Abstract 4481: First report of preclinical pharmacology of two novel potent AKT inhibitors and development of pharmacodynamic (PD) biomarkers in tumor and surrogate tissue
Authors: Yap Timothy A. , Walton Mike I. , Hunter Lisa , Grimshaw Kyla , Valenti Melanie , Eve Paul , Heaton Simon P. , Pickard Lisa , Caldwell John J. , Thompson Neil , de Bono Johann S. , Kaye Stan B. , Eccles Sue A. , Workman Paul , Collins Ian , Garrett Michelle D. 
Published: February 2011

Abstract
Deregulated AKT signalling is implicated in cancer. The preclinical characterization of AKT inhibitors and development of PD biomarkers are vital prior to clinical trials. Invasive techniques for clinical PD analyses pose ethical and logistical issues, hence hair follicles may represent a non-invasive option. The pyrazole AT7867 and pyrrolopyrimidine CCT128930 are novel ATP-competitive AKT inhibitors from different chemical series developed at The Institute of Cancer Research in collaboration with Astex Therapeutics (Cambridge, UK).
AT7867 and CCT128930 have IC50 values against AKT2 of 17nM and 6nM respectively. Growth inhibitory IC50 (GI50) values were 2.4μM and 6.3μM in PTEN-null U87MG glioblastoma cells as measured by SRB assay. Cellular studies of both compounds in U87MG cells were carried out by Meso Scale Discovery (MSD) ELISA, western blot (WB) and immunofluorescence. These showed an initial induction of phosphorylated (p) Ser473 AKT as expected with ATP-competitive AKT inhibitors, and inhibition of downstream AKT targets, including pSer9 GSK-3β, pThr246 PRAS40, pSer127 YAP and pSer235/236 S6RP, indicating AKT pathway blockade. Interestingly, CCT128930 inhibited phosphorylation of AKT targets GSK-3β, S6RP and PRAS40 at lower concentrations (0.5-1µM), compared to AT7867 (5-10µM) in U87MG cells. Phosphorylation was also inhibited at earlier time points with equipotent doses (3XGI50) of CCT128930 versus AT7867. CCT128930 also caused a predominant G0/G1 phase arrest, while AT7867 resulted in a predominant G2 arrest in U87MG cells at equipotent doses (3XGI50), using BrdU and PI staining and flow cytometry. We report for the first time in vivo efficacy with intraperitoneally (ip) or orally administered AT7867 in PTEN-null MES-SA uterine sarcoma and U87MG mouse xenograft models; and ip administered CCT128930 in HER2-overexpressing BT474 breast cancer and U87MG xenografts, which correlated with in vivo pharmacokinetics (PK) and PD biomarker modulation of pSer9 GSK-3β and pSer235/236 S6RP using WB and MSD.
Hair follicles were developed as a robust surrogate PD biomarker, with pThr246 PRAS40 as a PD readout. This assay was validated with healthy volunteer hair follicles, which were treated ex vivo with CCT128930. A significant decrease in pThr246 PRAS40 (p&lt;0.001) relative to total PRAS40 was observed with immunofluorescence, which was quantified using INCell Translator software. This assay is currently being utilized as a PD readout in the MK2206 Phase I AKT inhibitor trial, and will be used in future clinical studies of ATP-competitive AKT inhibitors.
In conclusion, by employing an integrated PK and PD biomarker-driven drug discovery strategy, we have developed 2 novel and potent AKT inhibitors with antitumor activity, and have validated a robust surrogate biomarker assay for AKT inhibition for use in clinical trials.
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 4481.