SummaryThe G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-014-0120-7) contains supplementary material, which is available to authorized users.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain K p,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. K p,uu increased 2.8-and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) K p,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 K i values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.

Purpose: Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) represents a promising therapeutic strategy. However, despite documented evidence of clinical activity, limited information is available on the optimal dosing strategy of CDK4/6 inhibitors. Here, we present an integrated semimechanistic pharmacokinetic/pharmacodynamic model to characterize the quantitative pharmacology of LY2835219, a CDK4/6 inhibitor, in xenograft tumors.Experimental Design: LY2835219 plasma concentrations were connected to CDK4/6 inhibition and cell-cycle arrest in colo-205 human colorectal xenografts by incorporating the biomarkers, phospho-(ser780)-Rb, topoisomerase II a, and phosphohistone H3, into a precursor-dependent transit compartment model. This biomarker model was then connected to tumor growth inhibition (TGI) by: (i) relating the rate of tumor growth to mitotic cell density, and (ii) incorporating a concentration-dependent mixed cytostatic/ cytotoxic effect driving quiescence and cell death at high doses. Model validation was evaluated by predicting LY2835219-mediated antitumor effect in A375 human melanoma xenografts.Results: The model successfully described LY2835219-mediated CDK4/6 inhibition, cell-cycle arrest, and TGI in colo-205, and was validated in A375. The model also demonstrated that a chronic dosing strategy achieving minimum steady-state trough plasma concentrations of 200 ng/mL is required to maintain durable cell-cycle arrest. Quiescence and cell death can be induced by further increasing LY2835219 plasma concentrations.Conclusions: Our model provides mechanistic insight into the quantitative pharmacology of LY2835219 and supports the therapeutic dose and chronic dosing strategy currently adopted in clinical studies.