PD 0332991 HCL

Biological Activity

PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. PD-0332991 selectively inhibits cyclin-dependent kinases (particularly Cdk4/cyclin D1 kinase), which may inhibit retinoblastoma (Rb) protein phosphorylation; inhibition of Rb phosphorylation prevents Rb-positive tumor cells from entering the S phase of the cell cycle (arrest in the G1 phase), resulting in suppression of DNA replication and decreased tumor cell proliferation. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

Product Citations

• 

  BMC Biol. 2021 May 20;19(1):108.

  Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans
  PD 0332991 HCL purchased from AbMole

• 

  Journal of Molecular Structure. 2014 Jan 6;Pages 209-215.

  A theoretical study of the structure and protonation of Palbociclib (PD 0332991)
  PD 0332991 HCL purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Breast Cancer Res (2009). Figure 4. PD 0332991 HCL
	Method	Proliferation assays
	Cell Lines	SUM-190 and SUM-225 cell line
	Concentrations	100 nM
	Incubation Time	48 h
	Results	There was no evidence of apoptosis in even the most sensitive cell lines when PD 0332991 was used as a single agent

	Source	Breast Cancer Res (2009). Figure 3. PD 0332991 HCL
	Method	Western blot
	Cell Lines	SUM-190 and SUM-225 cell line
	Concentrations	100 nM
	Incubation Time	48 h
	Results	These lines did not have a significant decrease in Rb phosphorylation with 100 nM PD 0332991.

Protocol

Cell Experiment
Cell lines	HCT116, SW480, Lovo and LS174T cells
Preparation method	Cell proliferation and colony formation assays Cells were seeded in a 96-well plate at 2000–6000 cells/well, incubated overnight at 37°C to allow adhesion, and then treated with inhibitors for 72 hours. Cell proliferation was determined using MTS solution (Promega), and formal assessment for synergy performed per the Chou Talalay method [64, 65] using CompuSyn (ComboSyn, Inc, Paramus, NJ). For colony formation assay, cells were seeded in a 6-well plate at 8000– 80,000 cells/well, incubated overnight at 37°C to allow adhesion, and then treated with inhibitors for 2–3 weeks. Cell colonies were fixed with ice-cold methanol and stained with 1% crystal violet. The density of colonies over the plate area was quantified by ImageJ (NIH) [66]. For detection of exposed phosphatidylserine residues reflective of apoptosis, Annexin V-FITC apoptosis assay kit (BD Biosciences) was used. Cells were seeded at 500,000 cells/ well in 6-well plates, incubated overnight at 37°C to allow adhesion, and then treated with inhibitors for 72 hours. Cells were washed and stained with annexin V-FITC and propidium iodide, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter) and analyzed with Kaluza flow analysis software (Beckman Coulter). To measure cell senescence, treated cells were stained for senescence-associated beta-galactosidase (Chemicon). Cells were seeded at 500,000 cells/well in 6-well plates, incubated overnight at 37°C to allow adhesion, and then treated with inhibitors for 72 hours. Cells were washed with PBS, fixed with glutaraldehyde and methanol, washed twice more, and then incubated overnight in the dark at 37°C under ambient atmospheric conditions with X-Gal. Subsequently, cells were washed, and 10 high-powered light microscopy images of each well were captured, and cells with blue staining were manually counted.
Concentrations	0~400nM
Incubation time	72 h

Animal Experiment
Animal models	Primary human-tumor xenograft models in NU/J6-week old female mice
Formulation	PBS
Dosages	100 mg/kg per os daily for 21 days
Administration	oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

[1] Lee MS, et al. Oncotarget. Efficacy of the combination of MEK and CDK4/6 inhibitors in vitro and in vivo in KRAS mutant colorectal cancer models.

[2] Liu F et al. Mol Cancer Ther. Cdk4/6 Inhibition Induces Epithelial-Mesenchymal Transition and Enhances Invasiveness in Pancreatic Cancer Cells.

[3] Dean JL et al. Cell Cycle. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors.

[4] Flaherty KT et al. Clin Cancer Res. Phase I, dose-escalation trial of the oral cyclin-dependent kinase 4/6 inhibitor PD 0332991, administered using a 21-day schedule in patients with advanced cancer.

[5] Smith D et al. J Chromatogr B Analyt Technol Biomed Life Sci. Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry.

[6] Katsumi Y et al. Biochem Biophys Res Commun. Sensitivity of malignant rhabdoid tumor cell lines to PD 0332991 is inversely correlated with p16 expression.