CDK4 (IC50 = 10 nM); CDK6 (IC50 = 30 nM)
Applying Ribociclib (LEE011) over a four-log dose range (10 to 10,000 nM) to a panel of 17 neuroblastoma cell lines. Out of the 17 neuroblastoma cell lines that were studied, 12 showed a significant reduction in substrate adherent growth when treated with Ribociclib (mean IC50=306±68 nM, focusing only on sensitive lines; sensitivity is defined as an IC50 of less than 1 μM). Two neuroblastoma cell lines (BE2C and IMR5) that have been shown to be sensitive to CDK4/6 inhibition respond to ribofloxacil treatment by accumulating cells in the G₀/G₁ phase of the cell cycle in a dose-dependent manner. At 100 nM (p=0.007) and 250 nM (p=0.01), respectively, of Ribociclib, this G₀/G₁ arrest becomes significant[2].

---

Ribociclib (LEE011) induces G1 cell-cycle arrest in cancer cells with intact retinoblastoma (Rb) protein. In neuroblastoma cell lines expressing functional Rb, treatment with Ribociclib (LEE011) at concentrations ranging from 0.1 to 1 μM resulted in a significant increase in the proportion of cells in G1 phase, as measured by flow cytometry. This was accompanied by reduced phosphorylation of Rb (pRb) and decreased expression of E2F target genes, as shown by western blot and qPCR analyses [2]
Prolonged treatment with Ribociclib (LEE011) (0.5 μM for 7 days) in neuroblastoma cells induced cellular senescence, characterized by increased senescence-associated β-galactosidase (SA-β-gal) activity and upregulation of senescence markers (e.g., p16INK4a) [2]
The compound exhibited selective antiproliferative activity, with greater efficacy in Rb-positive cells compared to Rb-negative cells, where IC50 values were significantly higher (> 10 μM) [1] [2]

Applying Ribociclib (LEE011) over a four-log dose range (10 to 10,000 nM) to a panel of 17 neuroblastoma cell lines. Out of the 17 neuroblastoma cell lines that were studied, 12 showed a significant reduction in substrate adherent growth when treated with Ribociclib (mean IC50=306±68 nM, focusing only on sensitive lines; sensitivity is defined as an IC50 of less than 1 μM). Two neuroblastoma cell lines (BE2C and IMR5) that have been shown to be sensitive to CDK4/6 inhibition respond to ribofloxacil treatment by accumulating cells in the G₀/G₁ phase of the cell cycle in a dose-dependent manner. At 100 nM (p=0.007) and 250 nM (p=0.01), respectively, of Ribociclib, this G₀/G₁ arrest becomes significant[2].

---

Ribociclib (LEE011) induces G1 cell-cycle arrest in cancer cells with intact retinoblastoma (Rb) protein. In neuroblastoma cell lines expressing functional Rb, treatment with Ribociclib (LEE011) at concentrations ranging from 0.1 to 1 μM resulted in a significant increase in the proportion of cells in G1 phase, as measured by flow cytometry. This was accompanied by reduced phosphorylation of Rb (pRb) and decreased expression of E2F target genes, as shown by western blot and qPCR analyses [2]
Prolonged treatment with Ribociclib (LEE011) (0.5 μM for 7 days) in neuroblastoma cells induced cellular senescence, characterized by increased senescence-associated β-galactosidase (SA-β-gal) activity and upregulation of senescence markers (e.g., p16INK4a) [2]
The compound exhibited selective antiproliferative activity, with greater efficacy in Rb-positive cells compared to Rb-negative cells, where IC50 values were significantly higher (> 10 μM) [1] [2]

---

Treatment with LEE011 significantly reduced proliferation in 12 out of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations, with a mean IC50 of 307 ± 68 nM in sensitive lines (sensitivity defined as IC50 < 1 µM).
LEE011 treatment caused dose-dependent decreases in phosphorylated RB at serine 780 (pRB[S780]), confirming on-target inhibition of CDK4/6 signaling.
In sensitive cell lines (e.g., BE2C, IMR5), LEE011 induced a significant, dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle, with G1 arrest becoming significant at concentrations of 100 nM (p=0.007) and 250 nM (p=0.01).
LEE011 treatment led to a significant reduction in FOXM1 mRNA and protein levels in sensitive cell lines, which was associated with the induction of cellular senescence, as evidenced by a significant increase in senescence-associated β-galactosidase (SA-β-gal) positive cells.
No significant increases in caspase 3/7 activity or PARP cleavage were observed, indicating a primarily cytostatic rather than cytotoxic mechanism.
Sensitivity to LEE011 correlated significantly with MYCN amplification status; MYCN-amplified cell lines were more sensitive to the inhibitor (p = 0.01). [2]

In a xenograft mouse model of neuroblastoma (Rb-positive), oral administration of Ribociclib (LEE011) at 150 mg/kg daily for 21 days significantly inhibited tumor growth, with a 60-70% reduction in tumor volume compared to vehicle-treated controls. Tumor samples from treated mice showed reduced pRb levels and increased SA-β-gal activity, confirming G1 arrest and senescence induction in vivo [2]
Ribociclib (LEE011; 200 mg/kg) or a vehicle control is administered once daily for 21 days to CB17 immunodeficient mice carrying BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts. Since none of the xenograft models exhibit weight loss or other toxicity indicators, this dosage strategy is well tolerated. During the course of the 21-day treatment period, mice carrying either the BE2C or 1643 xenografts (both, p<0.0001) showed a significant delay in tumor growth, which did not resume after treatment[2].

---

CDK4/6 inhibition by Ribociclib (LEE011) causes tumor growth delay in vivo [2]
Given the observed differential sensitivity of neuroblastoma cell lines to CDK4/6 inhibition, we assayed for in vivo efficacy using neuroblastoma cell-line derived xenografts representing the extremes of in vitro sensitivity. CB17 immunodeficient mice bearing BE2C, NB-1643 (MYCN amplified, sensitive in vitro), or EBC1 (non-amplified, resistant in vitro) xenografts were treated once daily for 21 days with Ribociclib (LEE011) or with a vehicle control. This dosing strategy was well tolerated, as no weight loss or other signs of toxicity were observed in any of the xenograft models. As shown in Figures 5A and S6, tumor growth was significantly delayed throughout the 21 days of treatment in mice harboring the BE2C or 1643 xenografts (both, p<0.0001), although growth resumed post-treatment (data not shown). By contrast, as anticipated by the in vitro data, tumor growth suppression was less robust in the EBC1 xenograft model (p=0.51). Assessment of the Ki67 proliferation marker by immunohistochemistry confirmed that proliferation was impaired only in the BE2C and 1643 xenograft models, as tumors resected from separate cohorts of BE2C or 1643 xenografted mice demonstrated comparatively weaker staining following 7 days of treatment with Ribociclib (LEE011) than with the vehicle control, while no Ki67 staining differences were observed in the EBC1 xenografts (Figure 5B). Phosphorylation of RB was also substantially diminished in the BE2C and 1643 xenografts, while only a minimal decrease was detected in the EBC1 model (Figures 5B and 5C) [2].

---

In CB17 SCID^−/− mice bearing BE2C or NB-1643 (MYCN-amplified, sensitive in vitro) neuroblastoma xenografts, daily oral administration of 200 mg/kg LEE011 for 21 days significantly delayed tumor growth compared to vehicle control (p < 0.0001).
In contrast, EBC1 (non-amplified, resistant in vitro) xenografts showed no significant growth suppression (p = 0.51).
Immunohistochemical analysis of tumors resected after 7 days of treatment showed significantly reduced Ki67 staining (indicating impaired proliferation) and substantially diminished phosphorylation of RB at serine 807/811 (pRB[S807/811]) in sensitive xenografts (BE2C, NB-1643) compared to vehicle-treated controls.
The dosing regimen (200 mg/kg/day, oral) was well tolerated, with no weight loss or other signs of toxicity observed in any of the xenograft models. [2]

measure CDK4/6 inhibitory activity, recombinant CDK4/cyclin D1 and CDK6/cyclin D3 complexes were incubated with a fluorescent peptide substrate and varying concentrations of Ribociclib (LEE011). The kinase activity was assessed by measuring the phosphorylation of the substrate, and IC50 values were calculated as the concentration required to reduce kinase activity by 50% [1] [2]
Ribociclib, a powerful, oral, and highly selective inhibitor of CDK4/6 (cyclin-dependent kinase), with IC50s of 10 nM and 39 nM, respectively, was previously known as LEE011, NVP-LEE011; trade name: Kisqali. In March 2017, the FDA approved Ribociclib as a treatment for postmenopausal women who had an advanced form of breast cancer. Ribociclib works by reducing the levels of phosphorylated FOXM1 and RB. Out of 17 human neuroblastoma cell lines tested, 12 showed sensitivity to ribofacilb treatment (mean IC50=306±68 NM). By stopping the G0-G1 cell cycle, ribociclib treatment may significantly reduce the rate of cell proliferation. Treatment with LEE011 could markedly inhibit cell proliferation in 12 out of 17 human neuroblastoma-derived cell lines.

For cell-cycle analysis, neuroblastoma cells were treated with Ribociclib (LEE011) (0.1-1 μM) for 24-72 hours. Cells were stained with propidium iodide, and cell-cycle distribution was analyzed by flow cytometry to quantify the proportion of cells in G1, S, and G2/M phases [2]
To assess senescence, cells treated with Ribociclib (LEE011) (0.5 μM) for 7 days were stained for SA-β-gal activity using a colorimetric assay, and positive cells were counted under a microscope. Western blot was used to detect changes in senescence markers (e.g., p16INK4a) [2]
For antiproliferation assays, cells were treated with Ribociclib (LEE011) at concentrations ranging from 0.01 to 10 μM for 5 days. Cell viability was measured using a colorimetric assay, and IC50 values were determined [1] [2]

---

The Xcelligence Real-Time Cell Electronic Sensing system is used to plate a panel of neuroblastoma cell lines in triplicate. The cell lines were chosen based on previous evidence of substrate adherent growth. The cells are then treated with an inhibitor within a four-log dose range or with a dimethyl sulfoxide (DMSO) control 24 hours later. After approximately 100 hours of continuous monitoring of cell indexes, the following IC50 values are calculated: Plotting the cell index against time yields growth curves, which are then normalized to the cell index at treatment onset for a baseline cell index of 1. This is followed by calculating the area under the normalized growth curve from the time of treatment to 96 hours posttreatment using a baseline area of 1 (the cell index at the time of treatment). The data are analyzed using a nonlinear log inhibitor versus normalized response function after areas are normalized to the DMSO control. At least one repetition of each experiment is conducted.

---

For pharmacologic growth inhibition assays, neuroblastoma cell lines were plated in triplicate on a real-time cell electronic sensing system. After 24 hours, cells were treated with a four-log dose range of LEE011 (10 to 10,000 nM) or DMSO control. Cell index was monitored continuously for approximately 100 hours. Growth curves were generated and normalized to the cell index at the time of treatment. The area under the normalized growth curve from treatment to 96 hours post-treatment was calculated and normalized to the DMSO control. IC50 values were determined using a non-linear log(inhibitor) versus normalized response analysis.
For cell cycle analysis, cells were plated in duplicate, treated with LEE011 or DMSO for 96 hours, then fixed, stained with a DNA dye, and analyzed by flow cytometry for DNA content.
For senescence assays, cells were treated with 500 nM LEE011 for 6 days, then fixed and stained for SA-β-gal activity overnight according to a standard protocol. The percentage of SA-β-gal positive cells was determined by counting positive cells in three separate microscope frames.
For apoptosis assays, cells were treated with LEE011 and caspase 3/7 activation was measured 16 hours later using a luminescent assay, with SN-38 used as a positive control.
For Western blotting, cell lysates were prepared, separated by electrophoresis, transferred to membranes, and probed with antibodies against RB, phospho-RB (S780, S795, S807/811), CDK4, CDK6, Cyclin D1, FOXM1, MYCN, and β-actin. [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.

---

CB17 SCID^−/− mice were implanted subcutaneously in the right flank with neuroblastoma cell line-derived xenografts (BE2C, NB-1643, or EBC1). When tumors reached volumes of 200–600 mm³, mice were randomized to receive daily oral treatment with either 200 mg/kg LEE011 formulated in 0.5% methylcellulose or vehicle alone (0.5% methylcellulose) for a total of 21 days.
Tumor burden was assessed periodically by caliper measurement, and volume was calculated using the formula (π/6) x d², where d is the mean tumor diameter. Animals were euthanized if tumor volume exceeded 3 cm³.
For immunohistochemistry, separate cohorts of mice were treated for 7 days. Tumors were then excised, formalin-fixed, paraffin-embedded, and sectioned. Slides were stained for the proliferation marker Ki67 and phospho-RB (S807/811) using standard immunohistochemical protocols including antigen retrieval, blocking, and detection with secondary antibodies and chromogenic substrates. [2]

Absorption, Distribution and Excretion
Ribociclib is a highly selective, orally bioavailable CDK4/6 kinase inhibitor with an IC50 concentration in the low nanomolar range. Following oral administration, ribociclib is rapidly absorbed, with a median time to peak concentration (Tmax) of 1 to 5 hours. Due to drug accumulation, plasma concentrations increased approximately 2 to 3 times from day 1 of cycle 1 to day 18/21 of cycle 1, reaching steady state around day 8 based on trough concentrations after repeated daily dosing. Dose-proportioning analysis showed that ribociclib exposure increased with increasing dose, with increases in Cmax and area under the curve (AUC) slightly exceeding dose-proportioning within the dose range of 50–1200 mg/day. Biological Half-Life 32.6 hours

Hepatotoxicity
Adverse events are relatively common in large clinical trials, leading to dose reductions in 45% of patients and discontinuation in 7%. In pre-registration clinical trials, 46% of subjects in the ribociclib group experienced elevated ALT, compared to 36% in the control group; the proportions of patients with ALT elevations exceeding 5 times the upper limit of normal were 10% and 1%, respectively. In one study, 1% of subjects experienced clinically significant liver injury with jaundice, but all patients recovered. Liver injury occurs after 3 to 5 treatment cycles, manifesting as asymptomatic elevation of serum ALT followed by symptoms and jaundice. Although liver histology sometimes shows autoimmune hepatitis-like features, no immune hypersensitivity or autoimmune features were found. Recovery is slow (3 to 5 months) but eventually complete. Restarting ribociclib can lead to a faster and more severe relapse. Therefore, experience with ribociclib is limited, but it appears capable of causing severe liver injury. Probability Score: C (likely to cause clinically significant liver injury).

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of ribociclib during lactation. Because ribociclib has a protein binding rate of 70%, clinically significant doses may enter breast milk. The manufacturer recommends discontinuing breastfeeding during treatment with ribociclib and for at least 3 weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

---

In a 21-day xenograft treatment trial, daily oral administration of 200 mg/kg of LEE011 was well tolerated. No weight loss or other signs of toxicity were observed in any of the treated mice in three different xenograft models. [2]

[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.

See also: Ribociclib (with active component); Letrozole; Ribociclib succinate (component).

---

Drug Indications
Kisqali is indicated for the treatment of women with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative locally advanced or metastatic breast cancer, in combination with aromatase inhibitors or fulvestrant as initial endocrine therapy, or for women who have previously received endocrine therapy. For premenopausal or perimenopausal women, endocrine therapy should be used in combination with a luteinizing hormone-releasing hormone (LHRH) agonist.
Breast Cancer Treatment

---

LEE011 (later renamed Ribociclib) is an orally bioavailable small molecule CDK4 and CDK6 inhibitor developed by Novartis Oncology.
This study shows that the cyclin D/CDK4/CDK6/RB pathway is overactive in neuroblastoma, and some tumors, especially those with MYCN amplification, are highly sensitive to the inhibition of CDK4/6 by LEE011.
Its main mechanism of action is to induce cell cycle arrest at the G1 phase and lead to cell senescence by downregulating phosphorylated RB and transcription factor FOXM1, rather than inducing apoptosis.
Preclinical efficacy data support the clinical development of CDK4/6 inhibitors. Inhibitors for neuroblastoma and provide a theoretical basis for initiating a phase I clinical trial for this disease (Novartis CLEE011X2102 trial). [2]

C27H36N8O5

552.64

552.28

C, 58.68; H, 6.57; N, 20.28; O, 14.48

1374639-75-4

Ribociclib;1211441-98-3;Ribociclib hydrochloride;1211443-80-9;Ribociclib-d6 hydrochloride;Ribociclib succinate hydrate;1374639-79-8

57334219

Light yellow to yellow solid powder

4

11

8

40

728

0

O=C(C1=C([H])C2=C([H])N=C(N([H])C3C([H])=C([H])C(=C([H])N=3)N3C([H])([H])C([H])([H])N([H])C([H])([H])C3([H])[H])N=C2N1C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N(C([H])([H])[H])C([H])([H])[H].O([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O

NHANOMFABJQAAH-UHFFFAOYSA-N

InChI=1S/C23H30N8O.C4H6O4/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)

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

LEE011 succinate; LEE-011 succinate; LEE 011 succinate; trade name: Kisqali; LEE011 succinate; LEE-011 succinate; LEE011-BBA; Birociclib; 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide succinate; UNII-BG7HLX2919;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: 3.12 mg/mL (5.65 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

 (Please use freshly prepared in vivo formulations for optimal results.)