Introduction: First generation reversible, ATP competitive inhibitors, gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers, in particular specific EGFR-activating mutations. However their initial efficacy is limited by the development of drug resistance mutations, including the gatekeeper T790M mutation. Second-generation irreversible EGFR inhibitors that were designed to overcome the drug resistance by T790M mutation have thus far had limited success. Our goal was then to develop a dual kinase EGFR inhibitor effective against gefitinib sensitive and resistant (T790M) mutations. Herein, we describe the biological and pharmacokinetic properties of a representative molecule, KL-ON113 from a series of novel and small molecule EGFR inhibitors as a clinical development for non-small cell lung cancer (NSCLC).
Experimental Procedures: Based on structure activity relationship (SAR) studies, we identified a novel dual kinase EGFR inhibitor, KL-ON113 effective against gefitinib sensitive and resistant (T790M) mutations. EGFR tyrosine kinase activity of KL-ON113 was determined using HTRF® KinEASE™ assay kit (CisBio, Bedford, MA) with modifications. EGF induced cell proliferation assay (XTT) was conducted to determine the growth inhibitory effect of KL-ON113 in EGFR expressing WT and mutant cell lines. Inhibition of EGF induced EGFR phosphorylation (Y1173) in A431 cells was measured by PathScan® Phospho-EGF Receptor (Tyr1173) Sandwich ELISA Kit (Cell Signaling Technology, Beverly, MA). Metabolic stability of the KL-ON113 was evaluated in microsomes obtained from mouse, rat, monkey, and human. Pharmacokinetic behavior of KL-ON113 in plasma after single dose oral administration or IV injection was determined in female Balb/c mice.
Results: KL-ON113 demonstrated remarkable potency against the purified EGFR/HER2 including EGFR (wt), EGFR (L858R), EGFR (delE746_A750), EGFR (T790M), EGFR (L858R/T790M) and HER2 with IC50 of 12nM, 1.3nM, 0.9nM, 49nM, 325nM and 14nM, respectively with 100-fold more active against gefitinib/erlotinib resistant L858R/T790M EGFR mutant. Additionally, KL-ON113 caused a significant reduction in viability & EGFR phosphorylation (Y1173) in A431 cells, while no effect observed in normal WI-38 lung fibroblast cells confirming its selectivity. Pharmacokinetic studies in female Balb/c mice indicated good oral bioavailability of 80% for KL-ON113. Further, KL-ON113 was metabolically stable across the species studied.
Conclusions: Our findings demonstrate that KL-ON113 as a potent dual kinase EGFR inhibitor effective against gefitinib sensitive and resistant (T790M) mutations with a favorable pharmacokinetic profile. KL-ON113 is also being tested for its in-vivo efficacy in NSCLC xenograft models harboring EGFR WT and EGFR L858R/T790M besides selectivity against other receptor tyrosine kinases.
Citation Format: Vijaybaskar Lakshmikanthan, Venkateswarlu Sommepalli, Ramamohan Lekkala, Kondababu Rasamsetti, Rama Raju Gokaraju, Kiran Bhupathiraju, Sudhakar Kasina. KL-ON113, a novel orally available dual EGFR kinase inhibitor targeting EGFR-activating and T790M mutants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2014-1729
