RG-7112 (RO-5045337)   中文名称: [(4R,5S)-4,5-双(4-氯苯基)-2-[4-(1,1-二甲基乙基)-2-乙氧基苯基]-4,5-二氢-4,5-二甲基-1H-咪唑-1-基][4-[3-(甲磺酰基)丙基]-1-哌嗪基]甲酮

MDM2 (Kd = 11 nM)

RG7112 是 MDM2 拮抗剂 nutlin 家族的有效且选择性成员，目前正处于 I 期临床研究中。在体外，MDM2 与 p53 的相互作用被 RG7112 与 MDM2 的高度特异性结合（KD 为 10.7 nM）所阻断。 RG7112-MDM2 复合物已结晶，表明该小分子通过与 MDM2 的 p53 口袋结合来模拟关键 p53 氨基酸残基的相互作用。通过激活 p53 通路，RG7112 导致表达野生型 p53 的癌细胞发生细胞周期停滞和凋亡。一组实体瘤细胞系对 RG7112 的抗肿瘤作用敏感。然而，这种药物的凋亡活性差异很大，具有MDM2基因扩增的骨肉瘤细胞表现出最好的反应。 [1]

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RG7112是p53-MDM2结合的强效抑制剂[2]。
RG7112在癌症细胞中稳定野生型p53并诱导p53信号传导[2]。
RG7112有效激活癌症细胞中的p53功能[2]。
半胱天冬酶抑制不影响RG7112诱导的细胞死亡的开始[2]。

在体内，RG7112 导致肿瘤细胞凋亡并激活 p53 通路。在无毒剂量下，对携带人类异种移植物的小鼠口服 RG7112 会导致增殖/凋亡生物标志物以及肿瘤抑制和消退发生剂量依赖性变化。值得注意的是，雄激素剥夺和 RG7112 在 LNCaP 异种移植肿瘤中具有强大的协同作用。 [1]

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以无毒浓度对携带人类异种移植物的小鼠口服RG7112，会引起增殖/凋亡生物标志物以及肿瘤抑制和消退的剂量依赖性变化。值得注意的是，RG7112与LNCaP异种移植物肿瘤中的雄激素剥夺具有高度协同作用。我们的研究结果提供了一个临床前的概念证明，即RG7112在治疗表达野生型p53的实体瘤方面是有效的。[2]

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RG7112处理的PDCL颅内异种移植物的PK分析表明，该化合物显著穿过血脑和血肿瘤屏障。最重要的是，MDM2扩增/TP53野生型PDCL衍生模型（皮下和原位）的治疗减少了肿瘤生长，具有细胞毒性，并显著提高了生存率。 结论：这些数据有力地支持了MDM2抑制剂的开发，用于MDM2扩增的GBM患者的临床试验。此外，在非MDM2扩增模型的一个子集中具有显著疗效，这表明必须确定对MDM2抑制剂反应的其他标志物。[3]

均匀时间分辨荧光（HTRF）测定测量两种成分在接近时产生的信号。p53-MDM2结合测定使用来源于p53的MDM2结合结构域的生物素化肽和含有p53结合结构域重组人GST标记的MDM2蛋白的截短N端部分。使用编码lacIq阻遏物和稀有tRNAArg[AGA/AGG]的辅助质粒pUBS 520在大肠杆菌BL21菌株中表达用于晶体结构研究的蛋白质。为了结晶，将冷冻的蛋白质解冻，并使用Centricon浓缩器（3000 MW截止）浓缩至9.8mg/mL。然后通过将蛋白质与稍摩尔过量的抑制剂（DMSO中的储备溶液为100 mmol/L）结合形成复合物，并将该溶液在4°C下静置4小时。在布鲁克海文国家实验室的国家同步辐射光源处，使用低温保存的晶体收集光束线X8C的衍射数据[2]。

通过四唑蓝（MTT）法评估细胞增殖/存活率。使用IncuCyte活细胞成像系统测量细胞生长动力学。对于细胞周期分析，将细胞在T75烧瓶中用适当的生长培养基（10 mL中106个细胞/条件）培养，并在37°C下孵育过夜。它们与测试化合物一起孵育，并如前所述进行处理。使用GuavaNexin凋亡检测试剂盒通过Annexin V测定法测定细胞凋亡，并按照制造商的方案使用Guava个人细胞分析仪测定细胞凋亡百分比[2]。

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抗增殖试验[3]
对于队列#1细胞系的药物敏感性测定，在37°C下用10μg/mL层粘连蛋白涂覆96孔板1小时。然后将三千个细胞/孔进行电镀RG7112作为10mM DMSO储备溶液重新悬浮，并在镀覆后24小时加入。添加药物72小时后，根据制造商的说明添加WST-1试剂。WST-1盐在活细胞中通过NAD（P）H依赖性反应裂解为可溶性甲氮染料。将板温育3小时，并在450nm波长下通过分光光度法读取。对于队列#2细胞系，细胞以384孔格式铺板，并使用针转移机器人将化合物溶液转移到每个孔中，每种条件有3个重复。通过CellTiter-Glo发光测定法在连续药物暴露72小时后测量细胞存活率。使用GraphPad®Prism 6通过最小二乘曲线拟合确定IC75、IC99和IC100（分别导致细胞存活率降低75%、99%和100%的浓度）。[3]

For SJSA-1, SJSA-1luc2, and MHM xenograft studies, female Balb/c nude mice were implanted subcutaneously in the right flank with 5 × 106 cells suspended in a 0.2 mL volume of a 1:1 mixture of Matrigel:PBS. For studies with hormone-dependent LNCaP xenografts, castrated male Balb/c nude were implanted with 12.5 mg sustained-release testosterone pellets 5 days before subcutaneous inoculation with 1 × 107 cells suspended in 0.2 mL of Matrigel:PBS. Mice were randomized into treatment groups (n = 10 per group) when mean tumor volume reached approximately 150 to 400 mm3. In all studies, mice received either vehicle (1% Klucel LF/0.1% Tween 80) or RG7112, administered as an oral suspension at the dose indicated (25–200 mg/kg). For assessment of androgen ablation treatment in combination with RG7112 in LNCaP xenograft-bearing mice, testosterone pellets were removed under ketamine/xylazine anesthesia. Tumor volume was monitored by caliper measurement and body weights were recorded 2 to 3 times weekly. Tumor volume (in mm3) was calculated as described previously [2].
For Western blot analysis, mice bearing established SJSA-1 subcutaneous xenografts received a single oral dose of vehicle or 50, 100, or 200 mg/kg RG7112, and tumors were harvested at 4 and 8 hours after dosing. Protein was extracted from tumor tissue with 1× radioimmunoprecipitation assay buffer containing protease inhibitors by homogenization. Equal amounts of total protein were resolved on 4% to 12% NuPAGE gradient gel and blotted with antibody dilutions as recommended by manufacturer. The chemiluminescent signal was generated with enhanced chemiluminescence Plus and detected with Fujifilm LAS-3000 imager. The densitometric quantitation of specific bands was determined using Multi Gauge Software. The complete methods can be found in the online Supplementary Information.[2]

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For the heterotopic (subcutaneous) model, 2x106 cells were resuspended in Hank’s Buffered Salt Solution, mixed with an equal volume of Matrigel and injected into both flanks of eight-week-old NU/NU mice. Animals were randomly assigned to treatment or vehicle arm when tumors measured a volume of 200 mm3. For both orthotopic and heterotopic models, animals were treated by gavage with 100 mg/kg of RG7112formulation (100 mg/mL RG7112, 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water) or vehicle once per day, 5 days/week for 3 weeks. For the evaluation of GBM blood-brain barrier (BBB) integrity only, 1.2 mg of Hoechst 33342 diluted in PBS was injected intravenously (iv) prior to termination. Mice were terminated by asphyxiation when they showed signs of tumor-associated illness or before reaching maximum subcutaneous tumor burden.[3]
Pharmacokinetics studies[3]
GBM cells were inoculated in the brain of Athymic Nude mice as described below and animals were assigned to different pharmacokinetics time points when bioluminescence signal reached 1.108 photon/second. This threshold was selected to ensure that tumor volumes were as significant as possible without causing symptoms of pain or illness. The dose treatment solution of RG7112 (100 mg/mL RG7112) was prepared in a vehicle composed of 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water . Mice were sacrificed at 0, 1h, 2h, 4h, 8h, 24h and 48h post-gavage (3 mice per time point). Blood was collected via live cardiac puncture in polyethylene tubes using a heparinized syringe. Samples were immediately centrifuged at 5000 rpm for 15 min and plasma was removed and stored at −80°C until analysis. Whole brains were collected, rinsed with 0.9% sodium chloride. The right and left brain hemispheres were harvested separately and labeled as tumor hemisphere and counter hemisphere, respectively, and were frozen at −80°C. RG7112 levels in mice plasma, and brains were measured using validated liquid chromatography coupled with mass tandem spectrometry methods. [3]

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1% Klucel LF/0.1% Tween 80; 200 mg/kg; oral taken

SJSA-1, SJSA-1luc2, and MHM xenografted Balb/c nude mice

[1]. ACS Med Chem Lett. 2013 Apr 2;4(5):466-9.

[2]. MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models. Cancer Res. 2013 Apr 15;73(8):2587-97.
[3]. Preclinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 1;22(5):1185-96.

RO-5045337 is under investigation in clinical trial NCT01164033 (A Study of RO5045337 in Patients With Solid Tumors).

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MDM2 Antagonist RO5045337 is an MDM2 (human homolog of double minutes-2; HDM2) antagonist with potential antineoplastic activity. RO5045337 binds to MDM2, thereby preventing the binding of the MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By preventing this MDM2-p53 interaction, the proteasome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored, which may result in the restoration of p53 signaling and thus the p53-mediated induction of tumor cell apoptosis. MDM2, a zinc finger protein, is a negative regulator of the p53 pathway; often overexpressed in cancer cells, it has been implicated in cancer cell proliferation and survival.

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The p53 tumor suppressor is a potent transcription factor that plays a key role in the regulation of cellular responses to stress. It is controlled by its negative regulator MDM2, which binds directly to p53 and inhibits its transcriptional activity. MDM2 also targets p53 for degradation by the proteasome. Many tumors produce high levels of MDM2, thereby impairing p53 function. Restoration of p53 activity by inhibiting the p53-MDM2 interaction may represent a novel approach to cancer treatment. RG7112 (2g) is the first clinical small-molecule MDM2 inhibitor designed to occupy the p53-binding pocket of MDM2. In cancer cells expressing wild-type p53, RG7112 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest, apoptosis, and inhibition or regression of human tumor xenografts.[1]

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MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.[2]

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Purpose: p53 pathway alterations are key molecular events in glioblastoma (GBM). MDM2 inhibitors increase expression and stability of p53 and are presumed to be most efficacious in patients with TP53 wild-type and MDM2-amplified cancers. However, this biomarker hypothesis has not been tested in patients or patient-derived models for GBM.[3]
Experimental design: We performed a preclinical evaluation of RG7112 MDM2 inhibitor, across a panel of 36 patient-derived GBM cell lines (PDCL), each genetically characterized according to their P53 pathway status. We then performed a pharmacokinetic (PK) profiling of RG7112 distribution in mice and evaluated the therapeutic activity of RG7112 in orthotopic and subcutaneous GBM models.[3]
Results: MDM2-amplified PDCLs were 44 times more sensitive than TP53-mutated lines that showed complete resistance at therapeutically attainable concentrations (avg. IC50 of 0.52 μmol/L vs. 21.9 μmol/L). MDM4-amplified PDCLs were highly sensitive but showed intermediate response (avg. IC50 of 1.2 μmol/L), whereas response was heterogeneous in TP53 wild-type PDCLs with normal MDM2/4 levels (avg. IC50 of 7.7 μmol/L). In MDM2-amplified lines, RG7112 restored p53 activity inducing robust p21 expression and apoptosis. PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival.[3]
Conclusions: These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2-amplified models suggests that additional markers of response to MDM2 inhibitors must be identified.[3]

C38H48CL2N4O4S

727.78

726.277

C, 62.71; H, 6.65; Cl, 9.74; N, 7.70; O, 8.79; S, 4.41

939981-39-2

57406853

White to off-white solid powder

1.2±0.1 g/cm3

790.4±70.0 °C at 760 mmHg

431.8±35.7 °C

0.0±2.8 mmHg at 25°C

1.598

6.67

90.9

C[C@]1([C@@](C2C=CC(Cl)=CC=2)(C)N=C(C2C=CC(C(C)(C)C)=CC=2OCC)N1C(N1CCN(CCCS(=O)(=O)C)CC1)=O)C1C=CC(Cl)=CC=1

QBGKPEROWUKSBK-QPPIDDCLSA-N

InChI=1S/C38H48Cl2N4O4S/c1-8-48-33-26-29(36(2,3)4)14-19-32(33)34-41-37(5,27-10-15-30(39)16-11-27)38(6,28-12-17-31(40)18-13-28)44(34)35(45)43-23-21-42(22-24-43)20-9-25-49(7,46)47/h10-19,26H,8-9,20-25H2,1-7H3/t37-,38+/m0/s1

[(4S,5R)-2-(4-tert-butyl-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazol-1-yl]-[4-(3-methylsulfonylpropyl)piperazin-1-yl]methanone

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)

配方 1 中的溶解度: ≥ 10 mg/mL (13.74 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 100.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 10 mg/mL (13.74 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 100.0 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 3 中的溶解度: ≥ 5 mg/mL (6.87 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 4 中的溶解度: ≥ 2.5 mg/mL (3.44 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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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配方 5 中的溶解度: 1% CMC Na : 14mg/mL

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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。