Imidazole ketone erastin

Ferroptosis; system Xc-

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]

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。

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。

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

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；
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