Ferroptosis; system Xc-

Imidazole ketone erastin (IKE) potently reduces DLBCL cell number[1]
The 18 DLBCL cell lines showed differential sensitivity to IKE inhibition, with cell lines exhibiting IC50 < 100 nM classified as sensitive cell lines, those with IC50 > 10 μM classified as resistant cell lines, and those with IC50 values between 100 nM to 10 μM classified having intermediate resistance (Fig. 1B). We further tested the degree of IKE -induced lethality upon co-treatment with the ferroptosis inhibitor fer-1, a radical-trapping antioxidant that inhibits lethal lipid peroxidation during ferroptosis (Skouta et al., 2014, Zilka et al., 2017). Co-treatment with fer-1 rescued cell death induced by IKE in DLBCL cell lines, indicating that IKE -induced lethality in these cell lines resulted from lipid peroxidation and ferroptosis.[1]
Previous studies found that IKE inhibited glutamate release, and the IKE parental analog erastin inhibited cystine uptake. Thus, we tested the cellular level of reduced glutathione (GSH), which requires cysteine for its biosynthesis, as a readout of IKE potency. A fluorometric method revealed dose-dependent GSH depletion by IKE (Fig. 1C); this effect was reversed by co-treatment with 10 μM β-ME, which reduces cystine to cysteine, allowing its import into cells through systems A, ASC, and L, thus circumventing inhibition of system xc−. The IC50 of GSH depletion by IKE was 34 nM (Fig. S1B) in SUDHL-6 cells, while, sulfasalazine’s IC50 for GSH depletion is in the millimolar range.[1]
While, co-treatment with DFO inhibited IKE -induced cell death in culture (Fig. S1E), it only partially eliminated lipidomic changes upon IKE treatment, possibly due to DFO inhibiting iron-mediated lipid peroxidation, but not enzyme-mediated lipid peroxidation, suggesting that only a subset of lipidomic changes are needed for inducing cell death.[1]

IKE pharmacokinetics (PK) and pharmacodynamics (PD) in vivo[1]
To determine the suitability of IKE for in vivo studies, we first evaluated multiple dosage routes by administrating a single dose of IKE (50 mg/kg, 5% DMSO in HBSS at pH 4) using intraperitoneal (IP), intravenous (IV), and oral (PO) routes in NOD/SCID mice. Determination of IKE concentration over a period of eight hours revealed IP to be the most effective and practical means of IKE administration (Table S1). Next, IKE concentration in plasma and tumor samples was determined after a single dose of IKE (50 mg/kg, 5% DMSO in HBSS at pH 4, IP) in SUDHL6-xenograft-bearing NCG mice over a period of 24 hours. IKE reached the highest plasma concentration of 5.2 μg/mL at 1.35 h, and the highest tumor accumulation of 2.5 μg/mL at 3.30 h (Fig. 3A, Table S2).[1]

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IKE untargeted lipidomic study in vivo[1]
We sought to investigate lipidomic changes caused by IKE treatment in vivo. We performed untargeted lipidomics on tumor tissue with a single dose of IKE at different time points. We identified significant (one-way ANOVA p < 0.05) increases in the relative abundance of free fatty acids, phospholipids, and diacylglycerols (DAG) upon IKE treatment (Fig. 3F and Fig. 3G). Differences with the cell culture experiment might stem from the different tumor microenvironment in vivo. The lipids identified were enriched in linoleic acid and arachidonic acid metabolism (Fig. S3A). The significant increase in the levels of DAGs and free fatty acids may result from ATGL-mediated-TAG hydrolysis (Fig. S3B). The increased fatty acids might in turn promote phospholipid remodeling to synthesize specific phospholipids, including PC and PE. To explore the free fatty acids effects on cells and ferroptosis, we performed a cell survival test of free fatty acids in the presence or absence of IKE. [1]

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IKE PEG-PLGA NPs have suitable properties to be applied in vivo[1]
IKE is soluble in acidic aqueous conditions, but not to the same degree in neutral aqueous conditions (Fig. 1A). To improve delivery of the compound, we sought to use a nanoparticle formulation. We selected biocompatible and biodegradable PEG-PLGA di-block copolymer-based nanoparticles as an IKE carrier system (Fig. 4A). The PEG block was used to create a deformable hydrating layer by tight associations with water molecules, which prevents clearance by the mononuclear phagocyte system (MPS), prolonging circulation lifetime. The PLGA block was used to form a hydrophobic core to incorporate IKE, which provides sustained release by diffusion and surface and bulk erosion.[1]

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IKE inhibits tumor growth in vivo and the PEG-PLGA NP formulation enhances its therapeutic index[1]
We investigated the efficacy of IKE in vivo in male NCG mice bearing SUDHL6 subcutaneous xenografts. Once tumor volumes reached 100 mm3, mice were randomized into five groups and treated with vehicle (5% DMSO in HBSS at pH 4), unfunctionalized PEG-PLGA NPs in water, 40 mg/kg free IKE (5% DMSO in HBSS at pH 4), 23 mg/kg free IKE (5% DMSO in HBSS at pH 4), or 23 mg/kg IKE NP (IKE PEG-PLGA NPs in water) via IP injection once daily. During the experimental period, mouse weight and tumor volume were measured daily to determine IKE’s antitumor effect and possible toxicity. Tumor growth was calculated as the fold change to original tumor volume on day 0 before the first dose (Fig. 4C). Administration of 40 mg/kg IKE, 23 mg/kg IKE, and 23 mg/kg IKE NPs caused a significant decrease in tumor growth starting from day 9 of treatment. The tumor growth inhibition effect was not significantly different between 23 mg/kg free IKE and 23 mg/kg IKE NP; however, IKE NPs showed less toxicity, as evidenced by weight loss (Fig. 4D). Compared to saline vehicle, free IKE-(5% DMSO in HBSS at pH 4)-treated mice started losing weight from day 9, which might be caused by the precipitation of IKE after administrated into the peritoneum, an environment with pH ranges of 7.5–8.0, causing damage to abdominal organs, or possible toxicity of systemic system xc− inhibition, or off-target toxicity of IKE. However, IKE-NP-treated mice had a similar weight as the saline vehicle and the NP vehicle groups; the lower toxicity of the IKE NP formulation might result from the NP’s capability to prevent the aggregation of hydrophobic drugs(Sun et al., 2014), or the NP EPR effect, which decreases the non-specific distribution and systemic toxicity associated with conventional hydrophobic drugs(Yue et al., 2013). By analyzing IKE tumor accumulation using LC-MS, we found that IKE NPs at 23 mg/kg had slightly enhanced tumor accumulation compared with free IKE at 23 mg/kg and were comparable to the free IKE 40 mg/kg treatment (Fig. S5A). Overall, the PEG-PLGA NP formulation increased IKE’s therapeutic window.[1]

Glutamate-­‐release assay. [2]
Human astrocytoma cells (CCF-­‐STTG1) were used as the source of the cystine-­‐glutamate antiporter (xc-­‐). Cells were grown in 96-­‐well plates. At >95% confluence, medium was removed and cells washed with Earle’s Balanced Salt Solution (EBSS) to remove the glutamate contained in the media. The cells were then incubated for 2 h at 37°C with either EBSS (Blanks) or EBSS containing cystine 80 μM (Totals) ± erastin (30 nM to 100 μM). Known inhibitors of the target, sulfasalazine (SAS) and (S)-­‐4-­‐carboxyphenylglycine (S-­‐4CPG), were used as positive controls in the assay. Following the incubation period, the glutamate released into the medium was detected 4 fluorometrically. Tris buffer (100 mM, pH 7.4) containing glutamate oxidase (0.04 U/mL), horseradish peroxidase (0.125 U/mL) and Amplex UltraRed (50 μM) were added to the plate and the rate of change of fluorescence followed (ex 530, em 590). The data were normalized to the Totals and Blanks ((1-­‐(Unknown-­‐Blanks)/(Totals-­‐Blanks))*100) and the half maximal inhibitory constant (IC50) of SAS, S-­‐4CPG, erastin, erastin metabolites and erastin analogs were determined as a function of the normalized fluorescence intensity values[2].

DLBCL Lines Sensitivity Measurement[1]
DLBCL cells were plated at 10,000 cells per well in white 384-well plates (32 μL per well) in technical duplicates and incubated overnight. The cells were then treated with 8 μL medium containing a two-fold dilution series of vehicle (DMSO), IKE (starting from 100 μM) with or without Fer-1 (starting from 200 μM). After 24 h incubation, 40 μL of 50% CellTiter-Glo 50% cell culture medium was added to each well and incubated at room temperature with shaking for 15 min. Luminescence was measured using a Victor X5 plate reader. [1]

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Flow Cytometry Assay [1]
0.20 million SUDHL-6 cells were seeded in six-well plates and treated with DMSO, IKE , or fer-1 at specific concentration. The final cell density was 0.05 million cells/mL. After 24 h, cells were harvested by centrifuging at 300 × g for 5 min. Cells were resuspended in 500 μL HBSS containing 2 μM C11-BODIPY (BODIPY 581/591 C11) and incubated at 37 °C for 15 min. Cells were pelleted and resuspended in HBSS. Fluorescence intensity was measured on the FL1 channel with gating to record live cells only (gate constructed from DMSO treatment group). A minimum of 10,000 cells were analyzed per condition.

Pharmacokinetic analysis in mice with three different administration routes[1]
NOD/SCID mice (12-weeks of age and ~28 g weight) were weighed before injection and divided into groups of 3 mice per cage. IKE was dissolved in 5% DMSO/95% Hank’s Balanced Salt Solution (HBSS), pH 4, to create a 5 mg/mL solution. 5% DMSO/95% HBSS at pH 4 solution (Vehicle 1) without IKE was used as vehicle. The solution was sterilized using a 0.22 μm Steriflip filter unit. Mice were dosed using three different routes, IP and PO with 50 mg/kg IKE, and IV with 17 mg/kg IKE.Samples were collected at 0, 1, 3, 4, and 8 h from three mice per time point. Additionally, three mice per group were used as controls by administration with equivalent amount of vehicle 1 by IP, PO, and IV, and samples were collected at 8 h. At the appropriate time, mice were sacrificed by CO2 asphyxiation for 3 min and ~0.5 mL of blood was collected via cardiac puncture. Blood was immediately put into K3 EDTA micro tube (SARSTEDT 41.1504.105) and placed on ice. Samples were centrifuged for 10 min at 2,100 × g at 4°C, then plasma was transferred to a clean tube. Plasma samples were flash frozen in liquid nitrogen and stored at −80°C. IKE was extracted from plasma by adding 900 μL acetonitrile to 100 μL plasma. Samples were mixed for at least 5 min by rotating at room temperature and were sonicated prior to concentration for 10 min at 4,000 × g and 4°C. The supernatant was removed and dried on a GeneVac evaporator overnight on an HPLC setting. After drying, the samples were re-suspended in 100 μL of methanol and analyzed on the liquid chromatography mass spectrometry (LC-MS), with each sample analyzed twice. Quality control standard samples were prepared by dissolving IKE in 100 μL water and extraction with the same procedures to ensure that the extraction was efficient.[1]

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Pharmacokinetic and pharmacodynamic analysis in NCG mice bearing SUDHL6 xenografts [1]
IKE was dissolved in 5% DMSO/95% HBSS at pH 4 to create a 5 mg/mL solution or 3 mg/mL solution. 5% DMSO/95% HBSS at pH 4 was used as vehicle 1. IKE PEG-PLGA nanoparticles and unfunctionalized PEG-PLGA nanoparticles (without IKE) (vehicle 2) prepared with a NanoAssemblr were dialyzed with deionized water overnight, and the water was changed at least twice. Dialyzed IKE-PEG-PLGA nanoparticles and unfunctionalized PEG-PLGA nanoparticles were concentrated by Amicon Ultra-15 Centrifugal Filter Units to create a solution with 80 mg/mL PEG-PLGA nanoparticles. [1]

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IKE efficacy study[1]
IKE was dissolved in 5% DMSO/95% HBSS at pH 4 to create a 4 mg/mL solution. 5% DMSO/95% HBSS at pH 4 was used as vehicle 1. IKE PEG-PLGA nanoparticles and unfunctionalized PEGPLGA nanoparticles (without IKE loading) (vehicle 2) prepared with a NanoAssemblr were dialyzed with deionized water overnight; the water was changed at least twice. Dialyzed IKE-PEG-PLGA nanoparticles and unfunctionalized PEG-PLGA nanoparticles were concentrated by Amicon Ultra-15 Centrifugal Filter Units to create a solution with 80 mg/mL PEG-PLGA nanoparticles.

[1]. Imidazole Ketone Erastin Induces Ferroptosis and Slows Tumor Growth in a Mouse Lymphoma Model. Cell Chem Biol. 2019 Jan 31. pii: S2451-9456(19)30030-3.

erroptosis is a form of regulated cell death that can be induced by inhibition of the cystine-glutamate antiporter, system xc-. Among the existing system xc- inhibitors, imidazole ketone erastin (IKE) is a potent, metabolically stable inhibitor of system xc- and inducer of ferroptosis potentially suitable for in vivo applications. We investigated the pharmacokinetic and pharmacodynamic features of IKE in a diffuse large B cell lymphoma (DLBCL) xenograft model and demonstrated that IKE exerted an antitumor effect by inhibiting system xc-, leading to glutathione depletion, lipid peroxidation, and the induction of ferroptosis biomarkers both in vitro and in vivo. Using untargeted lipidomics and qPCR, we identified distinct features of lipid metabolism in IKE-induced ferroptosis. In addition, biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticles were employed to aid in IKE delivery and exhibited reduced toxicity compared with free IKE in a DLBCL xenograft model.[1]

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Introducing a reactive carbonyl to a scaffold that does not otherwise have an electrophilic functionality to create a reversible covalent inhibitor is a potentially useful strategy for enhancing compound potency. However, aldehydes are metabolically unstable, which precludes the use of this strategy for compounds to be tested in animal models or in human clinical studies. To overcome this limitation, we designed ketone-based functionalities capable of forming reversible covalent adducts, while displaying high metabolic stability, and imparting improved water solubility to their pendant scaffold. We tested this strategy on the ferroptosis inducer and experimental therapeutic erastin, and observed substantial increases in compound potency. In particular, a new carbonyl erastin analog, termed IKE, displayed improved potency, solubility and metabolic stability, thus representing an ideal candidate for future in vivo cancer therapeutic applications.[2]

C35H35CLN6O5

655.1426

654.235

C, 64.17; H, 5.39; Cl, 5.41; N, 12.83; O, 12.21

1801530-11-9

91824786

White to yellow typically exists as solids

4.5

0

8

11

47

1120

0

PSPXJPWGVFNGQI-UHFFFAOYSA-N

InChI=1S/C35H35ClN6O5/c1-24(2)47-32-12-7-25(31(43)20-40-14-13-37-23-40)19-30(32)42-33(38-29-6-4-3-5-28(29)35(42)45)21-39-15-17-41(18-16-39)34(44)22-46-27-10-8-26(36)9-11-27/h3-14,19,23-24H,15-18,20-22H2,1-2H3

3-(5-(2-(1H-imidazol-1-yl)acetyl)-2-isopropoxyphenyl)-2-((4-(2-(4-chlorophenoxy)acetyl)piperazin-1-yl)methyl)quinazolin-4(3H)-one

Imidazole ketone erastin; IKE; Imidazole ketone erastin; 1801530-11-9; IKE; PUN30119; PUN-301193-(5-(2-(1H-imidazol-1-yl)acetyl)-2-isopropoxyphenyl)-2-((4-(2-(4-chlorophenoxy)acetyl)piperazin-1-yl)methyl)quinazolin-4(3H)-one; CHEMBL3629671; 2-({4-[2-(4-chlorophenoxy)acetyl]piperazin-1-yl}methyl)-3-{5-[2-(imidazol-1-yl)acetyl]-2-isopropoxyphenyl}quinazolin-4-one; Imidazole ketone erastinIKE; Ferroptosis inducer IKE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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