Trypanosoma cruzi; LDH (Lactate dehydrogenase) [1]
Nifurtimox’s primary mechanism involves the generation of reactive oxygen species (ROS) through redox cycling. The drug is reduced by nitroreductase enzymes in the target organism, producing a highly reactive nitro anion free radical. Autooxidation of this radical generates cytotoxic superoxide anions and other ROS that cause oxidative stress, DNA damage, and cellular structure disruption. It also affects lactate dehydrogenase (LDH) enzyme activity. These effects lead to parasite death, though the exact mechanism is not fully elucidated.

Lactate dehydrogenase (LDH) enzyme activity is impacted by furolimus. LDH enzymatic activity was measured 4 hours after 50 µg/mL Nifurtimox administration in order to differentiate between changes in pyruvate metabolism brought on by PDH activation and a decrease in LDH activity. When compared to the untreated control group, the LDH activity of LA-N-1 (P=0.005), IMR-32 (P=0.009), LS (P=0.0035), and SK-N-SH (P=0.0065) was significantly lower. ..In neuroblastoma cells, furtimox decreases cell viability and causes apoptosis and cell cycle arrest. Numerous studies were conducted on four different cell lines to assess the cytotoxic effects of nifurtimox on neuroblastoma. Cell viability for all four neuroblastoma cell lines (LA-N-1, IMR-32 LS, SK-N-, and SH) dropped to an average of 66%, 63%, 62%, and 75% after a 24-hour incubation period at 50 µg/mL. When compared to vehicle controls with DMSO (P<0.05) and untreated controls (P<0.01), this reduction was substantial for all cell lines [1].
In vitro, nifurtimox demonstrates potent activity against Trypanosoma cruzi, the causative agent of Chagas disease, with IC50 values in the low micromolar range. It reduces cell viability and induces cell cycle arrest and apoptosis in neuroblastoma cell lines. Studies have characterized its cytotoxic effects across multiple cancer cell lines. It also inhibits lactate dehydrogenase activity in biochemical assays. The compound shows concentration-dependent antiparasitic activity in axenic cultures, with efficacy correlated with nitroreductase expression levels in target organisms.

Nifurtimox could competently hinder the development of astroglioma in the mouse brain as compared to temozolomide, the first line of drug for brain tumors. Meanwhile the surviving rate, as well as the body-weight was dramatically upregulated upon nifurtimox treatment, as compared to that of temozolomide. These findings offered nifurtimox as a better alternative drug in treating astroglioma in vivo[2].
In vivo, nifurtimox is administered orally and has demonstrated efficacy in animal models of Chagas disease by reducing parasitemia and improving survival. It has been used clinically for the treatment of Trypanosoma cruzi infections. In neuroblastoma xenograft models, the compound shows tumor growth inhibition through its effects on N-Myc expression and aerobic glycolysis. Its in vivo activity is dose-dependent, with therapeutic efficacy balanced against toxicity. Clinical studies have confirmed its utility in treating acute and chronic Chagas disease, though treatment duration is prolonged and side effects are common.

Reactive oxygen species – DCF assay[1]
Neuroblastoma cells were treated with nifurtimox as described and were subjected to a flow cytometric analysis of intracellular ROS; 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) is taken up into the cells and metabolized to the fluorescent 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. Cells were dyed using the Cellular Reactive Oxygen Species Detection Kit according to the manufacturer's protocol and the intracellular amount of ROS was quantified with a flow cytometer.[1]

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Western Blot[1]
Neuroblastoma cells were treated with nifurtimox as described and indicated. For separation of mitochondrial (determination of (phosphorylated) pyruvate dehydrogenase (PDH, PDH-P)) and cytosolic fraction (determination of N-Myc), 5 × 107 treated cells were homogenized with a dounce tissue grinder. Mitochondria were subsequently extracted using the Mitochondria/Cytosol fractionation kit according to the manufacturer's instructions.
In vitro enzyme/receptor binding studies for nifurtimox typically focus on its interaction with nitroreductases, the enzymes responsible for its bioactivation. Assays measure the reduction of the nitro group by cellular or recombinant nitroreductases under anaerobic or aerobic conditions, monitoring the generation of reactive intermediates using spectroscopic or electrochemical methods. The compound also inhibits lactate dehydrogenase (LDH) activity, which can be assessed spectrophotometrically by measuring NADH oxidation rates in the presence of pyruvate. Standard enzyme kinetics protocols are employed to determine IC50 values and inhibition mechanisms.

Cell viability – MTS assay[1]
To assess the cell viability after incubation with nifurtimox at different concentrations (10 µg/mL up to 50 µg/mL or 34.8 µM to 174 µM, respectively in the supernatant growth medium) or the vehicle control with according concentrations, all neuroblastoma cell lines were subjected to an MTS assay. Stock solutions of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) were made at 480 µM in sterile filtered deionized water and stored at −20°C. Cells were grown to approximately 50% confluency, treated with nifurtimox, and incubated for 1 h with fresh media containing 12 µM MTS. The supernatant was subsequently removed and the cells were lysed with DMSO containing 10% (w/v) sodium dodecyl sulfate (SDS; Carl Roth, #0183) and 1% (v/v) glacial acetic acid. Purple formazan contents of each cell lysate were photometrically analyzed in triplicates at 570 nm (630 nm reference wave length) in 96 microtiter plates.

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Differentiation of apoptosis and necrosis[1]
50% confluent neuroblastoma cells were treated with nifurtimox as described and dyed with Annexin V (apoptotic cells) and Ethidium homodimer III (necrotic cells) using the Apoptotic/Necrotic Cells Detection Kit according to the manufacturer's instructions.

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Analysis of cell cycle profiles[1]
50% confluent neuroblastoma cells were treated with nifurtimox as described. Cells were stained using the Nuclear-ID® Green Cell Cycle Kit for flow cytometry according to the manufacturer's protocol. Dyed cells were analyzed in a flow cytometer
In vitro cellular assays for nifurtimox involve culturing Trypanosoma cruzi epimastigotes or amastigotes in appropriate media and treating with serial dilutions of the compound to determine IC50 values for parasite growth inhibition. For anticancer studies, neuroblastoma and other cancer cell lines are plated in 96-well plates, treated with nifurtimox for 48-72 hours, and cell viability is assessed using MTT or resazurin-based assays. Apoptosis is quantified by flow cytometry using Annexin V/PI staining, and cell cycle analysis is performed by propidium iodide staining.

Establishment and Bioluminescence Detection of Mice Tumor Model for Brain Orthotopic Transplantation [2]
Prior to implantation, the U251-luc2-GFP cells were harvested in the logarithmic growth phase, washed with PBS and re-suspended in PBS at the concentration of 2×107 cells/ml. Nude mice were anesthetized and fixed; 20 μl of the cell suspension/per mice (namely 4×105 cells) was injected slightly with a micro-syringe into the right side of the nude mouse brain. The IVIS Spectrum CT imaging system was used to detect bioluminescence at various time points. Briefly, mice were first anesthetized with 2% isoflurane for preparation, and then, transferred into the chamber of IVIS Spectrum CT machine while maintaining the mice with isofluran (0.5%) for sleeping. Bioluminescence images were taken and lastly, the detection signal was quantified with the imaging software coupled with the IVIS Spectrum CT system. 2.7.

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Design for the Treated Groups and Drug Administration Experiment[2]
7 days after surgical implantation, the tumor-bearing mice were re-imaged with IVIS Spectrum CT machine. A total of 50 mice were randomly divided into 5 groups with 10 mice per group: one normal control group (treated with sodium carboxymethyl cellulose solution), one positive control group (temozolomide 30 mg/kg) and three nifurtimox treated groups at the concentrations of 50 mg/kg, 100 mg/kg and 200 mg/kg. The drugs dissolved in sodium carboxymethyl cellulose solution were administered by gavage for 1 time/day at the dosage of 0.2mL/20g. All animals were daily checked for health conditions and weighed every other day. The bioluminescence signal for the tumor progression was planned for day 1 (7 days after injection), day 6 and day 9 according to the health condition of the individual mice. All treated animals were sacrificed after treatment, brain and tumor block were dissected, weighed and photographed.
In vivo animal studies for nifurtimox use murine models of Chagas disease, where mice are infected with Trypanosoma cruzi and treated orally with nifurtimox at various doses for several weeks. Parasitemia is monitored by microscopic examination of blood samples, and survival rates are recorded. In neuroblastoma studies, tumor-bearing mice are dosed orally or intraperitoneally, and tumor volume is measured over time. Standard protocols include assessment of drug efficacy, toxicity, and pharmacokinetic parameters, with comparison to untreated controls and reference drugs.

Absorption, Distribution and Excretion
The mean AUC of nifurushimil is estimated to be between 1676 and 2670 μg∙h/L. A pharmacokinetic study in healthy volunteers showed an AUC of 5430 ng∙ml⁻¹∙h. After a single 20 mg dose (postprandial) in adults, the Cmax ranged from 425 to 568 μg/L (26-50%). The Tmax was 4 hours, 2-8 hours postprandial. In a pharmacokinetic study in healthy volunteers, serum concentrations were low, likely due to the first-pass effect. 44% of the dose is excreted primarily as metabolites in the urine postprandially. Fecal and bile excretion of nifurushimil has not been investigated. Nifurushimil can cross the blood-brain barrier and the placenta. A pharmacokinetic study of nifurushimil showed a clearance of 193.4 L∙h⁻¹. In patients with normal renal function, the clearance rate is 99.7 L∙h⁻¹. Metabolism/Metabolites Nifurtimoxos is primarily metabolized by nitroreductases. Two major inactive metabolites have been identified: M-4 and M-6. The M-4 metabolite is a cysteine conjugate of Nifurtimoxos, while M-6 is likely formed by the hydrolytic cleavage of the Nifurtimoxos hydrazone moiety. Several other minor metabolites have also been found in human plasma. Biological Half-Life The elimination half-life of Nifurtimoxos is 2.4–3.6 hours. A pharmacokinetic study in healthy volunteers and patients with renal failure showed mean half-lives of 2.95 hours and 3.95 hours, respectively.
Pharmacokinetic properties of nifurtimox include good oral bioavailability after administration. It is metabolized extensively in the liver, primarily by nitroreduction, and its metabolites are excreted renally. Peak plasma concentrations are achieved within a few hours of oral dosing. The compound has a moderate half-life, supporting twice-daily dosing in clinical regimens. Its distribution includes penetration into tissues where Trypanosoma cruzi resides, including the heart and gastrointestinal tract. Metabolism varies among individuals due to genetic polymorphisms in nitroreductase enzymes, affecting efficacy and toxicity profiles.

Hepatotoxicity
In multiple prospective controlled trials, Nifurtimoxide treatment was not associated with elevated aminotransferase or bilirubin levels or clinically visible liver injury. Since Nifurtimoxide was approved for the treatment of Chagas disease, there have been no reported cases of liver injury associated with its use. Probability score: E (Unlikely to cause clinically visible liver injury with recommended Chagas disease treatment regimens). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Limited information suggests that maternal administration of up to 15 mg/kg of Nifurtimoxide daily does not cause any serious adverse effects in breastfed infants. Breast milk concentrations and computer simulations show that exclusively breastfed infants ingest doses via breast milk that are significantly lower than the doses used to treat Chagas disease in newborns. Other authors consider breastfeeding during Nifurtimoxide use not to be contraindicated.
◉ Impact of Breastfeeding on Infants
In the Democratic Republic of Congo, a cohort study of 33 infants involved hospitalized mothers taking nifurulimus who breastfed (feeding extent not specified). Thirty mothers completed 30 doses of oral nifurulimus (15 mg/kg/day), and all mothers received 14 doses of intravenous efornithine (400 mg/kg/day) for 7 days to treat human African trypanosomiasis (sleeping sickness). The breastfeeding mothers also received an average of four other concomitant medications, including amoxicillin, ciprofloxacin, metronidazole, trimethoprim-sulfamethoxazole, aspirin, and diclofenac (1 case each); hydrocortisone, promethazine, and quinine (2 cases each); levamisole (6 patients); sulfadoxine-pyrimethamine (8 patients); aminopyrine (13 patients); acetaminophen (16 patients); and mebendazole (17 patients). No serious adverse events were reported in any of the breastfed infants.
◉ Effects on breastfeeding and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Nifurulimus has a plasma protein binding rate of approximately 42%. It is primarily bound to albumin.
Toxicological data for nifurtimox indicate a significant side effect profile, including gastrointestinal disturbances (nausea, vomiting, anorexia), neurological symptoms (headache, dizziness, peripheral neuropathy), and hypersensitivity reactions. Long-term use is associated with dose-dependent toxicity, and treatment may need to be interrupted or dose-reduced in some patients. The compound’s mechanism involving ROS generation contributes to its cytotoxic effects on host cells as well as parasites. Carcinogenicity studies in animals have shown some concerns, but the drug remains a critical treatment for Chagas disease due to limited alternatives.

[1]. Nifurtimox reduces N-Myc expression and aerobic glycolysis in neuroblastoma. Cancer Biol Ther. 2015;16(9):1353-63.

Nifurtimox is a nitrofuran antibiotic. It is an antiprotozoal prescription drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of Chagas disease (American trypanosomiasis) caused by Trypanosoma cruzi, and is indicated for children under 18 years of age and weighing at least 2.5 kg (5.5 lbs). Chagas disease can be an opportunistic infection of HIV. Chagas disease is caused by a parasite called Trypanosoma cruzi and is a vector-borne disease that primarily affects animals and humans in the Americas. It is commonly known as American trypanosomiasis. The U.S. Centers for Disease Control and Prevention (CDC) estimates that approximately 8 million people in Central America, South America, and Mexico are infected with Trypanosoma cruzi but are asymptomatic. If Chagas disease is not treated promptly, it can lead to life-threatening sequelae. Nifurtimox, developed by Bayer, is a nitrofuran antiprotozoal drug used to treat Chagas disease. On August 6, 2020, the U.S. Food and Drug Administration (FDA) granted accelerated approval for its use in pediatric patients based on favorable results from the Phase III clinical trial. Continued approval will depend on subsequent confirmatory data. One advantage of this formulation manufactured by Bayer is that the scored tablets can be manually divided without the need for a cutter. Nifurtimox is a nitrofuran antibiotic used to treat Chagas disease (American trypanosomiasis), a chronic protozoan infection caused by Trypanosoma cruzi, which can lead to severe gastrointestinal and cardiac problems, resulting in disability or even death. Nifurtimox treatment rarely causes elevated serum transaminases, and no clinically significant cases of liver injury have been observed. Nifurtimox is a nitrofuran derivative with antiprotozoal activity and potential antitumor activity. Nifurtimox is reduced to a highly reactive nitro anion radical by cytoplasmic enzymes or flavin-containing microsomal enzymes; the auto-oxidation of this nitro anion radical generates cytotoxic superoxide anion (O₂⁻). Furthermore, Nifurtimoxose-derived nitro anion radicals can alkylate macromolecules such as nucleic acids and proteins, thereby disrupting their structure and function. Nifurtimoxose is a nitrofurantoin derivative used to treat trypanosomiasis. Indications: Nifurtimoxose is indicated for children under 18 years of age weighing at least 2.5 kg. Continued approval for this indication depends on the results of confirmatory clinical trials. Treatment of Chagas Disease: Mechanism of Action: The mechanism of action of Nifurtimoxose is not fully understood, but it is believed to involve the activation of nitroreductases to produce active metabolites that exert a series of harmful effects on Trypanosoma cruzi, the parasite that causes Chagas disease. Nifurtimoxose's antiprotozoal activity exists both intracellularly and extracellularly. Inhibition of parasite dehydrogenase activity is another potential mechanism of action for Nifurtimoxose and warrants further investigation. Pharmacodynamics: Nifurtimoxose exhibits trypanosome activity against Trypanosoma cruzi and can be used to treat Chagas disease. A study reported that nifurulimus and other benzofuran derivatives can reduce the activity of parasite dehydrogenases. Recent results from a phase III clinical trial showed that a large number of pediatric patients with acute or chronic Chagas disease treated with nifurulimus were negative for immunoglobulin G (IgG) antibodies, and in two IgG antibody tests against Trypanosoma cruzi antigens, optical density decreased by at least 20%.
Nifurtimox is approved for the treatment of Chagas disease in many countries, particularly in Latin America, and is included on the WHO Model List of Essential Medicines. It is typically used in combination with benznidazole in some regimens. The drug is also being investigated for repurposing in oncology, particularly for neuroblastoma. Its mechanism as a nitroreductase-activated prodrug makes it a valuable tool for studying ROS-mediated cytotoxicity. Clinical use requires careful monitoring due to its narrow therapeutic index and significant toxicity profile.

C10H13N3O5S

287.29232

287.058

C, 41.81; H, 4.56; N, 14.63; O, 27.85; S, 11.16

23256-30-6

Nifurtimox-d4

6842999

Light yellow to yellow solid powder

1.56g/cm3

550.3ºC at 760mmHg

177-183

286.6ºC

3.7E-12mmHg at 25°C

1.653

2.182

0

7

2

19

467

0

O=S1(CC(C)N(/N=C/C2=CC=C([N+]([O-])=O)O2)CC1)=O

ARFHIAQFJWUCFH-IZZDOVSWSA-N

InChI=1S/C10H13N3O5S/c1-8-7-19(16,17)5-4-12(8)11-6-9-2-3-10(18-9)13(14)15/h2-3,6,8H,4-5,7H2,1H3/b11-6+

(E)-3-methyl-4-(((5-nitrofuran-2-yl)methylene)amino)thiomorpholine 1,1-dioxide

BAY 2502; Bayer 2502; NIFURTIMOX; Lampit; Bayer 2502; 23256-30-6; Nifurtimoxum; BAY 2502; (+/-)-Nifurtimox; BAYER-2502; Lampit.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage. In solution, nifurtimox is critically unstable when exposed to light. Therefore, all stock solutions and standard solutions containing nifurtimox should be handled in light-proof containers and reaction tubes. Nifurtimox solutions should be discarded after 7 d.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 57~150 mg/mL (198.4~522.10 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)