Estrogen receptor[1]

ARV-471是一种雌激素受体（ER）α-PROTAC，是一种杂双功能分子，促进雌激素受体α与细胞内E3连接酶复合物之间的相互作用，导致雌激素受体通过蛋白酶体泛素化并随后降解。ARV-471以~2nM的半最大降解浓度（DC50）强力降解ER-阳性乳腺癌症细胞系中的ER。PROTAC介导的ER降解降低了经典调节的ER靶基因（PR、GREB1、TFF）的表达，并抑制了ER依赖性细胞系（MCF7、T47D）的细胞增殖。此外，ARV-471可降解临床相关的ESR1变体（Y537S和D538G），并抑制表达这些变体的细胞系的生长[2]。

在未成熟的大鼠子宫营养模型中，ARV-471会降解大鼠子宫ER，并且没有表现出激动剂活性。每日口服单药ARV-471（3、10和30 mpk）会导致雌二醇依赖性MCF7异种移植物的肿瘤体积显著缩小，并在研究结束时伴随肿瘤ER蛋白减少>90%。此外，当CDK4/6抑制剂在MCF7模型中与ARV-471联合使用时，观察到更明显的肿瘤生长抑制（约130%TGI），同时ER蛋白水平显著降低。在ESR1 Y537S，激素非依赖性患者来源的异种移植物模型中，10 mpk的ARV-471完全抑制了生长，也降低了突变ER蛋白水平。综上所述，ARV-471的临床前数据支持其作为同类最佳口服ER PROTAC降解剂的持续发展[2]。

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Vepdegestrant在MCF7原位异种移植物模型中实现了显著的TGI（87%-123%），优于ET药物氟维司群（31%-80%TGI）。在激素非依赖性（HI）突变ER Y537S患者源性异种移植物（PDX）癌症模型ST941/HI中，vepdegestrant实现了肿瘤消退，并且在ST941/HI/PBR帕博昔单抗耐药性模型（102%TGI）中同样有效。Vepdegestrant与CDK4/6抑制剂palbociclib、abemaciclib和ribociclib中的每一种联合使用可诱导稳健的肿瘤消退；mTOR抑制剂依维莫司；PI3K抑制剂alpelisib和inavolisib。 结论：与富司琼相比，维替格斯特在体内实现了更大的ER降解，这与TGI的改善有关，表明维替格斯特朗可能是治疗ER+/HER2-乳腺癌症患者更有效的骨干ET。

蛋白质印迹程序[3]
所有细胞系和子宫或异种移植物肿瘤组织在RIPA裂解缓冲液和Halt蛋白酶抑制剂中裂解/均质化。降解试验中的ER蛋白水平通过标准蛋白质印迹、细胞内蛋白质或在WES或JESS仪器上进行的数字蛋白质分析进行测量。完整方法见补充扩展方法。

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细胞生长抑制试验[3]
除非另有说明，否则在96孔板上以2000个细胞/孔的速度进行细胞生长抑制研究，使用3倍连续稀释的8点DRCs。在第5天，使用cell Titer Glo测量细胞存活率，并使用GraphPad分析CTG数据。活细胞成像增殖和剂量基质药物组合测定在补充扩展方法中进行了描述。

Immature rat uterotrophic assay [3]
This model was conducted as previously described using immature female rats younger than postnatal day (PND) 30. Sprague–Dawley (SD) rats at PND 18 were dosed with 30 mg/kg vepdegestrant or 10 mg/kg AZD-9496 in vehicle of PEG400/2% Tween80, by oral gavage (per os, po) once daily for 3 days (qdx3), or a single subcutaneous (sc) dose of 100 mg/kg fulvestrant in a vehicle of 10% w/v ethanol, 10% w/v benzyl alcohol, and 15% w/v benzyl benzoate, made up to 100% w/v with castor oil (EBB/castor oil). Five animals were used per arm. Animals were euthanized and tissues harvested 24-hours post-last dose or on day 4 for fulvestrant/sc arms. Uterine weights were measured, flash frozen in liquid nitrogen, and stored at −80°C. ER levels were determined by western blot.

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MCF7 orthotopic xenograft model[3]
Eight- to 10-week-old female NOD/SCID mice were surgically implanted with a 0.36 mg 90-day release 17β-estradiol pellet subcutaneously. One to 2 days later, each mouse was injected with 5 × 106/200 µL MCF7 cells (ATCC) into one mammary fat pad. Cells were prepared in a 50/50 RPMI-1640 phenol red-free media/Corning Matrigel Membrane Matrix mix at 25 × 106 cells/mL. Dosing was initiated once tumors reached an average of 200 mm3. When oral combinations were dosed, vepdegestrant was dosed first and the second agent 30 to 60 minutes later. All oral agents (vepdegestrant, palbociclib, abemaciclib, ribociclib, inavolisib, alpelisib, and everolimus) were dosed at 5 mL/kg volume once daily for 28 days (qdx28) unless otherwise stated. Fulvestrant sc was dosed at 4 mL/kg twice per week (biw) for 2 weeks plus once per week (qw) for 2 weeks (biwx2, qwx2). Vehicles for the various compounds dosed in vivo are listed in Supplementary Table S5. Tumor volumes were measured twice per week in efficacy studies and calculated using (width2 × length)/2, in which all measurements are in millimeters (mm), and the tumor volume is in mm3. Body weights were recorded twice per week. In some drug combinatorial efficacy studies, some single-day dosing holidays (small black arrows in Fig. 6D and ​andE)E) were implemented on all arms if any body weight loss approached 10%. At study termination, mice were euthanized 18 hours post-last dose, and harvested tissue was snap-frozen on dry ice. TGI was calculated as follows, with tumor volume being expressed in mm3:

[1]. Targeting estrogen receptor α for degradation with PROTACs: A promising approach to overcome endocrine resistance. Eur J Med Chem. 2020;206:112689.

[2]. Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer.

[3]. Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor-Positive Breast Cancer. Cancer Res. 2023 Oct 2;83(19):3284-3304.

Vepdegestrant is an orally available hetero-bifunctional molecule and selective estrogen receptor (ER) alpha-targeted protein degrader, using the proteolysis targeting chimera (PROTAC) technology, with potential antineoplastic activity. Vepdegestrant is composed of an ER alpha ligand attached to an E3 ligase recognition moiety. Upon oral administration,vepdegestrant targets and binds to the ER ligand binding domain on ER alpha. E3 ligase is recruited to the ER by the E3 ligase recognition moiety and ER alpha is tagged by ubiquitin. This causes ubiquitination and degradation of ER alpha by the proteasome. This decreases ER alpha protein levels, decreases the expression of ER alpha-target genes and halts ER-mediated signaling. This results in an inhibition of proliferation in ER alpha-overexpressing tumor cells. In addition, the degradation of the ER alpha protein releases the ARV-471 and can bind to additional ER alpha target proteins. ER alpha is overexpressed in a variety of cancers and plays a key role in cancer cell proliferation.

C45H49N5O4

723.9017

723.37845

C, 74.66; H, 6.82; N, 9.67; O, 8.84

2229711-68-4

2229711-08-2 (racemate)

134562533

White to off-white solid powder

6.4

96.4Ų

2

7

7

54

1310

3

O([H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C([H])([H])[C@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])[C@]2([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])N2C([H])([H])C([H])([H])N(C3C([H])=C([H])C4C(N([C@]5([H])C(N([H])C(C([H])([H])C5([H])[H])=O)=O)C([H])([H])C=4C=3[H])=O)C([H])([H])C2([H])[H])C([H])([H])C1([H])[H]

TZZDVPMABRWKIZ-XMOGEVODSA-N

InChI=1S/C45H49N5O4/c51-37-12-15-39-33(27-37)8-13-38(31-4-2-1-3-5-31)43(39)32-6-9-35(10-7-32)48-20-18-30(19-21-48)28-47-22-24-49(25-23-47)36-11-14-40-34(26-36)29-50(45(40)54)41-16-17-42(52)46-44(41)53/h1-7,9-12,14-15,26-27,30,38,41,43,51H,8,13,16-25,28-29H2,(H,46,52,53)/t38-,41+,43+/m1/s1

(3S)-3-[6-[4-[[1-[4-[(1R,2S)-6-hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl]phenyl]piperidin-4-yl]methyl]piperazin-1-yl]-3-oxo-1H-isoindol-2-yl]piperidine-2,6-dione

ARV-471; 2229711-68-4; Vepdegestrant; ARV-471 (protac); WC1U3R1YMI; ARV471; (S)-3-(5-(4-((1-(4-((1R,2S)-6-Hydroxy-2-phenyl-1,2,3,4-tetrahydronaphthalen-1-yl)phenyl)piperidin-4-yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

DMSO: 110 mg/mL (151.95 mM)

配方 1 中的溶解度: ≥ 5.5 mg/mL (7.60 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 55.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 2 中的溶解度: ≥ 2 mg/mL (2.76 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL 生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: 2 mg/mL (2.76 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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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。