Milciclib (PHA-848125) (PHA-848125) is a potent, ATP-competitive CDK inhibitor for CDK2 with IC50 of 45 nM. It is >3-fold more selective for CDK2 than CDK1, 2, 4, 5, and 7. Phase 2.

CDK2-CyclinA:45 nM

Milciclib以较低的效能抑制cyclin H/CDK7、cyclin D1/CDK4、p35/CDK5以及cyclin E/CDK2和cyclin B/CDK1的活性，其IC50值分别为0.15、0.16、0.265、0.363、0.398 μM。[1] Thropomyosin受体激酶A也在与CDKs相同的nM范围内被Milciclib抑制。在最敏感的Milciclib细胞系中，Milciclib引起浓度依赖性的G(1)阶段阻滞。[2]Milciclib还干扰了在CDK2和CDK4特异性位点上视网膜母细胞瘤蛋白的磷酸化，降低了视网膜母细胞瘤蛋白和cyclin A的水平，并提高了p21(Cip1)、p27(Kip1)和p53的表达。在添加TMZ后48小时将Milciclib加入细胞，之后再培养3天评估细胞生长。[2] TMZ、BG和Milciclib的化合物组合根据细胞系的不同，对细胞生长产生加和作用或协同作用。[2] 在没有BG的情况下，该组合在对TMZ中等敏感的细胞系中比单独化合物更活跃，但与仅用Milciclib治疗的两个TMZ抗性细胞系效果类似。当TMZ和BG与Milciclib联合使用针对培养的正常黑素细胞时，并未观察到协同或加和的抗增殖效应。[2]

在临床前异种移植的A2780人类卵巢癌模型中，Milciclib展现了良好的疗效并在连续日常治疗中表现出良好的耐受性。对K-Ras(G12D)LA2小鼠用Milciclib处理（每日两次，每次40 mg/kg，持续10天），在治疗结束时显著抑制了肿瘤生长，并伴随着细胞膜周转的减少。[3] 另一方面，经口给药后，Milciclib在多种人源异种移植物、致癌诱导的肿瘤以及分散性原发性白血病模型中显示出显著的抗肿瘤活性；啮齿动物的血浆浓度与抑制癌细胞增殖的浓度处于相同范围内。[4]

Biochemical kinase inhibition assays: Inhibition of kinase activity by PHA-848125 is assessed using a strong anion exchanger (Dowex 1X8 resin)–based assay in robotized format run on 384-well plates. In this assay, specific peptides or protein substrates are transphosphorylated by their specific kinase in the presence of ATP traced with [γ-33P]ATP using optimal buffers and cofactors. The potency of PHA-848125 toward CDKs and 38 additional kinases belonging to an in-house Kinase Selectivity Screening panel is evaluated, and the relevant IC50s are determined. For each enzyme, the absolute KM values for ATP and the specific substrate are calculated and each assay is then run at optimized ATP (2KM) and substrate (5KM) concentrations. This setting enables direct comparison of IC50 values of PHA-848125 across the panel for evaluation of its biochemical profile.

Melanoma cells are suspended in culture media at a concentration of 2 × 104 cells/mL, dispensed in 50 μL aliquots into flat-bottom 96-well plates and allowed to adhere overnight at 37 °C. Graded amounts of PHA-848125 or TMZ are then added to the wells (4 wells per point) in 50 μL of CM and the plates are incubated at 37 °C in a 5% CO2 humidified atmosphere for 5 days. The cytotoxic effects of TMZ are also evaluated in combination with the MGMT inhibitor BG. To this end, 10 μM BG is added to the plates 2 hours before TMZ and left in culture for the entire period of cell exposure to the drug. ContS1017rol groups are represented by untreated cells and cells treated with BG or DMSO alone. The growth of the cells treated with BG or DMSO alone does not differ from that of untreated cells. MGMT activity of BG-treated cells is undetectable 2 hours after the addition of the inhibitor PHA-848125 and remained essentially undetectable up to the end of the assay. Normal melanocytes are suspended in MGM at the concentration of 1.6 × 105 cells/mL, plated (50 μL/well) and exposed to TMZ + BG or to PHA-848125 as described for melanoma cells. At the end of the incubation period, cell growth is evaluated by the MTT assay. Briefly, 0.1 mg of MTT (in 20 μL of PBS) is added to each well and cells are incubated at 37 °C for 4 hours. Cells are then lysed with a buffer (0.1 mL/well) containing 20% SDS and 50% N,N-dimethylformamide, pH 4.7. After overnight incubation, the absorbance is read at 595 nm using a 3550-UV microplate reader. Cell sensitivity to drug treatment is expressed in terms of IC50 (drug concentration producing 50% inhibition of cell growth, calculated on the regression line in which absorbance values at 595 nm are plotted against the logarithm of drug concentration).(Only for Reference)