Rose T. Ajamie

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft

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 Kp,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. Kp,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) Kp,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 Ki 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.

Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of the Antitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6 Inhibitor, in Mice Bearing Human Tumor Xenografts

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 semi-mechanistic 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 α, 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. Clin Cancer Res; 20(14); 3763–74. ©2014 AACR.

A Novel CDK9 Inhibitor Shows Potent Antitumor Efficacy in Preclinical Hematologic Tumor Models

DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies. Mol Cancer Ther; 13(6); 1442–56. ©2014 AACR.

Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs.

Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.

Abstract B234: LY2835219, a potent oral inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6) that crosses the blood-brain barrier and demonstrates in vivo activity against intracranial human brain tumor xenografts.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. The blood-brain barrier (BBB) arises from both a structural barrier and drug efflux transporters that prevent most anti-cancer drugs from efficiently reaching brain tumors or metastases. The CDK4/6 pathway (CDK4/6-cyclin D1-Rb-CDKN2) plays a key role in regulating cellular proliferation. The importance of this pathway is highlighted by its inactivation in a majority of human tumors including glioblastoma multiforme. We have identified and characterized a potent and selective dual cdk4/6 inhibitor, LY2835219. Preclinical characterization was performed with the monomesylate salt (LY2835219-MsOH), which inhibits these kinases with IC50 values of 2 and 10 nM for CDK4 and CDK6, respectively. LY2835219-MsOH is a potent inhibitor of Rb phosphorylation in vitro and in vivo that induces G1 specific arrest and inhibition of tumor growth. To determine the potential of LY2835219-MsOH for the treatment of brain tumors and metastases, we assessed the ability of LY2835219-MsOH to cross the BBB and its interaction with the P-gp and BCRP efflux pumps that are expressed at the BBB. Using MDCK cells over-expressing either human ABCB1 (P-gp) or mouse abcg2 (Bcrp), LY2835219-MsOH and a second CDK4/6 inhibitor (PD0332991) are substrates for these two pumps, but each cross the murine BBB in vivo to a different degree. Unlike PD0332991, LY2835219-MsOH saturates BBB efflux with an unbound plasma IC50 of ∼95 nM (1.8 uM total plasma). The percent of dose in brain for LY2835219-MsOH is 0.5–3.9% and is comparable to that for temozolomide (1.9% plasma). In both a subcutaneous and intracranial human glioblastoma model (U87MG), LY2835219-MsOH suppressed tumor growth in a dose-dependent manner both as a single agent, and in combination with temozolomide. In summary, LY2835219-MsOH is a potent and selective oral CDK4/6 inhibitor that crosses the BBB and inhibits the growth of intracranial tumors alone or in combination with other agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B234.

Abstract B233: Identification and characterization of LY2835219: A potent oral inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6) with broad in vivo antitumor activity.

Dysregulation of the cell cycle, which normally regulates cell proliferation in response to mitogenic signaling and other extracellular stimuli, is a hallmark of cancer. The G1 restriction point is a primary mechanism controlling cell cycle progression and is controlled by the CDK4/6 pathway (CDK4/6-cyclin D1-Rb-CDKN2). The importance of this pathway is highlighted by inactivation of restriction point control in a majority of human tumors. Transition through the restriction point requires phosphorylation of Rb by CDK4/6, and these kinases are considered highly validated cancer drug targets. We have identified and characterized a potent and selective dual CDK4/6 inhibitor, LY2835219. Preclinical characterization was performed with the monomesylate salt (LY2835219-MsOH), which inhibits these kinases with a IC50 of 2 and 10 nM for CDK4 and CDK6, respectively. In vitro, LY2835219-MsOH is a potent inhibitor of Rb phosphorylation resulting in a G1 arrest, and its activity is specific for tumors that have functional Rb protein. In a multiplexed in vivo target inhibition assay (IVTI), LY2835219-MsOH is a potent inhibitor of Rb phosphorylation and induces complete cell cycle arrest 24 hrs after a single dose. In tumor-bearing mice, oral administration of LY2835219-MsOH inhibits tumor growth in a dose-dependent manner in colon (colo-205), glioblastoma (U87MG), acute myeloid leukemia (MV4–11), mantle cell lymphoma (Jeko-1), and lung (H460) xenografts. LY2835219-MsOH may be administered up to 56 days without adverse events or tumor outgrowth. LY2835219-MsOH enhances the in vivo activity of cytotoxic drugs, suggesting that this novel CDK4/6 inhibitor can be used in combination with these anti-neoplastic agents. In summary, we have identified an oral small molecule inhibitor of CDK4/6 that may provide therapeutic benefit to cancer patients with tumors that have functional Rb protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B233.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2–7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drugs half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344–56. ©2016 AACR.

Pharmacokinetic and screening studies of the interaction between mononuclear phagocyte system and nanoparticle formulations and colloid forming drugs

Studies have shown that nanoparticles (NPs) are cleared through the mononuclear phagocyte system (MPS). Pharmacokinetic studies of Doxil, DaunoXome, micellar doxorubicin (SP1049C) and small molecule (SM) doxorubicin were performed in SCID mice, Sprague-Dawley rats, and beagle dogs. An ex vivo MPS profiling platform was used to evaluate the interaction between the same agents, as well as colloid-forming and non-colloid forming SM drugs. In all species, the systemic clearance was highest for SP1049C and lowest for Doxil. With the exception of dog blood, the MPS screening results of mouse and rat blood showed that the greatest reduction in phagocytosis occurred after the ex vivo addition of SM-doxorubicin>SP1049C>DaunoXome>Doxil. The MPS profiling platform in rats, but not dogs, could differentiate between colloid forming and non-colloid forming drugs. The results of the MPS profiling platform were generally consistent with in vivo clearance rates of NP and SM anticancer drugs in mice and rats. This study suggests the MPS profiling platform is an effective method to screen and differentiate the important characteristics of NPs and colloid-forming drugs that affect their in vivo clearance. Implications of these findings on preclinical prediction of human clearance are discussed.

Abstract 352: Preclinical evaluation of LYS6K1, a novel, highly selective, orally bioavailable inhibitor of p70 S6 kinase currently in phase I clinical trials for cancer

PI3K/AKT/mTOR/S6K signaling pathway (AKT pathway) controls cell survival, cell-cycle progression, cell growth and metabolism through a cascade phosphorylation of a number of key substrates. This pathway is regulated by three well characterized tumor suppressors; pten, tsc2, and lkb1. Deletion of these genes results in activation of the AKT pathway and proliferative disorders. Similarly, activating mutations of the receptor tyrosine kinases or PI3 Kinase result in the activation of the pathway. Therefore, multiple nodes of the pathway have become drug targets. As part of a comprehensive drug discovery platform aimed at targeting the PI3K pathway, we have developed a potent small molecule inhibitor of p70 S6 kinase that is a downstream effector in this pathway. LYS6K1 is a potent, highly selective ATP competitive inhibitor against p70 S6 kinase with an IC50 of 0.002 uM. In vitro, LYS6K1 inhibits the phosphorylation of S6 ribosomal protein in HCT116 colon cancer cells with an IC50 of 0.2 uM and similar activity is seen in a broad range of other cell lines. In vivo, LYS6K1.tosylate demonstrates potent phospho-S6 inhibition in nude mice bearing HCT116 colon carcinoma cells, with an ED50 value of 2.2 mg/kg and a ED90 value of 10 mg/kg 4 hours after a single oral dose. In these studies, LYS6K1.tosylate did not show statistically significant elevation of phospho-AKT, in spite of potent p70 S6 kinase inhibition. Proportional dose, exposure, and pharmacodynamic relationships were observed for LYS6K1 in dose- and time- dependent studies. In vivo, LYS6K1.tosylate effectively inhibits the growth of HCT116 colon carcinoma xenografts in mice and the growth of U87MG glioblastoma tumor as a single agent at 2.5 mg/kg given twice daily. Overall, LYS6K1.tosylate has high permeability that resulted in good oral absorption and PK properties. Based on these pre-clinical observations, LYS6K1.tosylate has advanced to Phase I studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 352.