| In Vitro | In vitro activity: Dinaciclib is also a potent DNA replication inhibitor that blocks thymidine (dThd) DNA incorporation in A2780 cells with IC50 of 4 nM. Dinaciclib strongly suppresses phosphorylation of Rb on Ser 807/811 at concentrations>6.25 nM, which is in agreement with the observation that 4 nM concentrations are required for 50% inhibition of dThd DNA incorporation in the same cell model. Significantly, complete suppression of Rb phosphorylation is correlated with the onset of apoptosis, as indicated by the appearance of the p85 PARP cleavage product in cells exposed to>6.25 nM Dinaciclib. Dinaciclib is active against a broad spectrum of human tumor cell lines. Addition of Dinaciclib during hydroxyurea exposure also suppresses accumulation of γ-H2AX, in a dose-dependent manner. Dinaciclib inhibits melanoma cell proliferation, and drives melanoma cells into massive apoptosis. Dinaciclib induces the apoptosis of several osteosarcoma cell lines including those resistant to doxorubicin and dasatinib. Dinaciclib attenuates the phosphorylation of RNAP II at serine 2 and the phosphorylation of the CDK inhibitor p27Kip1 at threonine 187. Reductions in phosphorylation activity occurrs at 12 - 40 nM Dinaciclib (4 to 16 hours post-Dinaciclib addition). Dinaciclib also reduces the phosphorylation of Rb at serine 807/811. Dinaciclib induces the apoptosis of mock- and p53-depleted U2OS cells to a similar extent.
Kinase Assay: Recombinant cyclin/CDK holoenzymes are purified from Sf9 cells engineered to produce baculoviruses that express a specific cyclin or CDK. Cyclin/CDK complexes are typically diluted to a final concentration of 50 μg/mL in a kinase reaction buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, and 0.1 mM sodium orthovanadate. For each kinase reaction, 1 μg of enzyme and 20 μL of a 2-μM substrate solution (a biotinylated peptide derived from histone H1) are mixed and combined with 10 μL of diluted Dinaciclib. The reaction is started by the addition of 50 μL of 2 μM ATP and 0.1 μCi of 33P-ATP. Kinase reactions are incubated for 1 hour at room temperature and are stopped by the addition of 0.1% Triton X-100, 1 mM ATP, 5 mM EDTA, and 5 mg/mL streptavidin-coated SPA beads. SPA beads are captured using a 96-well GF/B filter plate and a Filtermate universal harvester. Beads are washed twice with 2 M NaCl and twice with 2 M NaCl containing 1% phosphoric acid. The signal is then assayed using a TopCount 96-well liquid scintillation counter.
Cell Assay: A2780 cells are maintained in DMEM plus 10% fetal bovine serum and passaged twice weekly by detaching the monolayer with trypsin-EDTA. One hundred microliters of A2780 cells (5 × 103 cells) are added per well to a 96-well Cytostar-T plate and incubated for 16 hours to 24 hours at 37 °C. Dinaciclib is serially diluted in complete media plus 2% 14C-labeled dThd. Media are removed from the Cytostar T plate; 200 μL of various Dinaciclib dilutions are added in quadruplicate; and the cells are incubated for 24 hours at 37 °C. Accumulated incorporation of radiolabel is assayed using scintillation proximity and measured on a TopCounTM. |
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| In Vivo | Dinaciclib i.p. administration at 8, 16, 32, and 48 mg/kg daily for 10 days results in tumor inhibition by 70%, 70%, 89%, and 96%, respectively. Dinaciclib MED (minimum effective dose) appears to be<8 mg/kg. Dinaciclib is well tolerated, and the maximum body weight loss in the highest dosage group is 5%. Dinaciclib has dose-dependent antitumor activity in vivo, and that nearly complete inhibition of tumor growth occurs at a dose level below the MTD (maximum tolerated dose). Dinaciclib has a short plasma half-life in mouse. |
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