Palbociclib Isethionate (PD 0332991 isethionate) is a selective inhibitor of CDK4/6 (IC50s: 11/16 nM). It exhibits no inhibition against a panel of 36 additional protein kinases.

CDK4-CyclinD3:9 nM (cell free), CDK6-CyclinD2:15 nM (cell free), CDK4-CyclinD1:11 nM (cell free)

Palbociclib 是一种高度特异性的 Cdk4（IC50, 0.011 micromol/L）和 Cdk6（IC50, 0.016 micromol/L）抑制剂。它对于体外的视网膜母细胞瘤（Rb）阳性肿瘤细胞具有强大的抗增殖作用，引发了独特的G1期阻滞，并伴随Rb蛋白上的phospho-Ser780/Ser795含量减少[1]。除KP-MRT-YM细胞系外，Palbociclib对所有细胞系的细胞增殖抑制率>50%（IC50值 0.01至0.6 μM），并诱导G1期细胞周期阻滞。MRT细胞系对Palbociclib的敏感性与p16表达成反比。KP-MRT-YM细胞过表达p16，对Palbociclib的生长抑制效果产生了抵抗[2]。代表性的内膜型雌激素受体阳性（ER+）亚型（包括那些HER2扩增的）细胞系对Palbociclib的生长抑制最为敏感，而非内膜型/基底型亚型最为耐药[3]。

向携带Colo-205人类结肠癌的小鼠口服给药PD 0332991能显著促进肿瘤退缩。PD 0332991的治疗剂量会导致肿瘤组织中磷酸化Rb和增殖标记物Ki-67的消除，并下调E2F转录控制下基因的表达[1]。

CDK assays for IC50determinations and kinetic evaluation were performed in 96-well filter plates. All CDK-cyclin kinase complexes were expressed in insect cells through baculovirus infection and purified as described previously. The substrate for the assays was a fragment (amino acids 792–928) of pRb fused to GST. The total volume for each well was 0.1 ml containing a final concentration of 20 mM Tris-HCl, pH 7.4, 50 mMNaCl, 1 mM dithiothreitol, 10 mMMgCl2, 25 μM ATP (for CDK4-cyclin D1, CDK6-cyclin D2, and CDK6-cyclin D3) or 12 μM ATP (for CDK2-cyclin E, CDK2-cyclin A, and CDC2-cyclin B) containing 0.25 μCi of [γ-32P]ATP, 20 ng of enzyme, 1 μg of GST·RB-Cterm, and appropriate dilutions of inhibitor. All components except the [γ-32P]ATP were added to the wells, and the plate was placed on a plate mixer for 2 min. The reaction was then started by adding the [γ-32P]ATP, and the plate was incubated at 25?°C for 15 min. The reaction was terminated by addition of 0.1 ml of 20% trichloroacetic acid, and the plate was kept at 4?°C for at least 1 h to allow the substrate to precipitate. The wells were then washed five times with 0.2 ml of 10% trichloroacetic acid, and radioactive incorporation was determined with a β plate counter. Kinase assays for PDGFr, FGFr, EGFr, SRC, and PKC kinases were performed as described previously [4].

Cells were seeded at 2 × 10^4 per well in a 96-well Cytostar T plate and incubated overnight to allow cells to attach. Varying concentrations of PD 0332991 were added to the wells and incubated for 24 hours at 37°C. [14C]thymidine (0.1 μCi) was added to each well and incorporation of the radiolabel was allowed to proceed for 72 hours. Incorporated radioactivity was determined with a β plate counter [1].

Mice (18–22 g) were randomized and then implanted s.c. with tumor fragments (～30 mg) into the region of the right axilla. Treatment was initiated when tumors reached 100 to 150 mg. PD 0332991 was given according to the schedule and dose indicated in the table and figure legends by gavage as a solution in sodium lactate buffer (50 mmol/L, pH 4.0) based on mean group body weight. In all experiments, there were 12 mice in the control group and 8 mice each in the treated groups. Additional details for each experiment are given in the table legends [1].