Martine J. Mellor

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

UNLABELLED First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle. SIGNIFICANCE We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.

AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.

Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of Both Sensitizing and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.

Novel thienopyrimidine and thiazolopyrimidine kinase inhibitors with activity against Tie-2 in vitro and in vivo.

The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase domain is consistent with the SAR. These compounds have moderate potency in cellular assays of Tie-2 inhibition, good physical properties, DMPK, and show evidence of in vivo inhibition of Tie-2.

Irreversible Inhibition of EGFR: Modeling the Combined Pharmacokinetic–Pharmacodynamic Relationship of Osimertinib and Its Active Metabolite AZ5104

Osimertinib (AZD9291) is a potent, selective, irreversible inhibitor of EGFR-sensitizing (exon 19 and L858R) and T790M-resistant mutation. In vivo, in the mouse, it is metabolized to an active des-methyl metabolite, AZ5104. To understand the therapeutic potential in patients, this study aimed to assess the relationship between osimertinib pharmacokinetics, the pharmacokinetics of the active metabolite, the pharmacodynamics of phosphorylated EGFR reduction, and efficacy in mouse xenograft models of EGFR-driven cancers, including two NSCLC lines. Osimertinib was dosed in xenografted models of EGFR-driven cancers. In one set of experiments, changes in phosphorylated EGFR were measured to confirm target engagement. In a second set of efficacy studies, the resulting changes in tumor volume over time after repeat dosing of osimertinib were observed. To account for the contributions of both molecules, a mathematical modeling approach was taken to integrate the resulting datasets. The model was able to describe the pharmacokinetics, pharmacodynamics, and efficacy in A431, PC9, and NCI-H1975 xenografts, with the differences in sensitivity described by the varying potency against wild-type, sensitizing, and T790M-mutant EGFR and the phosphorylated EGFR reduction required to reduce tumor volume. It was inferred that recovery of pEGFR is slower after chronic dosing due to reduced resynthesis. It was predicted and further demonstrated that although inhibition is irreversible, the resynthesis of EGFR is such that infrequent intermittent dosing is not as efficacious as once daily dosing. Mol Cancer Ther; 15(10); 2378–87. ©2016 AACR.

Abstract A109: AZD9291: an irreversible, potent and selective third generation tyrosine kinase inhibitor (TKI) targeting EGFR activating (EGFRm+) and resistance (T790M) mutations in advanced lung adenocarcinoma.

The first generation EGFR TKIs gefitinib and erlotinib provide significant clinical benefit in patients with advanced lung adenocarcinoma harbouring activating EGFR mutants (EGFRm+), but patients will ultimately develop disease progression due to acquired resistance. Acquisition of the EGFR T790M mutation is the most common mechanism of drug resistance, detected in more than 50% of gefitinib/erlotinib resistant patients. Current therapeutic strategies are limited for advanced lung adenocarcinoma patients with EGFR T790M (EGFRm+/T790M), so this remains a key area of unmet need. AZD9291 (structure to be disclosed at meeting) is an oral, irreversible, third generation, selective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations. The mechanistic and functional activity of AZD9291 was characterised in vitro and in vivo across a number of cell lines harbouring various EGFR-mutations or wild type EGFR. Presented data shows AZD9291 potently inhibits EGFR phosphorylation in EGFRm+ (e.g. PC9; 500nM). Consistently, AZD9291 showed significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. In addition, AZD9291 administered once daily orally at 5mg/kg caused profound regression of tumours across EGFRm+ (PC9; 178% growth inhibition) and EGFRm+/T790M (H1975; 119% growth inhibition) tumour models in vivo, after 14 days dosing. Furthermore 5mg/kg AZD9291 was sufficient to cause significant shrinkage of EGFRm+ and EGFRm+/T790M transgenic mouse lung tumours. Tumour growth inhibition was associated with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK. Chronic long-term treatment of PC9 and H1975 xenograft tumours with AZD9291 led to a complete and sustained macroscopic response, with no visible tumours after 40 days dosing, and being maintained beyond 100 days. Furthermore, pre-clinical data also indicates that AZD9291 could target tumours that have acquired resistance to the more recently identified HER2-amplification mechanism, thus potentially extending its benefit in TKI resistant patients. Taken together, preclinical data demonstrates that AZD9291 is a potent and effective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations whilst sparing wild-type EGFR. These data support the further clinical investigation of AZD9291 in advanced EGFR mutant lung adenocarcinoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A109. Citation Format: Darren Cross, Sue Ashton, Caroline Nebhan, Cath Eberlein, M. Raymond V. Finlay, Gareth Hughes, Vivien Jacobs, Martine Mellor, Monica Red Brewer, Catherine Meador, Jonathon Orme, Paula Spitzler, Steve Powell, Amar Rahi, Paula Taylor, Richard A. Ward, Paula Daunt, Anne Galer, Teresa Klinowska, Graham Richmond, William Pao. AZD9291: an irreversible, potent and selective third generation tyrosine kinase inhibitor (TKI) targeting EGFR activating (EGFRm+) and resistance (T790M) mutations in advanced lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A109.

Abstract 3568: Characterization of AZD4547: An orally bioavailable, potent and selective inhibitor of FGFR tyrosine kinases 1, 2 and 3

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signalling axis plays an important role in normal organ, vascular and skeletal development. Deregulation of FGFR signalling through genetic modification or over-expression of the receptors (or their ligands) has been observed in numerous tumour settings, whilst the FGF/FGFR axis also plays a key role in driving tumor angiogenesis. A growing body of data demonstrates that inhibition of FGFR signalling can result in anti-proliferative and/or pro-apoptotic effects, both in vitro and in vivo, thus confirming the validity of FGFR as a therapeutic target. AZD4547 is a small molecule inhibitor which competes with ATP for binding to FGF receptors 1, 2 and 3, thus inhibiting autophosphorylation and downstream signalling. Using in vitro kinase assays, we demonstrate that AZD4547 is a potent inhibitor of FGFR9s 1,2 and 3 with IC50 values of 0.2, 2.5 and 1.8nM respectively. AZD4547 is also highly selective vs a panel of over 70 other kinases (no activity at 10μM) and importantly vs Kinase Insert Domain Receptor (KDR) (>120-fold window in cellular assays vs. FGFR2). In vitro treatment of a large panel of cell lines (>100) with AZD4547 resulted in varying anti-proliferative responses, the most potent of which were almost exclusively confined to those cell-lines in which FGFR signalling was de-regulated. Treatment of these sensitive cell lines with AZD4547 for 1hr, demonstrated potent inhibition of phosphorylation of FGF receptors and downstream substrates including p44/42 MAPK. The anti-proliferative effects of AZD4547 appear to be cell line dependent and vary between potent G1-phase cell cycle arrest and apoptotic induction. Chronic in vivo dosing of AZD4547 up to 12.5mg/kg qd was well tolerated and resulted in dose-dependent growth inhibition in a KMS11 tumour xenograft model. This in vivo anti-tumor activity is correlated with exposure to AZD4547 and associated pharmacodynamic modulation of phospho-FGFR. These data confirm the novelty of AZD4547 as a potent and selective agent for the therapeutic treatment of tumors with deregulated FGF/FGFR signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3568. doi:10.1158/1538-7445.AM2011-3568

Abstract B94: Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor.

Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics--Oct 19-23, 2013; Boston, MA Small molecule inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations in the kinase domain such as T790M is emerging as a growing clinical issue. This presentation will describe the discovery of AZD9291, an orally bioavailable, irreversible EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 0.5 μM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be discussed for the first time including the medicinal chemistry program that led to the identification of AZD9291, details of significant in vivo oral activity in pre-clinical xenograft models (including tumor regression in the L858R/T790M double mutant setting at a dose of 5 mpk) and the first disclosure of the candidate drug structure. The pre-clinical findings from this work strongly supported selection of AZD9291 as a clinical candidate, and first dose in man was achieved with AZD9291 in March 2013. View this table: Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B94. Citation Format: M. Raymond V. Finlay, Mark Anderton, Susan Ashton, Peter G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A. E. Cross, Heather L. McFarland, Martine J. Mellor, Richard A. Ward, Mike J. Waring. Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B94.

Quantitation of endogenous metabolites in mouse tumors using mass-spectrometry imaging

Described is a quantitative-mass-spectrometry-imaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysis-stabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at -80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment.

Abstract 4744: Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291

Small molecule inhibitors of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations such as T790M, (mutation of the gatekeeper residue), is recognized as a clinical issue. This presentation will describe the discovery and evolution of one of our novel chemical series, leading ultimately to the identification of AZD9291, an orally bioavailable, covalent EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 0.5 uM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be presented for the first time including a broader description of the medicinal chemistry program that led to the identification of AZD9291. We shall also present previously undisclosed work on the identification of additional distinct chemical series and an update of recent data from ongoing AZD9291 Phase I clinical studies in NSCLC patients. Citation Format: Richard A. Ward, Susan Ashton, Mark Anderton, Pete G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A E Cross, M Ray V. Finlay, Heather L. McFarland, Martine Mellor, Mike J. Waring. Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291. [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 4744. doi:10.1158/1538-7445.AM2014-4744