Imidazole ketone erastin

Ferroptosis; system Xc-
System xc- (cystine-glutamate antiporter) inhibitor (induces ferroptosis via cysteine deprivation and subsequent glutathione depletion) [1]

Imidazole ketone erastin（IKE）能有效减少DLBCL细胞数量[1]
18个DLBCL细胞系对IKE抑制表现出不同的敏感性，IC50<100 nM的细胞系被归类为敏感细胞系，IC50>10μM的细胞系归类为耐药细胞系，而IC50值在100 nM至10μM之间的细胞系则被归类为中度耐药细胞系（图1B）。我们进一步测试了与铁下垂抑制剂fer-1共同治疗后，IKE诱导的致死程度，fer-1是一种自由基捕获抗氧化剂，可抑制铁下垂过程中的致命脂质过氧化（Skouta等人，2014，Zilka等人，2017）。与fer-1的联合治疗挽救了DLBCL细胞系中由IKE诱导的细胞死亡，表明IKE在这些细胞系中诱导的致死性是由脂质过氧化和铁中毒引起的。[1]
先前的研究发现，IKE抑制谷氨酸释放，而IKE亲本类似物erastin抑制胱氨酸摄取。因此，我们测试了细胞水平的还原型谷胱甘肽（GSH），其生物合成需要半胱氨酸，作为IKE效力的读数。荧光法显示IKE对GSH的剂量依赖性耗竭（图1C）；这种效应被10μMβ-ME的共同处理所逆转，它将胱氨酸还原为半胱氨酸，允许其通过系统A、ASC和L进入细胞，从而绕过系统xc−的抑制。在SUDHL-6细胞中，IKE对GSH耗竭的IC50为34 nM（图S1B），而柳氮磺胺吡啶对GSH耗尽的IC50在毫摩尔范围内。[1]
虽然与DFO的联合处理抑制了培养物中IKE诱导的细胞死亡（图S1E），但在IKE处理后，它仅部分消除了脂质代谢变化，这可能是由于DFO抑制了铁介导的脂质过氧化，而不是酶介导的脂质过氧化，这表明诱导细胞死亡只需要一部分脂质代谢变化。[1]

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Imidazole ketone erastin (IKE) 能有效降低18种弥漫性大B细胞淋巴瘤细胞系的细胞活性，敏感性各异。IC50值范围从<100 nM（敏感）到>10 µM（耐药）。在敏感的SUDHL-6细胞系中，IKE诱导的细胞死亡可被铁死亡抑制剂ferrostatin-1共处理所挽救，证实了铁死亡是其细胞死亡机制。[1]
在SUDHL-6细胞中，IKE处理（500 nM）导致还原型谷胱甘肽剂量依赖性耗竭，其GSH耗竭的IC50为34 nM。该效应可被β-巯基乙醇共处理逆转。[1]
IKE处理在SUDHL-6细胞中诱导了脂质过氧化，通过流式细胞术检测C11-BODIPY荧光增强证实。该增强可被ferrostatin-1共处理抑制。[1]
使用抗二氢吡啶-MDA-赖氨酸加合物抗体进行的免疫荧光染色显示，IKE处理的SUDHL-6细胞中该脂质过氧化加合物水平增加。[1]
SUDHL-6细胞的RT-qPCR分析显示，IKE处理（500 nM）显著上调了与铁死亡和系统 xc- 抑制相关的基因表达，包括 SLC7A11、PTGS2 和 CHAC1。PTGS2 的上调可被ferrostatin-1共处理抑制，而 CHAC1 的上调则不受影响。β-巯基乙醇共处理可阻止所有三个基因的上调。[1]
对IKE处理的SUDHL-6细胞进行的非靶向脂质组学分析显示，62种脂质物种发生显著改变，包括含多不饱和脂肪酸的磷脂酰胆碱、磷脂酰乙醇胺和三酰甘油的减少。这些变化可被β-巯基乙醇共处理逆转。Ferrostatin-1共处理增加了TAG水平并降低了单酰甘油水平。[1]
IKE处理上调了参与脂质从头合成、磷脂重塑和酶介导的脂质过氧化的基因的mRNA水平。这种上调可被ferrostatin-1或β-巯基乙醇共处理部分逆转。[1]

IKE体内药代动力学（PK）和药效学（PD）[1]
为了确定IKE在体内研究中的适用性，我们首先通过在NOD/SCID小鼠中使用腹膜内（IP）、静脉内（IV）和口服（PO）途径给药单剂量IKE（50mg/kg，5%DMSO，pH 4的HBSS）来评估多种给药途径。在8小时内测定IKE浓度表明IP是IKE给药的最有效和最实用的方法（表S1）。接下来，在携带SUDHL6异种移植物的NCG小鼠中，在24小时内单剂量服用IKE（50mg/kg，5%DMSO在pH 4的HBSS中，IP）后，测定血浆和肿瘤样本中的IKE浓度。IKE在1.35小时达到最高血浆浓度5.2μg/mL，在3.30小时达到最高肿瘤积聚2.5μg/mL（图3A，表S2）。[1]

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IKE体内非靶向脂质组学研究[1]
我们试图研究体内IKE治疗引起的脂质代谢变化。我们在不同时间点用单剂量IKE对肿瘤组织进行了非靶向脂质组学研究。我们发现IKE治疗后游离脂肪酸、磷脂和二酰基甘油（DAG）的相对丰度显著增加（单因素方差分析p<0.05）（图3F和图3G）。与细胞培养实验的差异可能源于体内不同的肿瘤微环境。鉴定的脂质富含亚油酸和花生四烯酸代谢（图S3A）。DAGs和游离脂肪酸水平的显著增加可能是由ATGL介导的TAG水解引起的（图S3B）。增加的脂肪酸可能反过来促进磷脂重塑以合成特定的磷脂，包括PC和PE。为了探索游离脂肪酸对细胞和铁下垂的影响，我们在有或没有IKE的情况下进行了游离脂肪酸的细胞存活测试。[1]

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IKE PEG-PLGA NP具有适合在体内应用的特性[1]
IKE在酸性水条件下可溶，但在中性水条件下溶解度不同（图1A）。为了改善化合物的递送，我们试图使用纳米粒子制剂。我们选择了基于生物相容性和可生物降解的PEG-PLGA二嵌段共聚物的纳米粒子作为IKE载体系统（图4A）。PEG块用于通过与水分子的紧密结合来创建可变形的水合层，这可以防止单核吞噬细胞系统（MPS）的清除，从而延长循环寿命。PLGA块用于形成疏水性核心，以结合IKE，IKE通过扩散和表面及本体侵蚀提供持续释放。[1]

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IKE抑制体内肿瘤生长，PEG-PLGA-NP制剂提高其治疗指数[1]
我们研究了IKE在携带SUDHL6皮下异种移植物的雄性NCG小鼠体内的疗效。一旦肿瘤体积达到100 mm3，将小鼠随机分为五组，每天通过IP注射一次，分别用载体（pH 4的HBSS中5%的DMSO）、水中未官能化的PEG-PLGA NP、40 mg/kg游离IKE（pH 4时HBSS中5%DMSO）、23 mg/kg游离IKE。在实验期间，每天测量小鼠体重和肿瘤体积，以确定IKE的抗肿瘤作用和可能的毒性。肿瘤生长计算为第一次给药前第0天原始肿瘤体积的倍数变化（图4C）。从治疗的第9天开始，施用40mg/kg IKE、23mg/kg IKE和23mg/kg IKE NP导致肿瘤生长显著减少。23mg/kg游离IKE和23mg/kg IKE NP的肿瘤生长抑制作用没有显著差异；然而，如重量减轻所示，IKE NP的毒性较小（图4D）。与生理盐水载体相比，游离IKE-（pH 4的HBSS中5%的DMSO）处理的小鼠从第9天开始减肥，这可能是由于在pH范围为7.5-8.0的腹膜环境中给药后IKE沉淀造成的，对腹部器官造成损伤，或可能对全身系统xc-抑制产生毒性，或IKE的脱靶毒性。然而，经IKE NP处理的小鼠体重与生理盐水载体组和NP载体组相似；IKE NP制剂的较低毒性可能是由于NP能够防止疏水性药物的聚集（Sun等人，2014），或NP EPR效应，这降低了与常规疏水性药物相关的非特异性分布和全身毒性（Yue等人，2013）。通过使用LC-MS分析IKE肿瘤积聚，我们发现与23mg/kg的游离IKE相比，23mg/kg的IKE NP略微增强了肿瘤积聚，与40mg/kg的游离IKE治疗相当（图S5A）。总体而言，PEG-PLGA-NP制剂增加了IKE的治疗窗口。

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在NCG小鼠的SUDHL-6皮下移植瘤模型中，每日一次腹腔注射Imidazole ketone erastin (IKE)（23 mg/kg 或 40 mg/kg）与溶媒对照组相比，在14天的治疗期内显著抑制了肿瘤生长。[1]
在移植瘤模型中，单次给药后，IKE处理从4小时开始导致肿瘤组织中GSH显著耗竭。[1]
在肿瘤组织中，IKE处理上调了 PTGS2、SLC7A11 和 CHAC1 mRNA的表达，从给药后3小时开始。[1]
肿瘤组织的免疫荧光分析显示，与溶媒对照组相比，IKE处理组中二氢吡啶-MDA-赖氨酸加合物和8-羟基-2'-脱氧鸟苷的水平增加，表明体内诱导了脂质过氧化和氧化应激。[1]
对来自IKE处理小鼠的肿瘤组织进行的非靶向脂质组学显示，游离脂肪酸、磷脂和二酰甘油的相对丰度显著增加。[1]
将IKE配制在可生物降解的聚乙二醇-聚（乳酸-共-乙醇酸）纳米粒中，所得制剂（IKE NP，含23 mg/kg IKE）抑制肿瘤生长的效果与游离IKE（23 mg/kg）相似，但毒性显著降低（小鼠体重减轻减少）。与同等剂量的游离IKE相比，IKE NP显示出轻微的肿瘤蓄积增强。[1]
疗效研究中的免疫荧光分析证实，在游离IKE或IKE NP处理的小鼠肿瘤中，COX-2蛋白、二氢吡啶-MDA-赖氨酸加合物和8-OH-dG的水平增加，而cleaved caspase-3未增加，表明细胞死亡是铁死亡性而非凋亡性。[1]

Glutamate-­‐release assay/谷氨酸释放测定。[2]
人星形细胞瘤细胞（CCF-STTG1）被用作胱氨酸-谷氨酸逆向转运蛋白（xc-）的来源。细胞在96孔板中生长。在>95%融合时，取出培养基，用Earle平衡盐溶液（EBSS）洗涤细胞，以去除培养基中含有的谷氨酸。然后将细胞在37°C下与EBSS（空白）或含有胱氨酸80μM（总计）±erastin（30 nM至100μM）的EBSS一起孵育2小时。已知的靶标抑制剂柳氮磺胺吡啶（SAS）和（S）-4-羧基苯甘氨酸（S-4CPG）用作试验中的阳性对照。在培养期后，用荧光法检测释放到培养基中的谷氨酸。将含有谷氨酸氧化酶（0.04 U/mL）、辣根过氧化物酶（0.125 U/mL）和Amplex UltraRed（50μM）的Tris缓冲液（100 mM，pH 7.4）加入板中，并跟踪荧光变化率（ex 530，em 590）。将数据标准化为总量和空白（（1-（未知-空白）/（总量-空白））100），并根据标准化荧光强度值确定SAS、S-4CPG、erastin、erastin代谢物和erastin类似物的半最大抑制常数（IC50）[2]。

DLBCL Lines Sensitivity Measurement [1]
将DLBCL细胞以每孔10000个细胞的速度铺在白色384孔板（每孔32μL）上，形成技术复制品，并孵育过夜。然后用8μL培养基处理细胞，该培养基含有两倍稀释的载体系列（DMSO），IKE（从100μM开始），有或没有Fer-1（从200μM开始。孵育24小时后，向每个孔中加入40μL 50%CellTiter-Glo 50%细胞培养基，在室温下摇动孵育15分钟。使用Victor X5平板读数器测量发光。[1]

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Flow Cytometry Assay [1]
将20万个SUDHL-6细胞接种在六孔板中，并用DMSO、特定浓度的IKE或fer-1处理。最终细胞密度为0.05百万个细胞/mL。24小时后，通过300×g离心5分钟收集细胞。将细胞重新悬浮在含有2μM C11-BODIPY（BODIPY 581/591 C11）的500μL HBSS中，并在37°C下孵育15分钟。将细胞沉淀并重新悬浮在HBSS中。用门控在FL1通道上测量荧光强度，仅记录活细胞（由DMSO处理组构建的门控）。每种情况下至少分析10000个细胞。

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DLBCL细胞敏感性测定： 将DLBCL细胞接种于384孔板中过夜培养。然后用Imidazole ketone erastin (IKE)的两倍稀释系列（从100 µM开始）处理细胞，可联合或不联合ferrostatin-1。孵育24小时后，使用发光法细胞活力检测试剂评估细胞活力。测量发光值，数据归一化至溶媒对照。计算剂量反应曲线和IC50值。[1]
脂质ROS流式细胞术检测： 用DMSO、IKE或IKE联合ferrostatin-1处理SUDHL-6细胞24小时。收集细胞，重悬于含有脂质过氧化探针C11-BODIPY的缓冲液中并孵育。通过流式细胞术在FL1通道测量荧光强度，圈定活细胞。[1]
还原型谷胱甘肽测量： 用IKE处理细胞24小时，可联合或不联合β-巯基乙醇。收集细胞，洗涤并计数。裂解细胞，上清液去蛋白。使用商业检测试剂盒，按照制造商说明书，通过荧光法测定GSH水平。[1]
定量PCR： 用IKE、ferrostatin-1或β-巯基乙醇处理细胞指定时间。提取RNA，反转录为cDNA，使用基因特异性引物和SYBR Green预混液进行定量PCR反应。mRNA水平以 HPRT1 为内参归一化，并使用ΔΔCt法计算。[1]
细胞免疫荧光： 处理后的细胞固定、透化并封闭。与一抗孵育过夜，然后与荧光标记的二抗孵育。染色细胞核。通过共聚焦显微镜捕获图像，并量化荧光强度。[1]
IKE PEG-PLGA NP的细胞活性测定： 接种细胞，并用游离IKE或IKE负载纳米粒的稀释系列处理。24小时后，使用发光法检测细胞活力。[1]

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.

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Pharmacokinetics in NOD/SCID Mice: Imidazole ketone erastin (IKE) was dissolved in 5% DMSO in Hank's Balanced Salt Solution (HBSS) at pH 4. Mice were administered a single dose of IKE (50 mg/kg for intraperitoneal and oral routes; 17 mg/kg for intravenous route). Blood samples were collected at various time points. Plasma was separated, and IKE was extracted with acetonitrile. Samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine IKE concentration over time. [1]
Pharmacokinetics/Pharmacodynamics in Xenograft Model: NCG mice bearing subcutaneous SUDHL-6 xenografts were administered a single dose of IKE (50 mg/kg, intraperitoneal, in 5% DMSO/HBSS pH4). Mice were euthanized at various time points. Plasma and tumor samples were collected. Tumor samples were homogenized, and IKE was extracted and quantified by LC-MS. Parallel tumor samples were used for GSH measurement, qPCR, immunofluorescence, and lipidomics. [1]
Efficacy Study in Xenograft Model: NCG mice bearing SUDHL-6 xenografts (tumor volume ~100 mm³) were randomized into groups. They were treated once daily for 14 days via intraperitoneal injection with: vehicle (5% DMSO/HBSS pH4), blank PEG-PLGA nanoparticles, free IKE (23 or 40 mg/kg in 5% DMSO/HBSS pH4), or IKE-loaded PEG-PLGA nanoparticles (23 mg/kg IKE equivalent in water). Tumor dimensions and mouse body weight were measured daily. Mice were euthanized 3 hours after the final dose, and tumors were collected for analysis (IKE concentration, biomarkers, lipidomics). [1]

In NOD/SCID mice, after a single intraperitoneal injection of the imidazolidin (IKE) (50 mg/kg), the plasma concentration (Cmax) peaked at 1.35 hours (Tmax) at 5.2 µg/mL. Intraperitoneal injection is considered the most effective and convenient route of administration compared with intravenous and oral routes. [1] In NCG mice carrying SUDHL-6 xenografts, after a single intraperitoneal injection of IKE (50 mg/kg), the plasma concentration peaked at 1.35 hours (Cmax) at 5.2 µg/mL, and the tumor concentration peaked at 3.30 hours (Cmax) at 2.5 µg/mL. The plasma half-life (T1/2) was 1.83 hours, and the tumor half-life was 3.50 hours. [1] Compared with plasma, IKE encapsulated in PEG-PLGA nanoparticles accumulated more in tumor tissues, and the tumor accumulation was slightly higher than that of an equal dose of free IKE. [1]
IKE has moderate water solubility, is soluble under acidic conditions, but has low solubility under neutral pH conditions. [1]

In efficacy studies, daily intraperitoneal injection of free imidazolidin (IKE) (23 mg/kg and 40 mg/kg, dissolved in 5% DMSO/HBSS pH4 solution) resulted in a significant decrease in body weight in mice, starting around day 9 of treatment. [1]
The observed toxicity (weight loss) of free IKE was attributed to potential precipitation of the drug in the peritoneal cavity (pH approximately 7.5–8.0) and/or systemic inhibition or off-target effects. [1]
Forming IKE into PEG-PLGA nanoparticles (IKE NP, equivalent to 23 mg/kg IKE) significantly reduced this toxicity, with no significant decrease in body weight observed in mice compared to the carrier control group. This reduction in toxicity was attributed to the nanoparticles' ability to prevent drug aggregation and enhance tumor targeting through enhanced permeability and retention (EPR) effects, thereby reducing systemic exposure. [1]
Administration of blank PEG-PLGA nanoparticles (750 mg/kg daily for two weeks) did not result in detectable weight loss or observable signs of toxicity. [1]

[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 regulated form of cell death that can be induced by inhibiting the xc-transporter system of the cysteine-glutamate reverse transporter. Among existing xc-inhibitors, the imidazole ion erastin (IKE) is a potent and metabolically stable xc-inhibitor and also an inducer of Erroptosis, showing potential for in vivo application. We investigated the pharmacokinetics and pharmacodynamics of IKE using a diffuse large B-cell lymphoma (DLBCL) xenograft model. Our results showed that IKE exerts its antitumor effect by inhibiting xc-, leading to glutathione depletion, lipid peroxidation, and inducing the expression of Erroptosis biomarkers in vitro and in vivo. We used non-targeted lipidomics and qPCR to identify the unique characteristics of lipid metabolism during IKE-induced Erroptosis. Furthermore, biodegradable polyethylene glycol-polylactic acid-glycolic acid copolymer nanoparticles were used to assist IKE delivery and showed lower toxicity than free IKE in the DLBCL xenograft model. [1]

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Introducing a reactive carbonyl group into a skeleton that does not originally have an electrophilic functional group to construct a reversible covalent inhibitor is a potentially effective strategy to improve the potency of a compound. However, the metabolic instability of aldehydes makes this strategy unsuitable for animal models or human clinical studies. To overcome this limitation, we designed a ketone functional group that can form a reversible covalent adduct, while also possessing high metabolic stability and improving the water solubility of its side chain skeleton. We tested this strategy with ferroptosis inducers and the experimental therapeutic erastin and observed a significant improvement in compound potency. In particular, a novel carbonyl erastin analog called IKE exhibited higher potency, solubility and metabolic stability, making it an ideal candidate for future in vivo cancer treatment. [2]

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Imidazolone erastin (IKE) is an analog of erastin designed to include a ketone group, an isopropoxy substituent and an imidazole moiety. Compared to erastin, these modifications endow IKE with nanomolar potency, higher metabolic stability and higher water solubility under acidic conditions. [1]
IKE can induce ferroptosis, an iron-dependent cell death driven by lipid peroxidation, whose main mechanism is through inhibition of the cysteine-glutamate antitransport system xc-, leading to cysteine deficiency, glutathione depletion, and glutathione peroxidase 4 (GPX4) inactivation. [1]
This study highlights the potential of ferroptosis as a treatment strategy for cysteine-dependent cancers such as diffuse large B-cell lymphoma (DLBCL). [1]
The study used biodegradable PEG-PLGA nanoparticles as a delivery system to enhance the therapeutic index of IKE by increasing tumor accumulation and reducing systemic toxicity. [1]
The study identified pharmacodynamic biomarkers of ferroptosis, including upregulation of PTGS2, SLC7A11, and CHAC1 mRNA, as well as elevated levels of lipid peroxidation adducts (MDA-lysine) and oxidative DNA damage (8-OH-dG) in vivo. [1]
Lipidomics analysis revealed significant changes in lipid metabolism during IKE-induced ferroptosis, including a reduction in PUFA-containing phospholipids and TAGs, as well as activation of lipid biosynthesis and remodeling pathways. [1]

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 solid powder

4.5

110Ų

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。