Strong reversal of the lung fibrosis disease signature by autotaxin inhibitor GLPG1690 in a mouse model for IPF Maté Ongenaert (Mechelen, Belgium), Maté Ongenaert, Sonia Dupont, Roland Blanqué, Reginald Brys, Ellen van der Aar, Bertrand Heckmann ERS - European Respiratory Society International Congress 2016. Session: Therapeutic horizons: novel targets and pharmacological models Background and objectives GLPG1690 is a novel potent autotaxin (ATX) inhibitor shown to be efficacious in the mouse bleomycin (BLM) lung fibrosis model. Here, we analyze the impact of GLPG1690 on the gene expression signature in mouse fibrotic lung tissue. Methods Lung fibrosis was induced by intranasal administration of BLM. Animals were treated with GLPG1690 or vehicle.Whole superior right lung was used for RNA extraction. Full transcriptome analysis was performed using the Agilent SurePrint G3 mouse chip. Analysis was performed using empirical Bayes methods and linear models. Public human IPF expression data were re-analyzed. Results GLPG1690 strongly reduced lung fibrosis as shown by reduction of Ashcroft scores and collagen content. Microarray analysis of the lungs revealed that GLPG1690 strongly reversed the impact of gene expression caused by BLM (367 out of the 2375 probes). As GLPG1690 treatment affects 395 probes, this treatment effect is highly relevant in the model. Gene clusters affected by BLM treatment and reverted by GLPG1690 are related to extracellular matrix (such as Tnc and Spp1), collagen (Col3a1) and cytokines/chemokines (Cxcl12). Several of the affected genes are known to be involved in the development or progression of lung fibrosis in IPF patients. Conclusions These data provide further mechanistic understanding of the efficacy of ATX inhibition in a pre-clinical lung fibrosis model, highlighting a role for extracellular matrix and inflammation biology. These data strongly suggest that GLPG1690 may be beneficial in treating IPF patients and support its evaluation in a clinical study

1. ©Copyright 2016 Galapagos NV
Strong reversal of the lung fibrosis disease
signature by autotaxin inhibitor GLPG1690
in a mouse model for IPF
Maté Ongenaert, PhD
Senior Scientist Bioinformatics
Sonia Dupont, Roland Blanqué, Reginald Brys, Ellen van der Aar, Bertrand Heckmann
ERS London, UK
September 6th 2016

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Disclosure
• I and other authors have the following real or percieved conflicts of
interest that relate to this presentation:
 All employees of Galapagos NV (Mechelen, Belgium) or Galapagos SASU (Romainville,
France)

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Outline
Autotaxin/LPA pathway background
Transcriptomics studies in BLM model
Mouse bleomycin (BLM) model - GLPG1690
performance and target engagement
Summary and outlook

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GT2645
ATX
Lysophosphatidylcholine (LPC)
Lysophosphatidic acid (LPA)
Autotaxin (ATX)
Target background
• Also known as ENPP2, secreted enzyme
• Widely expressed (highest in brain, lymph nodes, kidney and testis)
• Converts LPC to the bioactive lipid mediator LPA
• “LPA” covers a family of related molecules (i.e. LPA18:2, LPA20:4)
• ATX is main source of LPA in blood

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LPA signalling
• LPA acts through at least six distinct
G-protein-coupled receptors (LPA1–6)
• LPA controls activities like migration,
contraction & survival
• Studies with KO of LPA receptors
indicate a role in
 bone development
 fertility/reproduction
 neurogenesis
 formation of blood- and
lymphatic vessels
Stoddard and Chun, 2015

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ATX/LPA pathway and IPF
Preclinical and translational validation
• In modeled disease: bleomycin-exposed mice
 increased LPA in BALF (Tager et al, 2008)
 ATX levels increased in lungs (Oikonomou, 2012)
 inhibition of ATX attenuated lung fibrosis in mice (Oikonomou, 2012)
 genetic or pharmacological inhibition of LPAR1 attenuates development of
pulmonary fibrosis (Tager et al, 2008; Swaney et al, 2010)
• In patients with idiopathic pulmonary fibrosis (IPF)
 LPA levels increased in BALF (Tager et al, 2008)
 ATX levels elevated in lung (Oikonomou et al, 2012)
 LPA increased in exhaled breath condensate (Montesi et al, 2014)

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GLPG1690 reduces fibrosis in BLM models, reduces LPA levels in BALF
Mouse bleomycin model for lung
fibrosis (prophylactic)
Relative levels of LPA species in BALF of
mice subjected to BLM treatment
GLPG1690 in vivo activity
LPApeakarea/
LPA17:0peakarea
vehicle
BLM + vehicle
BLM + pirfenidone 50 mg/kg bid
BLM + ‘1690 30 mg/kg bid
Group
Ashcroftscore

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GLPG1690 - bleomycin model
Global transcriptomics (PCA)
vehicle
bleomycin
GLPG1690
Lung microarray profiling: GLPG1690 protects for BLM induced effects
on a global transcriptome level

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GLPG1690 - bleomycin model
Most affected genes
vehicle BLM ‘1690
• Top-40 most differentially expressed
genes
 perfect separation of the BLM cluster
from sham
 GLPG1690 partially reverses the BLM
effect

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GLPG1690 - bleomycin model
Relevance in model
‘1690 (log2 Fold Change)
BLM(log2Foldchange)
Strong negative correlation
(Spearman R=-0.74)
between BLM effect and
GLPG1690 effects
‘1690
(DOWN)
BLM
(UP)
1088
probes
259 18
^ Probes with both:
- |log2(FC)|>1
- FDR< 1%

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GLPG1690 - bleomycin model
Functions
Color: log2 FC
Area: log2 FC * -log10(p-value)
Tnc
Tnc
Cxcl12
Smad6
Smad6
Col5a1
Serpine2
Col1a2 Serpine2
Ednrb
Ccl2
immune response cytokines, Jak-Stat, TGFb
Extracellular matrix – focal adhesion
Col1a2
• Affected functions and
pathways by BLM and
counteracted by
GLPG1690 relevant in
BLM model and IPF
biology:
 extracellular matrix
(Tnc, Spp1)
 several collagens
(Col3a1, Col5a1)
 cytokines / chemokines
(Cxcl12, Ccl2)

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GLPG1690 - bleomycin model
BLM/GLPG1690 – IPF relevance
• Many of the genes identified are relevant in IPF and/or altered in
expression in IPF patients (assessed in 4 public transcriptomics studies)
• 52 genes upregulated in BLM and at least in ¾ IPF vs. normal
studies and strongly counteracted by ‘1690
• BLM: log2(FC)>1
• counteracted by ‘1690: log2(FC)< -1
• IPF studies: log2(FC)>0.5 in at least ¾ studies
Gene Symbols BLM mouse models GLPG1690 Human IPF (public data)
Mouse Human
BLM
this study
BLM
GSE40151
GLPG1690
this study
GSE32537 GSE10667 GSE53845 Yang et al.
Cxcl12 CXCL12 3.09 1.11 -1.4 1.17 2.21 1.96 1.5
Col3a1 COL3A1 2.51 1.15 -0.71 1.4 2.82 1.71 2.19
Spp1 SPP1 4.19 1.59 -1.56 1.61 3.67 3.49 1.77
Tnc TNC 4.6 2.54 -2.22 1.13 0.86 1.89 1.49

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GLPG1690: potent and selective inhibitor of autotaxin
Pharmacological and literature data support positioning in IPF
Strong anti-fibrotic effects in the mouse bleomycin model
IPF-related gene expression signature clearly affected after
GLPG1690 administration
Exploratory phase IIa study in IPF patients ongoing (FLORA)

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Many thanks to all involved Galapagos colleagues