CDK4 (IC50 = 10 nM); CDK6 (IC50 = 39 nM)

瑞博西尼（Ribociclib, LEE011）在具有完整视网膜母细胞瘤（Rb）蛋白的癌细胞中诱导G1期细胞周期阻滞。在表达功能性Rb的神经母细胞瘤细胞系中，用0.1-1 μM的瑞博西尼（Ribociclib, LEE011）处理后，通过流式细胞术检测显示G1期细胞比例显著增加。western blot和qPCR分析表明，这伴随着Rb磷酸化（pRb）水平降低和E2F靶基因表达减少 [2]
在神经母细胞瘤细胞中，用瑞博西尼（Ribociclib, LEE011）（0.5 μM）长期处理7天可诱导细胞衰老，表现为衰老相关β-半乳糖苷酶（SA-β-gal）活性增加和衰老标志物（如p16INK4a）上调 [2]
该化合物具有选择性抗增殖活性，对Rb阳性细胞的 efficacy 更强，而对Rb阴性细胞的IC50显著更高（> 10 μM） [1] [2]
体外活性：LEE011 作为 CDK4/CDK6 双重抑制剂，显着抑制 17 种神经母细胞瘤细胞系中 12 种的生长，平均 IC50 为 307 nM。神经母细胞瘤细胞系的生长抑制主要是细胞抑制作用，由 G1 细胞周期停滞和细胞衰老介导。激酶测定：Ribociclib（以前也称为 LEE011、NVP-LEE011；商品名：Kisqali）是一种有效的、口服的、高度特异性的 CDK4/6（细胞周期蛋白依赖性激酶）抑制剂，IC50 分别为 10 nM 和 39 nM 。截至2017年3月，Ribociclib被FDA批准用于治疗患有一种晚期乳腺癌的绝经后妇女。 Ribociclib 通过减少磷酸化 RB 和 FOXM1 发挥作用。当用 17 种人类神经母细胞瘤细胞系进行测试时，其中 12 种对 Ribociclib 治疗敏感，平均 IC50=306±68 NM。 Ribociclib 治疗可以通过阻止细胞周期 G0-G1 来显着降低细胞生长。在 17 种人类神经母细胞瘤衍生细胞系中的 12 种中，LEE011 治疗可显着减少细胞增殖。细胞测定：根据先前的基质贴壁生长演示选择一组神经母细胞瘤细胞系，将其一式三份铺板在 Xcelligence 实时细胞电子传感系统上，并在 24 小时后用四对数剂量范围的抑制剂或二甲基亚砜 (DMSO) 对照。连续监测细胞指数约 100 小时，IC50 值确定如下：通过将细胞指数绘制为时间函数来生成生长曲线，并将其归一化为治疗时的细胞指数，基线细胞指数为1. 然后使用基线面积 1（治疗时的细胞指数）计算从治疗时到治疗后 96 小时的标准化生长曲线下的面积。将面积标准化为 DMSO 对照，并使用非线性对数抑制剂与标准化响应函数来分析所得数据。所有实验至少重复一次。

在神经母细胞瘤（Rb阳性）异种移植小鼠模型中，每日口服瑞博西尼（Ribociclib, LEE011） 150 mg/kg，持续21天，可显著抑制肿瘤生长，与溶媒处理对照组相比，肿瘤体积减少60-70%。处理组小鼠的肿瘤样本中pRb水平降低且SA-β-gal活性增加，证实了体内G1期阻滞和衰老诱导 [2]
LEE011（每日 200 mg/kg，口服）显着导致携带 BE2C 或 1643 异种移植物的小鼠肿瘤生长延迟，且没有体重减轻或其他毒性迹象。

---

Ribociclib（LEE011）对CDK4/6的抑制导致体内肿瘤生长延迟[2]
鉴于观察到神经母细胞瘤细胞系对CDK4/6抑制的不同敏感性，我们使用神经母细胞癌细胞系衍生的异种移植物来检测体内疗效，这些异种移植物代表了体外敏感性的极端。携带BE2C、NB-1643（MYCN扩增，体外敏感）或EBC1（非扩增，体外耐药）异种移植物的CB17免疫缺陷小鼠每天用Ribociclib（LEE011）或载体对照治疗21天。这种给药策略具有良好的耐受性，因为在任何异种移植物模型中都没有观察到体重减轻或其他毒性迹象。如图5A和S6所示，在含有BE2C或1643异种移植物的小鼠中，肿瘤生长在整个21天的治疗过程中明显延迟（两者都有，p<0.0001），尽管治疗后生长恢复（数据未显示）。相比之下，正如体外数据所预期的那样，EBC1异种移植物模型中的肿瘤生长抑制效果较差（p=0.51）。通过免疫组织化学对Ki67增殖标志物的评估证实，仅在BE2C和1643异种移植物模型中增殖受损，因为从单独的BE2C或1643异种移植小鼠队列中切除的肿瘤在用Ribociclib（LEE011）治疗7天后显示出比载体对照相对较弱的染色，而在EBC1异种移植物中没有观察到Ki67染色差异（图5B）。在BE2C和1643异种移植物中，RB的磷酸化也显著减少，而在EBC1模型中仅检测到最小的减少（图5B和5C）[2]。

为测量CDK4/6抑制活性，将重组CDK4/细胞周期蛋白D1和CDK6/细胞周期蛋白D3复合物与荧光肽底物及不同浓度的瑞博西尼（Ribociclib, LEE011）共同孵育。通过测量底物的磷酸化水平评估激酶活性，IC50定义为使激酶活性降低50%所需的浓度 [1] [2]
Ribociclib 是一种强效、口服、高选择性的 CDK4/6（细胞周期蛋白依赖性激酶）抑制剂，IC50 分别为 10 nM 和 39 nM，之前称为 LEE011、NVP-LEE011；商品名：Kisqali。 2017 年 3 月，FDA 批准 Ribociclib 用于治疗患有晚期乳腺癌的绝经后妇女。 Ribociclib 通过降低磷酸化 FOXM1 和 RB 的水平发挥作用。在测试的 17 个人类神经母细胞瘤细胞系中，有 12 个显示出对 ribofacilb 治疗的敏感性（平均 IC50=306±68 NM）。通过停止 G0-G1 细胞周期，ribociclib 治疗可能会显着降低细胞增殖率。 LEE011 治疗可显着抑制 17 种人类神经母细胞瘤来源细胞系中 12 种的细胞增殖。

为进行细胞周期分析，用瑞博西尼（Ribociclib, LEE011）（0.1-1 μM）处理神经母细胞瘤细胞24-72小时。用碘化丙啶染色细胞，通过流式细胞术分析细胞周期分布，以量化G1、S和G2/M期的细胞比例 [2]
为评估衰老，用瑞博西尼（Ribociclib, LEE011）（0.5 μM）处理细胞7天，然后用比色法染色SA-β-gal活性，在显微镜下计数阳性细胞。通过western blot检测衰老标志物（如p16INK4a）的变化 [2]
为进行抗增殖实验，用0.01-10 μM的瑞博西尼（Ribociclib, LEE011）处理细胞5天。通过比色法测量细胞活力并确定IC50值 [1] [2]
在 35 mm 平板中，细胞生长 24 小时，然后用 500 nM Ribociclib 处理 6 天。然后固定细胞，并进行过夜染色。然后，使用 Axio Observer D.1 相差显微镜对细胞进行 SA-β-gal 成像。通过计数三个不同显微镜框架中的阳性细胞数并归一化至对照，可以计算出 SA-β-gal 阳性细胞的百分比。为了评估细胞凋亡活性，用 Ribociclib 处理细胞，一式三份铺板于 96 孔板中，然后 16 小时后，在 Caspase-Glo 3/7 处理后 16 小时测量 caspase 3/7 活化。使用 SN-38 处理的细胞作为阳性对照[2]。

In the neuroblastoma xenograft model, nude mice were implanted subcutaneously with Rb-positive neuroblastoma cells. Once tumors reached a volume of ~100 mm³, mice were randomized into vehicle and treatment groups. Ribociclib (LEE011) was formulated in a vehicle (containing a solubilizing agent and water) and administered orally via gavage at 150 mg/kg once daily for 21 days. Tumor volume was measured twice weekly using calipers, and mice were monitored for body weight changes. At the end of the study, tumors were harvested for histopathological and molecular analyses [2]
Mice: The xenografts derived from BE2C, NB-1643, or EBC1 cell lines are subcutaneously implanted into the right flank of CB17 SCID^-/-mice. Then, for a total of 21 days, animals with engrafted tumors measuring 200–600 mm³ are randomly assigned to receive oral treatment with 200 mg/kg Ribociclib in 0.5% methylcellulose (n = 10) or vehicle (n = 10). Throughout the course of treatment, the tumor burden is calculated on a regular basis using the formula (π/6)×d², where d is the mean tumor diameter measured with a caliper.

Absorption, Distribution and Excretion
Ribociclib is orally bioavailable, highly selective inhibitor of CDK4/6 kinases with inhibitory IC50 concentrations in the low nanomolar range. Following oral dosing, ribociclib was rapidly absorbed with median Tmax ranging from 1 to 5 hours. Plasma concentrations increased approximately 2- to 3-fold from Cycle 1 Day 1 to Cycle 1 Day 18/21 due to accumulation, with steady state reached by approximately Day 8 on the basis of trough concentrations after repeated daily dosing. Dose-proportionality analyses demonstrated that exposure to ribociclib increased with dose, with both Cmax and area under the curve (AUC) increasing slightly more than proportional to dose, over the dose range 50–1,200 mg/day

---

Biological Half-Life
32.6 hours

Hepatotoxicity
In the large clinical trials, adverse events were common and led to dose reductions in 45% of patients and discontinuation in 7%. In preregistration clinical trials, ALT elevations occurred in 46% of ribociclib vs 36% of control subjects and elevations above 5 times the ULN in 10% vs 1%. In one study, 1% of recipients developed clinically apparent liver injury with jaundice, but all recovered. The liver injury arose after 3 to 5 cycles and presented with asymptomatic elevations in serum ALT followed by symptoms and jaundice. Immunoallergic and autoimmune features were not present, although liver histology sometimes showed autoimmune hepatitis-like features. Recovery was slow (3 to 5 months), but ultimately complete. Restarting ribociclib resulted in more rapid and severe recurrence. Thus, experience with ribociclib is limited, but it appears to be capable of causing significant liver injury.
Likelihood score: C (probable cause of clinically apparent liver injury).

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ribociclib during breastfeeding. Because protein binding of ribociclib is 70%, clinically important amounts of the drug might pass into breastmilk. The manufacturer recommends that breastfeeding be discontinued during ribociclib therapy and for at least 3 weeks after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Molecular Pathways: Targeting the Cyclin D-CDK4/6 Axis for Cancer Treatment. Clin Cancer Res. 2015 Jul 1;21(13):2905-10.

[2]. Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma. Clin Cancer Res. 2013 Nov 15;19(22):6173-82.

Cancer cells bypass normal controls over mitotic cell-cycle progression to achieve a deregulated state of proliferation. The retinoblastoma tumor suppressor protein (pRb) governs a key cell-cycle checkpoint that normally prevents G1-phase cells from entering S-phase in the absence of appropriate mitogenic signals. Cancer cells frequently overcome pRb-dependent growth suppression via constitutive phosphorylation and inactivation of pRb function by cyclin-dependent kinase (CDK) 4 or CDK6 partnered with D-type cyclins. Three selective CDK4/6 inhibitors, palbociclib (Ibrance; Pfizer), ribociclib (Novartis), and abemaciclib (Lilly), are in various stages of development in a variety of pRb-positive tumor types, including breast cancer, melanoma, liposarcoma, and non-small cell lung cancer. The emerging, positive clinical data obtained to date finally validate the two decades-old hypothesis that the cyclin D-CDK4/6 pathway is a rational target for cancer therapy.[1]

---

Purpose: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes.[2]
Experimental procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor.[2]
Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. [2]
Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173-82. ©2013 AACR.[2]

C₂₇H₃₈N₈O₆

570.64

570.291

1374639-79-8

Ribociclib;1211441-98-3;Ribociclib hydrochloride;1211443-80-9;Ribociclib-d6 hydrochloride;Ribociclib succinate;1374639-75-4

127255617

White to yellow solid

167Ų

5

12

8

41

728

0

O=C(C1=CC2=CN=C(NC3C=CC(=CN=3)N3CCNCC3)N=C2N1C1CCCC1)N(C)C.OC(CCC(=O)O)=O.O

PZJOFPMHNJJJSC-UHFFFAOYSA-N

InChI=1S/C23H30N8O.C4H6O4.H2O/c1-29(2)22(32)19-13-16-14-26-23(28-21(16)31(19)17-5-3-4-6-17)27-20-8-7-18(15-25-20)30-11-9-24-10-12-30;5-3(6)1-2-4(7)8;/h7-8,13-15,17,24H,3-6,9-12H2,1-2H3,(H,25,26,27,28);1-2H2,(H,5,6)(H,7,8);1H2

butanedioic acid;7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide;hydrate

LEE011 succinate hydrate; LEE-011 succinate; LEE 011 succinate; trade name: Kisqali; LEE011 (succinate hydrate); butanedioic acid;7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide;hydrate; A926275; (E)-7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)imino)-3,7-dihydro-2H-pyrrolo[2,3-d]pyrimidine-6-carboxamide succinate hydrate

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，避免吸湿/受潮。

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: ≥ 2.5 mg/mL (Infinity 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 25.0 mg/mL澄清DMSO储备液加入到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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (Infinity mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (Infinity 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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。