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Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity. A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab. EGFR D770-N771InsSVD cell phospho IC50 (µM)EGFR WT (H2073) cell phospho IC50 (µM)Fold-EGFR WT margin (cell)afatinib0.0060.00330.5osimertinib0.0940.414.4poziotinib0.00340.00351TAS64170.0230.0672.9AZ62810.0972.223 Citation Format: Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc’h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4813.

Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a >1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 > 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models. Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1207.

This paper details the SAR of 1,5-biaryl pyrrole derivatives with substituents in the 2-, 4-, and 5-positions of the benzoic acid group as EP1 receptor antagonists. Substitution at the 2-position was poorly tolerated, whereas only fluorine was tolerated at the 4-position. In contrast, a range of substituents at the 5-position were discovered which enhanced the in vitro affinity and led to compounds with promising oral exposure. Three derivatives showed efficacy in a preclinical model of inflammatory pain when dosed orally to rats.

The preliminary SAR of a series of novel 1,5-biaryl pyrrole EP1 receptor antagonists derived from compound 1 is described. Replacement of the benzyl group of 1 with isosteric groups was investigated. The most effective replacement was found to be the isobutyl group. The cyclopentylmethyl and cyclohexylmethyl groups were also effective benzyl replacements. The cyclohexylmethyl derivative 19 demonstrated the lowest metabolic clearance within this series. In addition, several high affinity substituted benzyl analogues were also identified. Compound 39 was found to have good bioavailability in rats and demonstrated efficacy in the established FCA preclinical model of inflammatory pain with a calculated ED50 of 9.2 mg/kg.

This work examines the inter-relationship between the unbound drug fractions in blood and brain homogenate, passive membrane permeability, P-glycoprotein (Pgp) efflux ratio, and log octanol/water partition coefficients (cLogP) in determining the extent of central nervous system (CNS) penetration observed in vivo. The present results demonstrate that compounds often considered to be Pgp substrates in rodents (efflux ratio greater than 5 in multidrug resistant Madin-Darby canine kidney cells) with poor passive permeability may still exhibit reasonable CNS penetration in vivo; i.e., where the unbound fractions and nonspecific tissue binding act as a compensating force. In these instances, the efflux ratio and in vitro blood-brain partition ratio may be used to predict the in vivo blood-brain ratio. This relationship may be extended to account for the differences in CNS penetration observed in vivo between mdr1a/b wild type and knockout mice. In some instances, cross-species differences that might initially seem to be related to differing transporter expression can be rationalized from knowledge of unbound fractions alone. The results presented in this article suggest that the information exists to provide a coherent picture of the nature of CNS penetration in the drug discovery setting, allowing the focus to be shifted away from understanding CNS penetration toward the more important aspect of understanding CNS efficacy.

Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.

The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

The discovery of a series of selective EP1 receptor antagonists based on a 1,2-diarylcyclopentene template is described. After defining the structural requirements for EP1 potency and selectivity, heterocyclic rings were incorporated to reduce log D and improve in vitro pharmacokinetic properties. The 2,6-substituted pyridines and pyridazines gave an appropriate balance of potency, in vivo pharmacokinetic properties and a low potential for inhibiting a range of CYP450 enzymes. From this series, GW848687X was shown to have an excellent profile in models of inflammatory pain and was selected as a development candidate.

Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A 2 with sub-nanomolar potency. Cyclopentyl fused derivative 21 , SB-480848, showed an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.

The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble, CNS penetrant inhibitor suitable for intravenous administration.