Ferroptosis (EC50 = 60 nM)

Ferrostatin-1 inhibits the build-up of lipid and cytosolic ROS that is generated by erastin. Neurotoxicity caused by glutamate is inhibited in organotypic rat brain slices by ferrostatin-1 [1]. Rat organotypic hippocampal slice cultures (OHSC) are protected from glutamate (5 mM)-induced neurotoxicity by ferrostatin-1 (2 μM; 24 hours) [2]. Ferrostatin-1 suppresses lipid peroxidation, but not the permeability of lysosomal membranes or the production of reactive oxygen species in the mitochondria [2]. In cellular models of renal failure, periventricular leukomalacia (PVL), and Huntington's disease (HD), ferrostatin-1 reduces cell death [2]. In HT-1080 cells, ferrostatin-1 (1 μM; 6 hours) prevents unsaturated fatty acid oxidative degradation, which increases the quantity of healthy medium spiny neurons (MSNs) [3].

In mice with rhabdomyolysis, ferrostatin-1 (5 mg/kg; i.p.; single dosage, given 30 min before glycerol injection) improves renal function; however, this benefit is not shown in mice lacking in the pan-caspase inhibitor zVAD or RIPK3. Acute lung damage (ALI) caused by LPS can be efficiently treated with ferrostatin-1 (0.8 mg/kg; tail vein injection)[4]. Rhabdomyolysis-affected mice's renal function is improved by ferrostatin-1 (5 mg/kg; i.p.; C57BL/6J mice) [5].

Western blot[4]
In our study, the cell samples were lysed using radioimmunoprecipitation assay lysis buffer, and the total protein concentration of different groups was detected using the Pierce BCA Protein Assay Kit. In our study, the cell lysates (20 μg/lane) were separated using 10% SDS-PAGE gel and then transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat dried milk diluted in PBS, and further incubated with primary antibodies overnight at 4 °C. Herein, the different primary antibodies used were: anti-SLC7A11 (1:3000; Cell signaling, Cat #: 12691), anti-GPX4 (1:1000), anti-FTH (1:2000) and anti-GAPDH (1:3000). The secondary antibodies used were: Anti-mouse IgG (HRP-conjugated; 1:5000) and anti-rabbit IgG (HRP-conjugated; 1:5000). Finally, the protein bands in each lane were visualized using SuperSignal West Femto Maximum Sensitivity Substrate and ChemiDoc Imagers. The results were finally quantified using the ImageJ 1.x software. All of the raw, uncropped blots for images throughout the paper are shown in Supplementary Fig. 1. [4]
Evaluation of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and iron level[4]
In our study, to evaluate the ferroptosis level in different groups, the MDA, 4-HNE and iron levels were detected in each group. The MDA concentration, 4-HNE concentration and iron concentration in cell lysates were assessed using the Lipid Peroxidation (MDA) Assay Kit, Lipid Peroxidation (4-HNE) Assay Kit and Iron Assay Kit according to the manufacturer’s instructions.

Cell viability assay[4]
To evaluate cell viability, the CCK-8 method was used in our study as the references. In brief, BEAS-2B cells were seeded into a 96-well plate at the concentration of 5 × 104 cells/well. The cells were cultured for 24 h, then treated with LPS and Fer-1 in different concentrations for 16 h followed by the addition of 20 μl of CCK-8 solution directly into the medium (200 μl per well) and incubation at 37 °C for 4 h. The absorbances (Abs) in different groups were detected at 450 nm (n = 3). In the blank group, the well only contained medium, and the cells without any treatment were used as the control group. Herein, the cell viability = (Abs of experimental group-Abs of blank group)/(Abs of control group-Abs of blank group) × 100%.

Animal/Disease Models: Male C57BL/6 mice (LPS-induced ALI)[4]
Doses: 0.8 mg/kg
Route of Administration: Tail vein injection
Experimental Results: Exerted therapeutic action against LPS-induced ALI.

---

In our study, the male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8–10 weeks old, weight = 23–25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 μl of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively. After the treatments for 16 h, the mice in each group were euthanized and bronchoalveolar lavage (BAL) fluid was collected via lung lavage. To analyze the differential BAL cell counts, the cells were concentrated using a Cytospin 4. Cell staining was performed using the Shandon Kwik-Diff kit. Additionally, the total protein concentration and the levels of IL-6 and TNF-α in each sample were detected with the Pierce BCA Protein Assay Kit, IL-6 ELISA Kit ELISA kit and TNF-α ELISA Kit according to the manufacturer’s instructions. Lung tissues in different groups were collected for qPCR and western blot detection, and part of lung tissues was fixed using 10% buffered formalin, then the tissues were embedded in paraffin for histological analyses as the references. Herein, a scoring system of 0–4 was used for the evaluation of lung injury as the reference.

[1]. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-1072.

[2]. Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models. J Am Chem Soc. 2014;136(12):4551-4556.

[3]. Antifungal Activity of the Lipophilic Antioxidant Ferrostatin-1. Chembiochem. 2017;18(20):2069-2078.

[4]. Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibiting ferroptosis. Cell Mol Biol Lett. 2020;25:10. Published 2020 Feb 27.

[5]. FP282 FERROPTOSIS-MEDIATED CELL DEATH IS DECREASED BY CURCUMIN IN RENAL DAMAGE ASSOCIATED TO RHABDOMYOLYSIS, Nephrology Dialysis Transplantation, Volume 34, Issue Supplement_1, June 2019, gfz106.FP282.

[6]. Ferrostatin-1 attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis. Transl Pediatr. 2023 Nov 28;12(11):1944-1970.

[7]. Effect of deferoxamine and ferrostatin-1 on salivary gland dysfunction in ovariectomized rats. Aging (Albany NY). 2023 Apr 6;15(7):2418-2432.

Ferrostatin-1 is an ethyl ester resulting from the formal condensation of the carboxy group of 3-amino-4-(cyclohexylamino)benzoic acid with ethanol. It is a potent inhibitor of ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. It is also a radical-trapping antioxidant and has the ability to reduce the accumulation of lipid peroxides and chain-carrying peroxyl radicals. It has a role as a ferroptosis inhibitor, a radiation protective agent, an antioxidant, a radical scavenger, an antifungal agent and a neuroprotective agent. It is a substituted aniline, an ethyl ester and a primary arylamine.

---

Background: Ferroptosis is a newly recognized type of cell death, which is different from traditional necrosis, apoptosis or autophagic cell death. However, the position of ferroptosis in lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored intensively so far. In this study, we mainly analyzed the relationship between ferroptosis and LPS-induced ALI. Methods: In this study, a human bronchial epithelial cell line, BEAS-2B, was treated with LPS and ferrostatin-1 (Fer-1, ferroptosis inhibitor). The cell viability was measured using CCK-8. Additionally, the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron, as well as the protein level of SLC7A11 and GPX4, were measured in different groups. To further confirm the in vitro results, an ALI model was induced by LPS in mice, and the therapeutic action of Fer-1 and ferroptosis level in lung tissues were evaluated. Results: The cell viability of BEAS-2B was down-regulated by LPS treatment, together with the ferroptosis markers SLC7A11 and GPX4, while the levels of MDA, 4-HNE and total iron were increased by LPS treatment in a dose-dependent manner, which could be rescued by Fer-1. The results of the in vivo experiment also indicated that Fer-1 exerted therapeutic action against LPS-induced ALI, and down-regulated the ferroptosis level in lung tissues. Conclusions: Our study indicated that ferroptosis has an important role in the progression of LPS-induced ALI, and ferroptosis may become a novel target in the treatment of ALI patients.[4]

---

Background: Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. Methods: The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. Results: The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Conclusions: Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.[6]

---

The mechanism underlying xerostomia after menopause has not yet been fully elucidated. This study aimed to investigate the mechanism of xerostomia and the effect of the ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (FER) on salivary gland dysfunction in a postmenopausal animal model. Twenty-four female Sprague-Dawley rats were randomly divided into four groups: a SHAM group (n = 6, sham-operated rats), an OVX group (n = 6, ovariectomized rats), an FER group (n = 6, ovariectomized rats injected intraperitoneally with FER), and a DFO group (n = 6, ovariectomized rats injected intraperitoneally with DFO). GPX4 activity, iron accumulation, lipid peroxidation, inflammation, fibrosis, and salivary gland function were analyzed. Recovery of GPX4 activity and a decrease in iron accumulation and cytosolic MDA + HAE were observed in the DFO group. In addition, collagen I, collagen III, TGF-β, IL-6, TNF-α, and TGF-β levels were decreased in the DFO group compared to the OVX group. Recovery of GPX4 activity and the morphology of mitochondria, and reduction of cytosolic MDA + HAE were also observed in the FER group. In addition, decreased expression of inflammatory cytokines and fibrosis markers and increased expression of AQP5 were observed in both the DFO and FER groups. Postmenopausal salivary gland dysfunction is associated with ferroptosis, and DFO and FER may reverse the postmenopausal salivary gland dysfunction after menopause. DFO and FER are hence considered promising treatments for postmenopausal xerostomia.[7]

C15H22N2O2

262.35

262.168

C, 68.67; H, 8.45; N, 10.68; O, 12.20

347174-05-4

4068248

Gray to gray purple solid

1.1±0.1 g/cm3

437.3±35.0 °C at 760 mmHg

218.3±25.9 °C

0.0±1.1 mmHg at 25°C

1.595

3.9

2

4

5

19

290

0

O(C([H])([H])C([H])([H])[H])C(C1C([H])=C([H])C(=C(C=1[H])N([H])[H])N([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O

UJHBVMHOBZBWMX-UHFFFAOYSA-N

InChI=1S/C15H22N2O2/c1-2-19-15(18)11-8-9-14(13(16)10-11)17-12-6-4-3-5-7-12/h8-10,12,17H,2-7,16H2,1H3

3-amino-4-(cyclohexylamino)-benzoic acid, ethyl ester

Frer-1; 3-amino-4-(cyclohexylamino)-benzoic acid, ethyl ester; Ferrostatin-1; 347174-05-4; Ethyl 3-amino-4-(cyclohexylamino)benzoate; Fer-1; Ferrostatin-1 (Fer-1); Ferrostatin 1; ferrrostatin 1; MFCD08072959;

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.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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.

---

Solubility in Formulation 2: 2.5 mg/mL (9.53 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 900 μL of corn oil and mix evenly.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (7.93 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 4: 0.2 mg/mL (0.76 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 5: 2% DMSO+50% PEG 300+5% Tween 80+ddH2O: 5mg/mL

---

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