Trypanosoma cruzi; LDH (Lactate dehydrogenase) [1]

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].

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].

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.

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

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.

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.

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.

[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%.

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.)