Biopharmaceutical Characteristics of Nifurtimox Tablets for Age- and Body Weight-Adjusted Dosing in Patients With Chagas Disease.

Treatment of Chagas disease with nifurtimox requires age- and body weight-adjusted dosing, resulting in complex dosing instructions. Appropriate formulations are needed for precise and compliant dosing, especially in pediatric patients. We characterized the biopharmaceutical features of a standard nifurtimox 120-mg tablet and a 30-mg tablet developed to improve dose accuracy. Two open-label, randomized crossover studies were conducted in adult outpatients with Chagas disease. One study investigated whether 4 × 30-mg tablets and 1 × 120-mg tablet were bioequivalent and whether tablets can be administered as an aqueous slurry without affecting bioavailability. The second study investigated the effect of a high-calorie/high-fat diet versus fasting on the absorption of nifurtimox after a single 4 × 30-mg dose. Interventions were equivalent if the 90% confidence interval (CI) of their least-squares (LS) mean ratios for both AUC0-tlast and Cmax were in the range of 80%-125%. The 4 × 30-mg and 1 × 120-mg tablet doses were bioequivalent (AUC0-tlast : LS mean ratio, 104.7%; 90%CI, 99.1%-110.7%; Cmax : LS mean ratio, 101.7%; 90%CI, 89.4%-115.6%; n = 24). Exposure when giving the 4 × 30-mg dose as a slurry or as tablets was comparable, with an AUC0-tlast ratio of 93.2% (84.2%-103.1%; n = 12) and a slightly decreased Cmax ratio for the slurry of 76.5% (68.8%-85.1%). Food improved the bioavailability of nifurtimox substantially (AUC0-tlast ratiofed/fasted , 172%; 90%CI, 154%-192%; Cmax ratiofed/fasted , 168%; 90%CI, 150%-187%). The data indicate that the 30- and 120-mg tablets are suitable for dosing adult and pediatric patients accurately; nifurtimox should be administered under fed conditions.

Experimental and Clinical Treatment of Chagas Disease: A Review.

Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi that infects a broad range of triatomines and mammalian species, including man. It afflicts 8 million people in Latin America, and its incidence is increasing in nonendemic countries owing to rising international immigration and nonvectorial transmission routes such as blood donation. Since the 1960s, the only drugs available for the clinical treatment of this infection have been benznidazole (BZ) and nifurtimox (NFX). Treatment with these trypanocidal drugs is recommended in both the acute and chronic phases of CD. These drugs have low cure rates mainly during the chronic phase, in addition both drugs present side effects that may result in the interruption of the treatment. Thus, more efficient and better-tolerated new drugs or pharmaceutical formulations containing BZ or NFX are urgently needed. Here, we review the drugs currently used for CD chemotherapy, ongoing clinical assays, and most-promising new experimental drugs. In addition, the mechanism of action of the commercially available drugs, NFX and BZ, the biodistribution of the latter, and the potential for novel formulations of BZ based on nanotechnology are discussed. Taken together, the literature emphasizes the urgent need for new therapies for acute and chronic CD.

Nitroheterocyclic drugs cure experimental Trypanosoma cruzi infections more effectively in the chronic stage than in the acute stage.

The insect-transmitted protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, and infects 5-8 million people in Latin America. Chagas disease is characterised by an acute phase, which is partially resolved by the immune system, but then develops as a chronic life-long infection. There is a consensus that the front-line drugs benznidazole and nifurtimox are more effective against the acute stage in both clinical and experimental settings. However, confirmative studies have been restricted by difficulties in demonstrating sterile parasitological cure. Here, we describe a systematic study of nitroheterocyclic drug efficacy using highly sensitive bioluminescence imaging of murine infections. Unexpectedly, we find both drugs are more effective at curing chronic infections, judged by treatment duration and therapeutic dose. This was not associated with factors that differentially influence plasma drug concentrations in the two disease stages. We also observed that fexinidazole and fexinidazole sulfone are more effective than benznidazole and nifurtimox as curative treatments, particularly for acute stage infections, most likely as a result of the higher and more prolonged exposure of the sulfone derivative. If these findings are translatable to human patients, they will have important implications for treatment strategies.

Enantiomers of nifurtimox do not exhibit stereoselective anti-Trypanosoma cruzi activity, toxicity, or pharmacokinetic properties.

With the aim of improving the available drugs for the treatment of Chagas disease, individual enantiomers of nifurtimox were characterized. The results indicate that the enantiomers are equivalent in their in vitro activity against a panel of Trypanosoma cruzi strains; in vivo efficacy in a murine model of Chagas disease; in vitro toxicity and absorption, distribution, metabolism, and excretion characteristics; and in vivo pharmacokinetic properties. There is unlikely to be any therapeutic benefit of an individual nifurtimox enantiomer over the racemic mixture.

Presente y Futuro en el Descubrimiento de Fármacos para la Enfermedad de Chagas / The Present and Future of Drug Discovery for Chagas Disease

La enfermedad de Chagas, también conocida como Tripanosomiasis Americana es una enfermedad parasitaria causada por el Trypanosoma cruzi. Se estima que alrededor de 7,7 millones de personas se encuentran infectadas y padecen la enfermedad de Chagas. Esta enfermedad silenciosa que esta estrechamente relacionada con la pobreza, es transmitida a los humanos por unos insectos que se encuentran exclusivamente en el continente americano, principalmente en áreas rurales con muy deficientes condiciones de salubridad. Los fármacos existentes (nifurtimox y benznidazol), no siempre disponibles, constituyen un tratamiento paliativo, pero no curan la enfermedad y no son aceptables desde un punto de vista terapéutico debido a sus efectos secundarios indeseables y a su falta de eficacia. Por tanto, es necesario el desarrollo urgente de nuevos tratamientos y por tanto, sería muy conveniente la utilización del diseño racional en todas las etapas. El diseño de fármacos es una tarea compleja que requiere la colaboración interdisciplinar de muchos especialistas en diferentes campos de la ciencia. El presente trabajo describe de manera estructurada las diferentes estrategias que se han utilizado y las que se pueden utilizar en el futuro para el descubrimiento de nuevos fármacos para la enfermedad de Chagas. Se recogen las estrategias más clásicas como el diseño de análogos, el cribado sistemático o el basado en la información biológica y los métodos más novedosos basados en lo que se conoce como quimioinformática(AU)

The Present and Future of Drug Discovery for Chagas DiseaseChagas disease, also known as American trypanosomiasis, is caused by infection with the Trypanosoma cruzi. The Pan American Health Organization (PAHO) estimates that 7.7 million persons currently have T. cruzi infection in the 21 endemic countries. This disease can be transmitted to humans by insect vectors that are found only in the American continent, mainly, in rural areas with unhealthy housing conditions where poverty is a general concern. Nifurtimox and benznidazole are the only drugs used against this disease, but sometimes they are not available. The treatment of Chagas disease with nifurtimox or benznidazole is unsatisfactory because of their limited efficacy on the prevalent chronic stage of the disease and their toxic side effects. It is, therefore, necessary the development of new effective antichagasic drugs for the suitable treatment of this disease. The development of new drugs for Chagas disease requires a multidisciplinary approach involving diverse disciplines such as molecular and cellular biology, chemistry, bioinformatics, biochemistry, pharmacology and toxicology. This revision describes the different strategies used for drug discovery on Chagas disease treatment. The most classic strategies as the design of analogous, the systematic screening or that one based on the biological information, together the most recent methods based on chemiinformatics, are presented(AU)

The transport of nifurtimox, an anti-trypanosomal drug, in an in vitro model of the human blood-brain barrier: evidence for involvement of breast cancer resistance protein.

Human African trypanosomiasis (HAT) is a parasitic disease affecting sub-Saharan Africa. The parasites are able to traverse the blood-brain barrier (BBB), which marks stage 2 (S2) of the disease. Delivery of anti-parasitic drugs across the BBB is key to treating S2 effectively and the difficulty in achieving this goal is likely to be a reason why some drugs require highly intensive treatment regimes to be effective. This study aimed to investigate not only the drug transport mechanisms utilised by nifurtimox at the BBB, but also the impact of nifurtimox-eflornithine combination therapy (NECT) and other anti-HAT drug combination therapies (CTs) on radiolabelled-nifurtimox delivery in an in vitro model of drug accumulation and the human BBB, the hCMEC/D3 cell line. We found that nifurtimox appeared to use several membrane transporters, in particular breast-cancer resistance protein (BCRP), to exit the BBB cells. The addition of eflornithine caused no change in the accumulation of nifurtimox, nor did the addition of clinically relevant doses of the other anti-HAT drugs suramin, nifurtimox or melarsoprol, but a significant increase was observed with the addition of pentamidine. The results provide evidence that anti-HAT drugs are interacting with membrane transporters at the human BBB and suggest that combination with known transport inhibitors could potentially improve their efficacy.

Drug resistance in human African trypanosomiasis.

Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.

A phase 1 study of nifurtimox in patients with relapsed/refractory neuroblastoma.

The primary aim of this phase 1 study was to determine the maximum tolerated dose (MTD) and evaluate the safety of nifurtimox alone and in combination with cyclophosphamide and topotecan in multiple relapsed/refractory neuroblastoma pediatric patients. The secondary aim was to evaluate the pharmacokinetics of nifurtimox and the treatment response. To these ends, we performed a phase 1 dose escalation trial of daily oral nifurtimox with toxicity monitoring to determine the MTD, followed by 3 cycles of nifurtimox in combination with cyclophosphamide and topotecan. Samples were collected to determine the pharmacokinetic parameters maximum concentration, time at which maximum concentration is reached, and area under the curve between 0 and 8 hours. Treatment response was evaluated by radiographic and radionuclide (I-metaiodobenzylguanidine) imaging, measurement of urinary catecholamines, and clearance of bone marrow disease. We determined the MTD of nifurtimox to be 30 mg/kg/d. The non-dose-limiting toxicities were mainly nausea and neuropathy. The dose-limiting toxicities of 2 patients at 40 mg/kg/d were a grade 3 pulmonary hemorrhage and a grade 3 neuropathy (reversible). Overall, nifurtimox was well tolerated by pediatric patients at a dose of 30 mg/kg/d, and tumor responses were seen both as a single agent and in combination with chemotherapy. A Phase 2 study to determine the antitumor efficacy of nifurtimox is currently underway.

The distribution of nifurtimox across the healthy and trypanosome-infected murine blood-brain and blood-cerebrospinal fluid barriers.

Nifurtimox, an antiparasitic drug, is used to treat American trypanosomiasis (Chagas disease) and has shown promise in treating central nervous system (CNS)-stage human African trypanosomiasis (HAT; sleeping sickness). In combination with other antiparasitic drugs, the efficacy of nifurtimox against HAT improves, although why this happens is unclear. Studying how nifurtimox crosses the blood-brain barrier (BBB) and reaches the CNS may clarify this issue and is the focus of this study. To study the interaction of nifurtimox with the blood-CNS interfaces, we used the in situ brain/choroid plexus perfusion technique in healthy and trypanosome-infected mice and the isolated incubated choroid plexus. Results revealed that nifurtimox could cross the healthy and infected blood-brain and blood-cerebrospinal fluid (CSF) barriers (K(in) brain parenchyma was 50.8 ± 9.0 µl · min(-1) · g(-1)). In fact, the loss of barrier integrity associated with trypanosome infection failed to change the distribution of [(3)H]nifurtimox to any significant extent, suggesting there is not an effective paracellular barrier for [(3)H]nifurtimox entry into the CNS. Our studies also indicate that [(3)H]nifurtimox is not a substrate for P-glycoprotein, an efflux transporter expressed on the luminal membrane of the BBB. However, there was evidence of [(3)H]nifurtimox interaction with transporters at both the blood-brain and blood-CSF barriers as demonstrated by cross-competition studies with the other antitrypanosomal agents, eflornithine, suramin, melarsoprol, and pentamidine. Consequently, CNS efficacy may be improved with nifurtimox-pentamidine combinations, but over time may be reduced when nifurtimox is combined with eflornithine, suramin, or melarsoprol.

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.

Is use of nifurtimox for the treatment of Chagas disease compatible with breast feeding? A population pharmacokinetics analysis.

INTRODUCTION: Women with Chagas disease receiving treatment with nifurtimox are discouraged from breast feeding. Many patients who would receive treatment with nifurtimox live in extreme poverty, have limited access to resources such as clean water and baby formula and may not have safe alternatives to breast milk. AIM: We aimed to estimate, using limited available pharmacokinetics data, potential infant exposure to nifurtimox through breast milk. METHODS: Original nifurtimox plasma concentrations were obtained from published studies. Pharmacokinetic parameters were estimated using non-linear mixed-effect modelling with NONMEM V.VI. A total of 1000 nifurtimox plasma-concentration profiles were simulated and used to calculate the amount of drug that an infant would be exposed to, if breast fed 150 ml/kg/day. RESULTS: Breast milk concentrations on the basis of peak plasma levels (1361 ng/ml) and milk-plasma ratio were estimated. We calculated infant nifurtimox exposure of a breastfed infant of a mother treated with this drug to be below 10% of the maternal weight-adjusted dose, even if milk-plasma ratio were overestimated. Simulation led to similar estimates. DISCUSSION: Risk for significant infant exposure to nifurtimox through breast milk seems small and below the level of exposure of infants with Chagas disease receiving nifurtimox treatment. This potential degree of exposure may not justify discontinuation of breast feeding.

Pediatric clinical pharmacology studies in Chagas disease: focus on Argentina.

Chagas disease is a neglected parasitic disease endemic in the Americas. It mainly affects impoverished populations and the acute phase of the infection mostly affects children. Many cases have also been detected in nonendemic countries as a result of recent migratory trends. The chronic phase is relatively asymptomatic, but 30% of patients with chronic infection eventually develop cardiac and digestive complications that commonly lead to death or disability. Only two drugs are available for the treatment of Chagas disease, benznidazole and nifurtimox. These drugs have been shown to be effective in the treatment of both acute and early chronic phases in children, but the pharmacokinetics of these drugs have never been studied in this population. We have set out to conduct a pharmacokinetics study of benznidazole in a pediatric population with Chagas disease. The results of this study are expected to allow better estimation of the optimal doses and schedule of pharmacotherapy for Chagas disease in children.

Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects.

Benznidazole (Bz) and Nifurtimox (Nfx) have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects

Benznidazole (Bz) and Nifurtimox (Nfx) have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

Toxic side effects of drugs used to treat Chagas' disease (American trypanosomiasis).

Chagas' disease (American trypanosomiasis) is an endemic parasitic disease in some areas of Latin America. About 16-18 million persons are infected with the aetiological agent of the disease, Trypanosoma cruzi, and more than 100 million are living at risk of infection. There are different modes of infection: (1) via blood sucking vector insects infected with T. cruzi, accounting for 80-90% of transmission of the disease; (2) via blood transfusion or congenital transmission, accounting for 0.5-8% of transmission; (3) other less common forms of infection, eg, from infected food or drinks or via infected organs used in transplants. The acute phase of the disease can last from weeks to months and typically is asymptomatic or associated with fever and other mild nonspecific manifestations. However, life-threatening myocarditis or meningoencephalitis can occur during the acute phase. The death rate for persons in this phase is about 10%. Approximately 10-50% of the survivors develop chronic Chagas' disease, which is characterized by potentially lethal cardiopathy and megacolon or megaoesophagus. There are two drugs available for the aetiological treatment of Chagas' disease: nifurtimox (Nfx) and benznidazole (Bz). Nfx is a nitrofurane and Bz is a nitroimidazole compound. The use of these drugs to treat the acute phase of the disease is widely accepted. However, their use in the treatment of the chronic phase is controversial. The undesirable side effects of both drugs are a major drawback in their use, frequently forcing the physician to stop treatment. The most frequent adverse effects observed in the use of Nfx are: anorexia, loss of weight, psychic alterations, excitability, sleepiness, digestive manifestations such as nausea or vomiting, and occasionally intestinal colic and diarrhoea. In the case of Bz, skin manifestations are the most notorious (e.g., hypersensitivity, dermatitis with cutaneous eruptions, generalized oedema, fever, lymphoadenopathy, articular and muscular pain), with depression of bone marrow, thrombocytopenic purpura and agranulocytosis being the more severe manifestations. Experimental toxicity studies with Nfx evidenced neurotoxicity, testicular damage, ovarian toxicity, and deleterious effects in adrenal, colon, oesophageal and mammary tissue. In the case of Bz, deleterious effects were observed in adrenals, colon and oesophagus. Bz also inhibits the metabolism of several xenobiotics biotransformed by the cytochrome P450 system and its reactive metabolites react with fetal components in vivo. Both drugs exhibited significant mutagenic effects and were shown to be tumorigenic or carcinogenic in some studies. The toxic side effects of both nitroheterocyclic derivatives require enzymatic reduction of their nitro group. Those processes are fundamentally mediated by cytochrome P450 reductase and cytochrome P450. Other enzymes such as xanthine oxidoreductase or aldehyde oxidase may also be involved.

Nifurtimox biotransformation to reactive metabolites or nitrite in liver subcellular fractions and model systems.

Liver microsomal (mic); nuclei (N) and mitochondria (mit) anaerobically nitroreduce Nifurtimox (Nfx) in the presence of NADPH generating system. Simultaneous formation of small amounts of nitrite was observed in microsomes and nuclei but not in mitochondria. The microsomal nitroreductase activity was enhanced by the presence of flavine-adenine-dinucleotide disodium salt (FAD), was not inhibited by CO and was significantly inhibited by diphenyleneiodonium (DPI). In the microsomal NADPH-dependent fraction nitrite formation was null in the presence of FAD, DPI and under air and was partially inhibited by pure CO. Pure human cytochrome P450 reductase in the presence of NADPH significantly nitroreduced Nfx and produced small amounts of nitrite. The nitroreductive process was significantly enhanced by FAD but the nitrite formation became null. FAD itself was able to chemically nitroreduce Nfx without production of nitrite. NADPH generating system enhanced the FAD nitroreductive effect and led to small production of nitrite. Formation of reactive metabolites and nitric oxide during Nfx metabolism might contribute to its toxicity.

Enzymatic reduction studies of nitroheterocycles.

The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.

Rat liver nuclear nifurtimox nitroreductase activity.

Nifurtimox (Nfx) 4-[(5-nitrofurfurylidine)amino-3-methylthiomorpholine-1-1-di oxide] is a drug that is being used to treat American Trypanosomiasis (Chagas' disease). Nfx has serious toxic effects including mutagenic, reproductive and carcinogenic actions. Its toxicity has been linked to NADPH dependent nitroreductive metabolic biotransformation with production of oxygen reactive species. In this study is reported that rat liver nuclei exhibit Nfx-nitroreductase activity (Nfx-ase). This activity is null under oxygen and partially inhibited under CO. Nfx does not promote a lipid peroxidation process. Results suggest that Nfx is biotransformed partially at a cytochrome P450 level but mostly by NADPH P450 reductase. Formation of reactive metabolites nearby DNA and nuclear proteins might be related to long term deleterious effects of this drug.

Disposition of nifurtimox and metabolite activity against Trypanosoma cruzi using rat isolated perfused liver.

Nifurtimox disposition was investigated using the rat isolated perfused-liver method after administration of 25 micrograms mL-1 nifurtimox, and its disappearance was monitored by analysing the perfusate sample at various times. Biliary excretion was also measured. The drug concentration profile underwent a biexponential decline over the 2-h study period, with a terminal half-life of 62.76 +/- 17.56 min. Nifurtimox is a high clearance compound (15.23 +/- 5.53 mL min-1). The extraction ratio was 0.621 +/- 0.159. Biliary excretion accounted for 0.05% of the dose, the remainder consisting of highly polar metabolites. By 2 h, a minimal fraction of unchanged nifurtimox was recovered from the perfusate. Nifurtimox activity against Trypanosoma cruzi (clone CA-1) during the perfusion was also determined. Epimastigotes isolated from continuous culture were exposed to the samples of perfusate at different perfusion times in a microtitre plate. After an incubation time of 72 h at 27 degrees C, the parasite number in each well was counted under a microscope. From 0 to 75 min after the perfusion, the anti-trypanosomal activity decreased, but an increase in activity was observed at the later times. These findings show that active metabolites are formed during the perfusion.