Reginald Brys

Preclinical Characterization of GLPG0634, a Selective Inhibitor of JAK1, for the Treatment of Inflammatory Diseases

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors. In a search for JAK small molecule inhibitors, GLPG0634 was identified as a lead compound belonging to a novel class of JAK inhibitors. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ∼30-fold for JAK1- over JAK2-dependent signaling. GLPG0634 dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. GLPG0634 was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of GLPG0634 in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by GLPG0634 treatment. Efficacy of GLPG0634 in the collagen-induced arthritis models was comparable to the results obtained with etanercept. In conclusion, the JAK1 selective inhibitor GLPG0634 is a promising novel therapeutic with potential for oral treatment of rheumatoid arthritis and possibly other immune-inflammatory diseases.

Discovery and Optimization of an Azetidine Chemical Series As a Free Fatty Acid Receptor 2 (FFA2) Antagonist: From Hit to Clinic

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

Triazolopyridines as Selective JAK1 Inhibitors: From Hit Identification to GLPG0634

Janus kinases (JAK1, JAK2, JAK3, and TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1, resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in phase 2B development for RA and phase 2A development for Crohns disease (CD).

Discovery of 2-[[2-Ethyl-6-[4-[2-(3-hydroxyazetidin-1-yl)-2-oxoethyl]piperazin-1-yl]-8-methylimidazo[1,2-a]pyridin-3-yl]methylamino]-4-(4-fluorophenyl)thiazole-5-carbonitrile (GLPG1690), a First-in-Class Autotaxin Inhibitor Undergoing Clinical Evaluation for the Treatment of Idiopathic Pulmonary Fibrosis

Autotaxin is a circulating enzyme with a major role in the production of lysophosphatic acid (LPA) species in blood. A role for the autotaxin/LPA axis has been suggested in many disease areas including pulmonary fibrosis. Structural modifications of the known autotaxin inhibitor lead compound 1, to attenuate hERG inhibition, remove CYP3A4 time-dependent inhibition, and improve pharmacokinetic properties, led to the identification of clinical candidate GLPG1690 (11). Compound 11 was able to cause a sustained reduction of LPA levels in plasma in vivo and was shown to be efficacious in a bleomycin-induced pulmonary fibrosis model in mice and in reducing extracellular matrix deposition in the lung while also reducing LPA 18:2 content in bronchoalveolar lavage fluid. Compound 11 is currently being evaluated in an exploratory phase 2a study in idiopathic pulmonary fibrosis patients.

Discovery of a series of imidazopyrazine small molecule inhibitors of the kinase MAPKAPK5, that show activity using in vitro and in vivo models of rheumatoid arthritis

MAPKAPK5 has been proposed to play a role in regulation of matrix metalloprotease expression and so to be a potential target for intervention in rheumatoid arthritis. We present here the identification of a series of compounds against this target which are effective in both biochemical and cell assays. The expansion of the series is described, along with early SAR and pharmacokinetics for some representative compounds.

Discovery, Structure–Activity Relationship, and Binding Mode of an Imidazo[1,2-a]pyridine Series of Autotaxin Inhibitors

Autotaxin (ATX) is a secreted enzyme playing a major role in the production of lysophosphatidic acid (LPA) in blood through hydrolysis of lysophosphatidyl choline (LPC). The ATX-LPA signaling axis arouses a high interest in the drug discovery industry as it has been implicated in several diseases including cancer, fibrotic diseases, and inflammation, among others. An imidazo[1,2-a]pyridine series of ATX inhibitors was identified out of a high-throughput screening (HTS). A cocrystal structure with one of these compounds and ATX revealed a novel binding mode with occupancy of the hydrophobic pocket and channel of ATX but no interaction with zinc ions of the catalytic site. Exploration of the structure-activity relationship led to compounds displaying high activity in biochemical and plasma assays, exemplified by compound 40. Compound 40 was also able to decrease the plasma LPA levels upon oral administration to rats.

AB0506 Identification of a Gene Signature and Response Biomarkers in Circulating Leukocytes of RA Patients After Treatment with the JAK1-Selective Inhibitor Filgotinib (GLPG0634)

Background The 4 Janus kinases (JAK1, JAK2, JAK3 and TYK2) are cytoplasmic tyrosine kinases that mediate intracellular signaling of cytokines (e.g. certain interleukins and interferons) and growth factors (e.g. erythropoietin). Filgotinib (formerly GLPG0634) is the first JAK inhibitor that displays a high selectivity for JAK1 over the 3 other JAK family members in biochemical and cellular assays. It showed a favorable safety and efficacy profile in two 4-week Phase 2a studies in rheumatoid arthritis (RA) patients. Objectives To assess the effect of RA disease effects, characterize the disease-relevant treatment responses to filgotinib and identify response biomarkers, we compared the gene expression profile of circulating leukocytes of RA patients, before and after up to 4 weeks of once-daily treatment with 200mg of filgotinib, with healthy volunteers. Methods RA patients participated in the Phase 2a Proof of concept, a randomized, double-blind, placebo-controlled study enrolling 24 patients with insufficient response to MTX. They were orally treated with placebo or 200 mg QD filgotinib for 4 weeks. Blood was sampled in PAXgene tubes at pre-dose and at the last day of treatment. Non-matched healthy volunteers were also sampled. mRNA was extracted, labeled and profiled using Affymetrix U219 micro-arrays. Data analysis was performed in R/BioConductor using linear regression models (limma). Results The leukocyte gene signature of 12 healthy subjects was first compared to the one obtained from the 24 RA patients prior to placebo or filgotinib treatment. Genes showing differential expression compared to healthy subjects allowed for definition of a disease signature, which includes novel players as well as genes already described in RA (TFPI or ITGA2B). Four weeks of treatment with filgotinib impacted the signal levels for 1005 probes (254 downregulated, 751 upregulated) in RA patient samples (p-value <0.05 and absolute log2-fold change >0.5 compared to pre-dose), while the signal levels of only 161 probes (86 down- and 75 upregulated) were impacted to the same extent in the placebo group. Several disease-relevant genes (CETP, CTSD, FSTL1, HTRA1, MYL9) were affected by filgotinib treatment. This was confirmed by qPCR. Response biomarkers could be identified by comparing strong responders and weaker responders based on percentage change in disease activity score 28 (DAS28). Functional classification of these biomarkers showed enrichment towards key RA pathways including regulation of immune cells, neutrophil recruitment, inositol phosphate metabolism and phosphatidylinositol signaling (PI3K). In addition, in the best responders, ECM-receptor interaction and focal adhesion pathways were enriched in the genes affected by filgotinib while in the weaker responders, regulation of actin cytoskeleton and TGF-beta signalling were affected by filgotinib. Conclusions Blood transcriptome analysis showed that 4-week filgotinib treatment partially reverses the disease effect and allows for the identification of treatment response biomarkers in circulating leukocytes of RA patients. Additional information to be obtained from the Darwin studies currently ongoing should provide more insights. Disclosure of Interest M. Ongenaert Grant/research support from: AbbVie, Employee of: Galapagos, S. Dupont Grant/research support from: AbbVie, Employee of: Galapagos, B. Vayssière Grant/research support from: AbbVie, Employee of: Galapagos, L. Meuleners Grant/research support from: AbbVie, Employee of: Galapagos, R. Brys Grant/research support from: AbbVie, Employee of: Galapagos, R. Galien Grant/research support from: AbbVie, Employee of: Galapagos

Abstract 1753: GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer

Receptor tyrosine kinases define a clinically relevant class of targets in the field of cancer. Here we report the discovery of a pre-clinical drug candidate directed against the EPH receptor family. Members of this family of receptor tyrosine kinases are over-expressed in diverse cancer types. GLPG1790 is a small molecule, nanomolar inhibitor of various EPH receptors kinases. The compound displayed a remarkable efficacy by once-daily oral administration in a mouse EPHA2 expressing xenograft model (MDA-MB-231). A rapid dose-dependent reduction of tumor growth was achieved, with full inhibition at the oral dose of 30 mg/kg/d. GLPG1790 efficacy at this dose was similar to that of Paclitaxel administered at its maximum tolerated dose. A series of experiments was initiated to confirm the mechanism of action of this compound. GLPG1790 inhibits human EPHA2 kinase activity with an IC 50 of 11 nM in a biochemical assay. In the human MDA-MB-231 breast cancer cell line, that expresses a high level of EPHA2 protein, GLPG1790 inhibits receptor phosphorylation with an IC 50 of 260 nM, and anchorage-independent growth with similar potency. In addition, in vivo target engagement was demonstrated in the mouse MDA-MB-231 xenograft model. GLPG1790 inhibited EPHA2 receptor phosphorylation after single oral administration of 30 and 100 mg/kg doses. The extent of the effects observed on EPHA2 phosphorylation correlated with intra-tumoral GLPG1790 concentration. Moreover, the MAPK pathway, known to be a major driver of proliferation of this cell line, was inhibited both in in vitro cellular assays and in xenograft target engagement studies. Flow cytometric analyses revealed a cell cycle arrest at the G0/G1 phase for MDA-MB-231 cells treated with GLPG1790. EPHA2 knock-down-based experiments further support the EPH-driven mode of action of GLPG1790. All together these data stimulate the development of GLPG1790 in triple negative breast cancer. This novel mechanism of action is under investigation in other cancer types overexpressing EPH9s (melanoma, pancreatic, ovarian, prostatic and colorectal cancers). Citation Format: Philippe Pujuguet, Filip Beirinckx, Carole Delachaume, Jacques Huck, Ellen Van der Aar, Reginald Brys, Luc Van Rompaey, Piet Wigerinck, Laurent Saniere. GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer. [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 1753. doi:10.1158/1538-7445.AM2014-1753

THU0116 Biological Effects of the JAK1 Selective Inhibitor GLPG0634 on Inflammation Markers in Arthritic Mice

Background Non-selective Janus kinase (JAK) inhibitors have shown long-term efficacy in treating rheumatoid arthritis (RA). However, tolerated doses and thereby efficacy are limited due to JAK2-driven side effects. Selective inhibition of JAK1 may be associated with an improved safety profile while maintaining clinical efficacy. Currently available information for JAK1 inhibitors is limited. GLPG0634 is a JAK inhibitor which displays a high selectivity for JAK1 over JAK2 in human whole blood assays. It is currently being developed for RA treatment and displayed good efficacy and tolerability in a 4-week Phase 2a study. Objectives Characterize the mode of action of a JAK1-selective inhibitor by measuring the impact of GLPG0634 on JAK pathway activity and inflammation parameters in blood and paws in the mouse CIA model. Methods Mice with established collagen-induced arthritis (CIA) were treated with GLPG0634 (50 mg/kg po, bid) or etanercept (10 mg/kg ip, 3 times a week). Disease progression was evaluated using clinical score and histological parameters. Expression of genes related to JAK pathways and markers of inflammation were measured in hind paws as well as in circulating leukocytes (WBC) using qRT-PCR. Luminex technology was used to measure a panel of 35 cytokines and chemokines in mouse serum. Results Oral treatment with GLPG0634 decreased inflammation in CIA in mice as measured by clinical scores and paw histology, with the same efficacy as etanercept. In paws of arthritic mice, an increase of 20- to more than 200-fold in the expression of inflammation markers (IL-6, IL-1β, TNFα, CXCL1) was found. A 30% to 45% decrease of their expression was observed in GLPG0634-treated arthritic mice. A similar expression pattern was observed for the osteoclast differentiation factor RANKL and for proteases linked to inflammation (MMP3, MMP13). These findings corroborate the bone and cartilage protective effects of GLPG0634 observed by histological analysis of paws. In addition, decreases from 30 to 70% in serum levels of cytokines and chemokines (IL-6, IP-10/CXCL10, MCP-1) in GLPG0634-treated arthritic mice confirmed the immunomodulation and anti-inflammatory effects. Of interest, the increased expression of JAK1-induced genes Mx1 and Mx2 in arthritic mice both in WBC and hind paws was abolished after treatment of animals with GLPG0634, whereas expression of the JAK2-dependent gene HRH4 was not altered by GLPG0634 treatment. These findings show that the decrease in inflammation markers was associated with inhibition of JAK1 but not JAK2. Conclusions These data show that oral GLPG0634 administration reduces inflammation in the mouse CIA model to the same extent as parenteral etanercept. Effects on several inflammation markers in paws and serum demonstrate the anti-inflammatory activity of GLPG0634. These effects were achieved through selective JAK1 inhibition. These data establish that selective JAK1 inhibition by GLPG0634 is sufficient to mediate strong efficacy in an established arthritis model and provide some useful potential markers to follow pathology progression in arthritic mice and humans. Disclosure of Interest B. Vayssiere Employee of: Galapagos SASU, S. de Vos Employee of: Galapagos NV, D. Merciris Employee of: Galapagos SASU, M. Auberval Employee of: Galapagos SASU, S. Dupont Employee of: Galapagos SASU, N. Vandeghinste Employee of: Galapagos NV, L. Lepescheux Employee of: Galapagos SASU, P. Clement-Lacroix Employee of: Galapagos SASU, P. Delerive Employee of: Galapagos SASU, L. van Rompaey Employee of: Galapagos NV, R. Brys Employee of: Galapagos NV, R. Galien Employee of: Galapagos SASU

THU0138 Glpg0634m1, A Major Metabolite of the Jak1-Selective Inhibitor Glpg0634, is Also Jak1-Selective and Efficient in the Rat CIA Model

Background GLPG0634 is a JAK inhibitor displaying a high selectivity for JAK1 over JAK2 in human whole blood assays. It is currently being developed as an oral treatment for rheumatoid arthritis (RA) and showed good efficacy and tolerability in two 4-week Phase 2a RA studies. GLPG0634 has a major metabolite, GLPG0634m1, with a half-life which might contribute to its clinical efficacy in RA patients. Objectives Characterize the pharmacological properties of the GLPG0634m1, its activity on JAK-driven pathways and its efficacy in the rat CIA (collagen-induced arthritis) model. Methods Selectivity of the metabolite GLPG0634m1 was measured in vitro in radiometric recombinant kinase assays and ex vivo in whole blood assays (human, dog and monkey) by monitoring STAT phosphorylation by flow cytometry. Rats with established arthritis were treated with the GLPG0634m1 molecule (60 mg/kg po, QD) or etanercept (10 mg/kg ip, 3 times a week). The clinical score as well as histological parameters were used to monitor/quantify disease progression. Plasma concentration of the molecule was quantified by LC-MS/MS. Results Biochemical analysis of potency of GLPG0634m1 on recombinant JAK kinases showed that this compound is 10-fold less active against JAK1 and JAK2 than GLPG0634 with IC50s of 546 nM and 624 nM, respectively. Potency on JAK3 and TYK2 over 3 μM indicates that this molecule is more selective for JAK1 and JAK2 compared to JAK3 and TYK2. In human whole blood assays (WBA), GLPG0634m1 inhibited a JAK1-dependent event (IL-6-induced STAT1 phosphorylation) with an IC50 of 11.9 μM and a JAK2-dependent event (GMCSF-induced STAT5 phosphorylation) with an IC50 exceeding 100 μM, revealing the >10-fold selectivity of this molecule for JAK1 over JAK2. The JAK1 potency was confirmed with assays using IL2-induced STAT5 and IFNα-induced STAT1 phosphorylation, respectively triggering the JAK1/JAK3 and JAK1/TYK2 pathways. When given orally to rats with established arthritis, GLPG0634m1 displayed a pronounced efficacy comparable to etanercept, strongly reducing the impact of disease on e.g. paw swelling and Larsen score. These effects were confirmed at histological level with decreased pannus severity, bone and cartilage lesion as well as cell infiltration indexes. Plasma levels of GLPG0634m1 exceeded its WBA-derived JAK1 IC50 but were far below its WBA-derived JAK2 IC50, implying that the metabolite efficacy in the CIA model is driven by JAK1 inhibition, as observed previously with the parent molecule. Conclusions While displaying a lower potency compared to its parent molecule, GLPG0634m1 has a similar JAK1 selectivity. Oral administration of this metabolite, albeit at a 10-times higher dose than GLPG0634, reduces inflammation in the rat CIA model to the same extent as parenteral etanercept, an effect that is supported by JAK1 inhibition but appears independent of JAK2 inhibition. These findings, together with the high exposure and long half-life of this metabolite observed in phase 1 and phase 2 clinical studies in humans, strongly suggest that it may contribute to the clinical efficacy of the parent compound GLPG0634 in RA. Disclosure of Interest : C. Belleville-Da-Costa Grant/research support: Abbvie, Employee of: Galapagos SASU, D. Merciris Grant/research support: Abbvie, Employee of: Galapagos SASU, B. Vayssière Grant/research support: Abbvie, Employee of: Galapagos SASU, N. Houvenaghel Grant/research support: Abbvie, Employee of: Galapagos NV, A. Monjardet Grant/research support: Abbvie, Employee of: Galapagos SASU, L. Lepescheux Grant/research support: Abbvie, Employee of: Galapagos SASU, S. Dupont Grant/research support: Abbvie, Employee of: Galapagos SASU, T. Christophe Grant/research support: Abbvie, Employee of: Galapagos NV, M. Borgonovi Grant/research support: Abbvie, Employee of: Galapagos SASU, P. Clément-Lacroix Grant/research support: Abbvie, Employee of: Galapagos SASU, C. Menet Grant/research support: Abbvie, Employee of: Galapagos NV, L. Van Rompaey Grant/research support: Abbvie, Employee of: Galapagos NV, R. Brys Grant/research support: Abbvie, Employee of: Galapagos NV, R. Galien Grant/research support: Abbvie, Employee of: Galapagos SASU DOI 10.1136/annrheumdis-2014-eular.4291