Structural and Mechanistic Investigation of the Unusual Metabolism of Nifurtimox.

The oral antiparasitic drug nifurtimox has been used to treat Chagas disease for more than 50 years. Historical studies determined that very little nifurtimox is excreted unchanged, but contemporaneous preclinical studies of radiolabeled nifurtimox found almost all of the radiolabel was rapidly excreted, suggesting that metabolism is extensive. Attempts to study nifurtimox metabolism have had limited success, yet this knowledge is fundamental to characterizing the pharmacokinetics and pharmacodynamics of the drug. We conducted in vitro studies using hepatic and renal sources with 14C-labeled nifurtimox as substrate and obtained samples of urine, plasma, and feces from rats administered 2.5 mg/kg [14C]-nifurtimox, and samples of human urine and plasma from phase 1 clinical studies in which participants received a single dose of 120 mg nifurtimox. Analysis of metabolites was done by high-performance liquid chromatography (HPLC)-high-resolution mass spectrometry (HRMS) and HRMS/MS with offline liquid scintillation counting of radiolabeled samples. Surprisingly, only traces of a few metabolites were identified from in vitro incubations with hepatocytes and subcellular fractions, but more than 30 metabolites were identified in rat urine, mostly with atypical mass changes. We developed an HRMS scouting method for the analysis of human samples based on the sulfur atom in nifurtimox and the natural abundance of 34S, as well as a characteristic tandem mass spectrometry (MS/MS) fragmentation of nifurtimox and metabolites. Fragmentation patterns on HRMS/MS were used to propose structures for 18 metabolites (22 including stereoisomers), and based on these structures, the six most abundant products were synthesized and the structures of the synthetic forms were confirmed by HRMS and two-dimensional nuclear magnetic resonance (2D NMR). Overall, we determined that the metabolism of nifurtimox is almost certainly not mediated by typical hepatic and renal drug-metabolizing enzymes, and instead is rapidly metabolized mainly by reduction or nucleophilic attack, with some evidence of oxidation. Knowledge of the most abundant metabolites of nifurtimox affords the possibility of future studies to investigate levels of exposure and possible drug-drug interactions.

Negligible exposure to nifurtimox through breast milk during maternal treatment for Chagas Disease.

BACKGROUND: Treatment with nifurtimox (NF) for Chagas disease is discouraged during breast-feeding because no information on NF transfer into breast milk is available. NF is safe and effective for paediatric and adult Chagas disease. We evaluated the degree of NF transfer into breast milk in lactating women with Chagas disease. PATIENTS AND METHODS: Prospective study of a cohort of lactating women with Chagas disease. Patients were treated with NF for 1 month. NF was measured in plasma and milk by high performance liquid chromatography (HPLC). Breastfed infants were evaluated at admission, 7th and 30th day of treatment (and monthly thereafter, for 6 months). RESULTS: Lactating women with chronic Chagas disease (N = 10) were enrolled (median age 28 years, range 17-36). Median NF dose was 9.75 mg/kg/day three times a day (TID). Six mothers had mild adverse drug reactions (ADRs), but no ADRs were observed in any of the breastfed infants. No interruption of breastfeeding was observed. Median NF concentrations were 2.15 mg/L (Inter quartil range (IQR) 1.32-4.55) in milk and 0.30 mg/L (IQR 0.20-0.95) in plasma. Median NF milk/plasma ratio was 16 (range 8.75-30.25). Median relative infant NF dose (assuming a daily breastmilk intake of 150 mL/kg/day) was 6.7% of the maternal dose/kg/day (IQR 2.35-7.19%). CONCLUSIONS: The low concentrations of NF in breast milk and the normal clinical evaluation of the breastfed babies imply that maternal NF treatment for Chagas disease during breastfeeding is unlikely to lead to clinically relevant exposures in the breastfed infants. TRIAL REGISTRATION: Clinical trial registry name and registration number: ClinicalTrials.gov NCT01744405.

Development of an ionic-liquid-based dispersive liquid-liquid microextraction method for the determination of antichagasic drugs in human breast milk: Optimization by central composite design.

Chagas disease constitutes a major public health problem in Latin America. Human breast milk is a biological sample of great importance for the analysis of therapeutic drugs, as unwanted exposure through breast milk could result in pharmacological effects in the nursing infant. Thus, the goal of breast milk drug analysis is to inquire to which extent a neonate may be exposed to a drug during lactation. In this work, we developed an analytical technique to quantify benznidazole and nifurtimox (the two antichagasic drugs currently available for medical treatment) in human breast milk, with a simple sample pretreatment followed by an ionic-liquid-based dispersive liquid-liquid microextraction combined with high-performance liquid chromatography and UV detection. For this technique, the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate has been used as the "extraction solvent." A central composite design was used to find the optimum values for the significant variables affecting the extraction process: volume of ionic liquid, volume of dispersant solvent, ionic strength, and pH. At the optimum working conditions, the average recoveries were 77.5 and 89.7%, the limits of detection were 0.06 and 0.09 µg/mL and the interday reproducibilities were 6.25 and 5.77% for benznidazole and nifurtimox, respectively. The proposed methodology can be considered sensitive, simple, robust, accurate, and green.

Follicular atresia in the prepubertal spiny mouse (Acomys cahirinus) ovary.

This study was designed to determine follicular atresia in the newborn and the prepubertal spiny mouse. We analyzed the processes of follicle loss using classical markers of apoptosis (TUNEL reaction, active caspase-3) and autophagy (Lamp1). Numerous small clear vacuoles and autophagosomes as well as strong Lamp1 staining were observed in dying oocytes of all follicle types, especially of the primordial and primary ones. Active caspase 3 and the TUNEL reaction were detected only in the granulosa cells of large secondary and antral follicles. The expression of apoptosis and autophagy markers was also changing during the prepubertal period. Western blot analysis indicated that at the moment of birth, females undergo an increased rate of follicular atresia mediated by autophagy, while apoptosis is the dominant form of ovarian atresia in consecutive postnatal days. On the basis of these observations, we concluded that apoptosis and autophagy are involved in follicular atresia and these processes are cell and developmental stage-specific.

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 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.