidate the mechanism of the biological activation of nitrofurans, the interaction of these compounds with lipoamide dehydrogenase (LipDH)** was investigated. LipDH catalysed one-electron reduction of several nitrofuran derivatives. The reaction could be demonstrated spectroscopically and was enhanced by cadmium, arsenite and anaerobiosis. The role of flavin in the nitroreductase activity was supported by (a) the nitrofuran effect on the spectral properties of anaerobic, arsenite-inhibited, NADH-reduced LipDH; (b) FAD catalytic activity in a NADH-nitrofuran model system; and (c) the nitroreductase activity of LipDH monomer. Two-electron nitrofuran reduction to less oxidized products was inhibited by cadmium, arsenite and NAD+. The possible role of reactive nitrosofuran derivatives as intermediates of the nitrofuran reduction sequence was supported by the LipDH capability for catalysing 2-nitroso-1-naphthol redox-cycling. The nitroso naphthol reduction was inhibited by cadmium and arsenite, like the two-electron nitrofuran reduction.

Comparative studies of nifurtimox uptake and metabolism by drug-resistant and susceptible strains of Trypanosoma cruzi.

1. Nifurtimox uptake and metabolism by epimastigote forms of three strains of Trypanosoma cruzi (Basileu, Y, YuYu) with different drug responsiveness in mice experimental infections were compared. 2. Statistical analysis of the results demonstrated no correlation between the ability of the strains to catalyze nifurtimox redox-cycling (Basileu = Y = YuYu) nor nifurtimox multiple electron reduction (Basileu = Y greater than Y) and drug susceptibility (Basileu greater than Y greater than YuYu). 3. A partial correlation however, was observed between drug responsiveness and nifurtimox uptake (Basileu greater than Y = YuYu). 4. The results suggest that drug uptake may be more important than drug metabolism in modulating resistance to nifurtimox in T. cruzi strains.

Ultrastructural effects of Nifurtimox on rat adrenal cortex related to reductive biotransformation.

Nifurtimox (Nfx) (4(5-nitrofurfurylidene)amino)-3-methylthiomorpholine-1, 1-dioxide) is a drug used against Chagas' disease, a parasitic sickness afflicting several million Latin Americans. Nfx administration to Sprague-Dawley male rats (220-250 g) at a dose of 100 mg/kg caused pronounced alterations in the adrenal cortex involving the fasciculata and reticularis zones but which were not evident in the glomerulosa. Alterations observed involved mitochondria, nuclei, Golgi apparatus, and the endoplasmic reticulum but were more intense in the mitochondria. There is Nfx nitroreductase activity in the adrenal microsomal, mitochondrial, and cytosolic-rich fractions but most of it is in the mitochondrial-rich fraction. Activity in the first two fractions requires NADPH and that in the cytosol is only observed in the presence of hypoxanthine as substrate. Enzymatic activity in all fractions is inhibited by oxygen. CO does not inhibit mitochondrial Nfx nitroreductase and inhibits only 10% of the microsomal enzyme activity. Hypoxanthine-dependent cytosolic activity is inhibited by allopurinol. Present results suggest that Nfx is activated to damage-producing reactive metabolites by nitroreductive biotransformation in rat adrenal organelles. Mitochondrial and microsomal bioactivation would occur at the level of the flavoenzyme P-450 reductase rather than at P-450 itself, and cytosolic bioactivation would be mediated by xanthine oxidase. Epidemiological studies on adrenal function in patients undergoing Nfx treatment would be necessary to establish the potential toxicological relevance of these findings.

Restricted bioreductive metabolism of a nitroimidazole-thiadiazole derivative with curative action in experimental Trypanosoma cruzi infections.

The bioreductive activation of megazol [2-amino-5(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole] promoted by ferredoxin: NADP+ oxidoreductase, rat liver microsomes and cellular fractions of Trypanosoma cruzi, Y strain, was investigated. Direct ESR detection and characterization by computer simulation of the megazol nitro anion radical were possible in the presence of NADPH and ferredoxin: NADP+ oxidoreductase under anaerobic conditions. By contrast, the megazol nitro anion radical was not detected in the presence of either rat liver microsomes or cellular fractions of T. cruzi under conditions where the corresponding nifurtimox anion radical was observed. The inefficiency of rat liver microsomes in catalyzing megazol reduction was also attested by visible light absorption spectroscopy. In the presence of cellular fractions of T. cruzi supplemented with NAD(P)H, megazol marginally affected oxygen consumption and decreased the yield of oxyradicals that can be spin-trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Our results indicate a restricted bioreductive metabolism of megazol and suggest that its trypanocidal activity is unrelated to a redox-cycling process.

Activation of anti-Trypanosoma cruzi drugs to genotoxic metabolites promoted by mammalian microsomal enzymes.

Salmonella typhimurium TA100 and its nitroreductase-deficient derivative, TA100 NR, were used to reevaluate the mutagenic activities of benznidazole and nifurtimox. Mutagenicity and toxicity of nifurtimox were abolished in the TA100 NR tester strain under aerobic or anaerobic conditions and addition of rat liver extracts did not alter the results. However, benznidazole showed a significant mutagenicity and toxicity to the nitroreductase-deficient strain TA100 NR under hypoxic conditions. Addition of rat liver extracts enhanced the observed mutagenicity and toxicity of benznidazole even more. In the presence of O2 the genotoxic activities of benznidazole to the TA100 NR tester strain were eliminated. These results lead us to conclude that bacterial enzymes were responsible for the previously observed genotoxic effects of nifurtimox and benznidazole on S. typhimurium TA100. Moreover, under anaerobic conditions, only benznidazole could be metabolized by mammalian nitroreductases into a mutagenic derivative.

The reductive metabolism of nifurtimox and benznidazole in Crithidia fasciculata is similar to that in Trypanosoma cruzi.

1. Addition of nifurtimox or benznidazole to the NADH-containing mitochondrial fraction of Crithidia fasciculata led to the appearance of the characteristic ESR spectra corresponding to their nitro anion radicals, suggesting that the nitro anion radical is a necessary intermediate in the reduction of both nitro compounds. 2. Nifurtimox anion radical generation by the mitochondrial fraction was insensitive to rotenone and antimycin A but was enhanced by KCN. 3. The nifurtimox anion radical reacted with oxygen under aerobic conditions leading to an increase in the cyanide-insensitive respiration of the intact cells and in the rate of O2- and H2O2 production by the C. fasciculata mitochondrial fraction. 4. In contrast, generation of O2- and H2O2 was not stimulated with pharmacological concentrations of benznidazole. Furthermore, benznidazole inhibited the cyanide-insensitive respiration of the intact cells.

Evaluation of genotoxic activity in the blood and urine of guinea pigs treated with nifurtimox and benznidazole.

1. The mutagenicity of serum and urine from guinea pigs treated with a single oral dose (500 mg/kg) of benznidazole and nifurtimox was assayed using the Salmonella/plate incorporation test with strain TA 100 and a nitroreductase-deficient derivative, TA 100NR. 2. The urine and blood of animals treated with nifurtimox were not mutagenic for either tester strain. 3. The urine and blood of animals receiving benznidazole were mutagenic to the TA 100 but not to the TA 100NR strain. Similar results were obtained with nitrofurantoin-treated animals. Maximum mutagenicity values were obtained in serum and urine of treated animals 90 min and 24 h after administration, respectively. 4. Mutagenicity induced by benznidazole in the serum and urine of treated animals was not altered when assayed in anaerobic environments. 5. These results indicate that benznidazole and nifurtimox are not metabolized by the mammalian host into stable mutagenic derivatives detectable by the Ames test. Based on these data, we suggest that the potential cancer risk to patients treated with these drugs is small but should be further evaluated.