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

This information is current as Luc Van Rompaey, René Galien, Ellen M. van der Aar, of October 2, 2021. Philippe Clement-Lacroix, Luc Nelles, Bart Smets, Liên Lepescheux, Thierry Christophe, Katja Conrath, Nick Vandeghinste, Béatrice Vayssiere, Steve De Vos, Stephen Fletcher, Reginald Brys, Gerben van 't Klooster, Jean H. M. Feyen and Christel Menet

J Immunol published online 4 September 2013 Downloaded from http://www.jimmunol.org/content/early/2013/09/04/jimmun ol.1201348 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/09/04/jimmunol.120134 Material 8.DC1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 4, 2013, doi:10.4049/jimmunol.1201348 The Journal of Immunology

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

Luc Van Rompaey,* Rene´ Galien,† Ellen M. van der Aar,* Philippe Clement-Lacroix,† Luc Nelles,* Bart Smets,* Lieˆn Lepescheux,† Thierry Christophe,* Katja Conrath,* Nick Vandeghinste,* Be´atrice Vayssiere,† Steve De Vos,* Stephen Fletcher,*,1 Reginald Brys,* Gerben van ’t Klooster,* Jean H. M. Feyen,* and Christel Menet*

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 of many type I and type II inflammatory receptors. In a search for JAK small molecule Downloaded from 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, http://www.jimmunol.org/ bone and cartilage degradation, and levels of inflammatory 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. The Journal of Immunology, 2013, 191: 000–000.

he JAKs are cytoplasmic tyrosine critical for in- IFN-g, IL-12, IL-23, and GM-CSF (2, 5). Hence, JAKs have been tracellular signal transduction of many cytokines, growth targeted for their therapeutic potential in immune-inflammatory T factors, and hormones. Four human JAKs have been de- disorders. In fact, small-molecule JAK inhibitors proved effica- by guest on October 2, 2021 scribed: JAK1, JAK2, JAK3, and TYK2. JAKs bind to the intra- cious in a range of animal disease models and have already shown cellular moieties of type I and type II receptors, and JAK homo- or promise in the clinic for organ transplant rejection, rheumatoid heterodimers become activated upon ligand binding. The JAKs arthritis (RA), psoriasis, dry eye disease, myelofibrosis, inflamma- phosphorylate each other followed by phosphorylation of tyrosine tory bowel disease, and asthma (5–10). JAK2-mediated side effects residues on the intracellular domains of the receptors. These phos- observed in clinical trials such as anemia, neutropenia, and throm- phorylated residues serve as docking sites for STAT transcription bocytopenia appear to be the main cause for not fully exploiting the factors. JAK phosphorylation of the STAT proteins results in their pharmacodynamic potential of these JAK inhibitors (11–13). Recent nuclear translocation and provides transcriptional output for the findings suggest that JAK1 dominates JAK1/JAK3/gc signaling, cytokine ligands (1, 2). JAKs play important roles in the functioning suggesting that JAK1 inhibition might be largely responsible for of the immune system. Mouse and human genetics studies linked the in vivo efficacy of JAK inhibitors in immune-inflammatory deficiencies of JAK1 and JAK3 to severe combined immune defi- diseases (14–16). These results indicate that a selective JAK1 in- ciency and TYK2 to increased susceptibility to infections (3, 4). hibitor could provide an increased therapeutic window allowing for JAK2 serves signal transduction for inflammatory cytokines such as higher dosing and efficacy while avoiding dose-limited pharma- cology as observed for the pan-JAK inhibitors. To exploit the therapeutic potential of JAK1 for the treatment of *Departement of In Vitro Pharmacology, Galapagos NV, 2800 Mechelen, Belgium; and †Departement of In Vivo Pharmacology and Translational Sciences, Galapagos immune-inflammatory diseases, we set out to identify selective JAK1 SASU, 93230 Romainville, France inhibitors. One of the lead compounds, GLPG0634, was shown to 1Current address: BioFocus, Essex, U.K. selectively inhibit JAK1-dependent signaling in cellular and whole Received for publication May 14, 2012. Accepted for publication July 29, 2013. blood assays (WBAs) and showed remarkable efficacy in collagen- Address correspondence and reprint requests to Luc Van Rompaey, Galapagos NV, induced arthritis (CIA) disease models for RA in both mouse and rat. Generaal de Wittelaan L11 A3, 2800 Mechelen, Belgium. E-mail address: luc. [email protected] The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: CII, collagen type II; CIA, collagen-induced ar- Small-molecule inhibitors thritis; Cmax, maximum blood concentration; EPO, ; OSM, ; PRL, prolactin; RA, rheumatoid arthritis; RT, room temperature; siRNA, small Focused kinase collections were sourced from BioFocus (Essex, U.K.). interfering RNA; WBA, whole blood assay. GLPG0634 was synthesized by Galapagos medicinal chemists. and were sourced from Shanghai Haoyuan Chemexpress (Shang- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 hai, China) and Charnwood Molecular (Loughborough, U.K.), respectively.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201348 2 JAK1 INHIBITOR GLPG0634 IS EFFICACIOUS IN RODENT CIA MODELS

Biochemical assays Knockdown experiments. HeLa and HCT116 cells obtained from the American Type Culture Collection were transfected with 50 nM ON-TARGETplus IC determination. Recombinant JAK1, TYK2 (Invitrogen), JAK2, and JAK3 50 SMARTpool small interfering RNA (siRNA) for human JAK1, JAK2, JAK3, or (Carna Biosciences) were used to develop activity assays in 50 mM HEPES TYK2, or with nontargeting or GAPDHnegative control siRNAs (Dharmacon) (pH 7.5), 1 mM EGTA, 10 mM MgCl ,2mMDTT,and0.01%Tween20.The 2 using Lipofectamine RNAiMAX transfection reagent from Invitrogen. amount of JAK protein was determined per aliquot, maintaining initial ve- Four days after transfection cells were starved overnight and stimulated locity and linearity over time. The ATP concentration was equivalent to 43 with IL-6/sIL-6R (both 250 ng/ml) for 20 min and pSTAT1 levels were the experimental K value and the substrate concentration (ULight-conjugated m determined using AlphaScreen technology (PerkinElmer) according to the JAK-1(Tyr1023) peptide; PerkinElmer) corresponded to the experimentally manufacturer’s protocol. determined Km value. After 90 min incubation at room temperature (RT), the amount of phosphorylated substrate was measured by addition of 2 nM T cell differentiation studies. PBMCs were isolated from buffy coats of europium-anti-phosphotyrosine Ab (PerkinElmer) and 10 mM EDTA in healthy donors (Blood Transfusion Center, Red Cross, Leuven, Belgium) using density gradient centrifugation on Lymphoprep. Naive CD4+ T cells Lance detection buffer (PerkinElmer). Compound IC50 values were deter- were further isolated by depletion of non–T helper and memory CD4+ mined by preincubating the with compound at RT for 60 min, prior + to the addition of ATP. T cells using a naive CD4 T cell isolation II (Miltenyi Biotec). Isolated naive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 mg/ml) K determination. Dissociation constants were determined at Proteros d and anti-CD28 (5 mg/ml) Abs in the presence of cytokines that drive Biostructures (Martinsried, Germany). Proprietary fluorescently labeled ATP differentiation into Th1, Th2, or Th17 Th subsets. For Th1 cell polarization, mimetics with fast dissociation rates (PRO13, PRO14, and PRO13 for JAK1, cells were cultured in the presence of 10 mg/ml anti–IL-4 Ab (MAB204; JAK2, and JAK3, respectively) were incubated with JH1 domains of purified R&D Systems), 10 ng/ml IL-2 (R&D Systems), and 10 ng/ml IL-12 (R&D JAKs in 20 mM MOPS (pH 7.5), 1 mM DTT, 0.01% Tween 20, and 500 mM Systems) (17). For Th2 cell polarization, cells were cultured in the presence hydroxyectoine (JAK3 only) for 30 min. Compounds (concentrations ranging of 10 mg/ml anti–IFN-g Ab (Becton Dickinson), 25 ng/ml IL-4 (R&D from 520 pM to 1.1 mM) were added in 100% DMSO and time depen- Systems), and 10 ng/ml IL-2 (17). For Th17 cell polarization, a mix of the dency of reporter displacement was measured. IC values corresponding 50 following cytokines was used: 10 ng/ml IL-6 (R&D Systems), 10 ng/ml to 50% probe displacement were obtained and K values were calculated Downloaded from d IL-1b (R&D Systems), 1 ng/ml TGF-b (PeproTech), and 100 ng/ml IL- according to the Cheng–Prusoff equation. 23 (R&D Systems) (18). To monitor effects of compounds on T cell dif- ferentiation, compounds were added at indicated concentrations at the start Cellular assays of T cell differentiation. After 5 d, RNA was extracted using an RNeasy Mini STAT6 phosphorylation induced by IL-4. THP-1 cells (ATCC TIB-202) kit (Qiagen), reverse transcribed, and the extent of Th subset differentiation were preincubated with compound at RT for 1 h, incubated with IL-4 was monitored by determining expression of IFN-g (Th1 marker), IL-13 (10 ng/ml) at RT for 60 min, and processed for flow cytometry. Cells (Th2 marker), or IL-17F (Th17 marker) using real-time PCR on the ViiA7 were fixed in Cytofix/Cytoperm (BD Biosciences) buffer and permeabilized thermocycler with predesigned TaqMan Assay-on-Demand gene expression http://www.jimmunol.org/ in Phosflow perm buffer III (BD Biosciences) on ice for 30 min. After primer/probe sets (Applied Biosystems). Gene expression was normalized to blocking (Fc blocking reagent; Miltenyi Biotec), pSTAT6 was detected with 18S and expressed as DCt values, with DCt = Ctgene 2 Ct18S or expressed as 2 mouse anti-human PE-labeled anti-pSTAT6 Ab (BD Biosciences). relative mRNA level of specific gene expression as obtained using the 2 DCt STAT5 phosphorylation induced by IL-2, IL-3, and erythropoietin. NK-92 method. cells (ATCC CRL-2407) were IL-2 starved overnight, preincubated with Human WBAs compound at 37˚C for 1 h, stimulated with IL-2 (1 ng/ml) at RT for 20 min, and processed for AlphaScreen analysis. TF1 cells (Sigma-Aldrich, catalog Human blood was collected from healthy volunteers, who gave informed no. 93022307) were starved overnight in RPMI 1640 with 0.1% FBS, pre- consent, into sodium heparin vacutainer tubes by venipuncture. After in- incubated with compound at RT for 1 h, stimulated with IL-3 (30 ng/ml) at cubation with compounds at 37˚C for 30 min, blood was triggered with RT for 20 min, and processed for AlphaScreen analysis. UT-7-erythropoietin either recombinant human IL-6 (10 ng/ml; R&D Systems), recombinant by guest on October 2, 2021 (EPO) cells (EPO-dependent derivative of UT-7; Centocor) were preincubated human IL-2 (4 ng/ml; R&D Systems), universal IFN-a (1000 U/ml; PBL with compound at RT for 1 h, stimulated with EPO (1 U/ml) for 20 min, Biomedical Laboratories), recombinant human GM-CSF (20 pg/ml; Pepro- and processed for AlphaScreen analysis. pSTAT5 was measured using Tech), or vehicle (PBS plus 0.1% [w/v] BSA) at 37˚C for 20 min and treated AlphaScreen technology essentially according to the manufacturer’s protocol. with prewarmed 13 lysis/fix buffer (BD Biosciences) to lyse RBCs and fix STAT1 phosphorylation induced by IFN-a and IFN-g. STAT1 U2OS cells leukocytes. Cells were permeabilized with 100% methanol and incubated (Invitrogen, catalog no. K1469) were preincubated with compound at 37˚C with anti-pSTAT1 and anti-CD4 (IL-6– and IFN-a–triggered samples), anti- for 1 h, treated with 30,000 U/ml IFN-aB2 (PBL IFN source, catalog no. pSTAT5 and anti-CD4 Abs (IL-2–triggered samples), or anti-pSTAT5 and 11115-1) or 20 ng/ml IFN-g (PeproTech, catalog no. 300-02, lot 010827) anti-CD33 Abs (GM-CSF–triggered samples) (all Abs were from BD Bio- at 37˚C for 1 h, lysed (lysis buffer containing 2 nM Tb-Ab; Invitrogen) ac- sciences) at 4˚C for 30 min, washed once with PBS 13, and analyzed on cording to manufacturer’s protocol, and incubated at RT for 60 min. pSTAT1 a FACSCanto II flow cytometer. was detected by time-resolved fluorescence resonance energy transfer (Per- kinElmer). Pharmacokinetics STAT5 phosphorylation induced by prolactin. 22Rv1 cells (ATCC CW22Rv) Formulations. GLPG0634 was formulated in polyethyleneglycol 200/0.9% were starved overnight, preincubated with compound, triggered with prolactin NaCl (60/40; v/v) for i.v. administration and in 0.5% (v/v) methylcellulose (PRL; 500 ng/ml human PRL for 20 min), lysed in 10 mM Tris-HCl (pH 7.5), for oral administration for all in vivo studies described. Compound purity 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 50 mM NaF, 30 mM sodium was .95% as measured by HPLC. pyrophosphate, 10% glycerol buffer containing phosphatase/protease inhibitor Animals. Male Sprague Dawley rats (180–200 g) and CD1 mice (23–25 g) cocktails, and centrifuged. Cell lysate (180 mg) was used for STAT5 im- were obtained from Janvier and Harlan (France), respectively. Two days munoprecipitation (anti-STAT5 polyclonal Abs, Santa Cruz Biotechnology, before administration of compound, rats underwent surgery to place a cath- C-17; protein A-Sepharose beads, GE Healthcare). Total and phosphorylated eter in the jugular vein under isoflurane anesthesia. Animals were deprived STAT5 were measured by densitometric analysis after Western blotting (anti- of food for at least 16 h before oral dosing until 4–6 h after. Before oral pSTAT5 mAb, AX-1, Advantex). dosing, animals were deprived of food for at least 12 h before compound IL-3/JAK2–induced proliferation of Ba/F3 cells. Ba/F3 cells (provided by administration until 4 h after administration. All in vivo experiments were V. Lacronique, Paris, France), which are dependent on IL-3 and JAK2 sig- carried out in a dedicated pathogen-free facility (22˚C). Animal care was in naling, were incubated with compound at 37˚C for 40 h, after which cell accordance with the French guidelines about the use of animals in scien- proliferation was analyzed by measuring ATP content (ATPlite; PerkinElmer). tific research. All procedures involving animals, including housing and Oncostatin M-induced STAT1 reporter assay in HeLa cells. HeLa cells (ATCC care, method of euthanasia, and experimental protocols, were conducted in CCL-2) were transfected with a pSTAT1 reporter construct (Panomics, catalog accordance with a code of practice established by the local ethical com- no. LR0127). After transfection for 24 h, cells were incubated for 1 h with mittee (Galapagos). compound and triggered with oncostatin M (OSM; 33 ng/ml; Peprotech, Pharmacokinetic studies. GLPG0634 was orally dosed as a single esoph- catalog no. 300-10). After 20 h incubation, the cells were lysed and luciferase ageal gavage at 5 mg/kg (dosing volume of 5 ml/kg) and i.v. dosed as a bolus activity was determined with the luciferase SteadyLite kit according to the via the caudal vein at 1 mg/kg (dosing volume of 5 ml/kg). In the rat study, supplier’s recommendations (PerkinElmer, catalog no. 6016759). In parallel, each group consisted of three rats and blood samples were collected via the b-galactosidase activity was measured in the presence of 4 mg/ml 2-nitro- jugular vein. In the mouse study, each group consisted of 21 mice (n = 3/time phenyl b-D-galactopyranoside (Sigma-Aldrich, catalog no. N1127). point) and blood samples were collected by intracardiac puncture under The Journal of Immunology 3 isoflurane anesthesia. Lithium heparin was used as anticoagulant and blood analysis and related to the total number of cells. Statistical analysis was wastakenat0.05,0.25,0.5,1,3,5,and8h(i.v.route)and0.25,0.5,1,3,5,8, performed by using an ANOVA unpaired test with *p , 0.05, **p , 0.01, and24h(bymouth). and ***p , 0.001 versus vehicle group. GLPG0634 plasma concentrations were determined by liquid chroma- Gene expression and pharmacokinetic/pharmacodynamic modeling in rodent tography–tandem mass spectrometry with a lower limit of quantification of blood. Fed male Sprague Dawley rats (180–200 g) received four daily oral 2 ng/ml. Pharmacokinetic parameters were calculated by noncompartmental doses of vehicle or GLPG0634 at 1 or 10 mg/kg. Blood samples were taken analysis using WinNonlin software (Pharsight, version 5.2). on the fourth day of dosing at 0.5, 1, 2, 6, and 24 h after dose via decapitation (n = 3/time point). GLPG0634 plasma concentrations were determined by In vivo pharmacology liquid chromatography–tandem mass spectrometry. Each blood sample (2 ml) Rodent CIA models. Animals. Dark Agouti rats (females, 7–8 wk old) and was divided in two tubes and either left untreated (control) or treated with DBA/1J mice (male, 6 wk old) were obtained from Janvier (Laval, France). 520 U/ml rat IFN-a at 37˚C for 1 h. RBCs were lysed (buffer EL; Qiagen) Materials. CFA and IFA were purchased from Difco (Detroit, MI). Bovine and the WBCs pelleted, dissolved, and homogenized in 350 mLbufferRLT collagen type II (CII) was obtained from Chondrex (Redmond, WA). All (Qiagen). Total RNA was extracted using the QIAamp RNA Blood Mini kit other reagents used were of reagent grade and all solvents were of analytical (Qiagen) and 500 ng was reverse transcribed using TaqMan RT (Applied grade. Biosystems) with oligo(dT) priming. Five microliters of 53 diluted cDNA preparations was used for real-time quantitative PCR (TaqMan technology, CIA. One day before the start of the experiment, CII solution (2 mg/ml) was using a StepOnePlus thermocycler; Applied Biosystems) with gene-specific prepared with 0.05 M acetic acid and stored at 4˚C. Just before the im- munization, equal volumes of IFA and CII were mixed by a homogenizer probes and primers designed according to standard procedures. in a precooled glass bottle in an ice water bath. For rat CIA experiments, For the analysis of gene expression in mouse whole blood cells (cir- the emulsion (0.2 ml) was injected intradermally at the base of the tail at culating leukocytes), blood was sampled in RNAprotect tubes (Qiagen) and day 1 and again at day 8. This immunization method was modified from processed using the RNeasy protect animal blood kit (Qiagen). Total RNA (300 ng) was reverse transcribed using a high-capacity cDNA synthesis kit published methods (19). The in vivo efficacy of GLPG0634 was deter- (Applied Biosystems) with random hexamers. Quantitative PCR reactions mined after daily oral administration for a period of 14 d after onset of Downloaded from disease (average clinical score at onset, 2.5 6 0.3; 10 rats/treatment group) were performed using QuantiFast SYBR Green PCR Master mix (Qiagen) b over the dose range 0.1–30 mg/kg. The TNF-a blocker etanercept (Wyeth and gene-specific primer pairs for -actin (Eurogentec) and QuantiTect Pharmaceuticals, Taplow, U.K.) was administered three times per week at primer assays for all other genes analyzed (Qiagen). Reactions were car- 10 mg/kg by i.p. injection. A fully active dose was reported to require ried out with a denaturation step at 95˚C for 5 min followed by 40 cycles repeated dosing in the 3–9 mg/kg range (20). In our model of Dark Agouti (95˚C for 10 s, 60˚C for 1 min) in a ViiA7 real-time PCR system (Applied Biosystems). Real-time PCR data for each target gene were expressed as female rats, disease normalization was reached for 10 mg/kg etanercept DCt, corresponding to Ct obtained for the gene of interest normalized with dosed three times a week i.p. as measured by clinical score, inflammation, b http://www.jimmunol.org/ bone resorption, pannus, and cartilage damage. At day 7 or 11, 200 ml the Ct of the -actin gene. blood was collected by retro-orbital puncture with lithium heparin as an- Gene expression analysis in mouse paws. Hind paws were dissected by ticoagulant at predose and 1, 3, and 6 h (n = 2 or 3/time point) for steady- cutting above the ankle joint and removing the digits. The remaining tissue state pharmacokinetics analysis. At sacrifice, hind paws were removed for was transferred into 2 ml homogenization tubes (Quality Scientific Plastics) x-ray analysis and histological examination. A Tukey multiple comparison containing 1 mm zirconium beads (BioSpec Products) and 750 mlTRIzol test was used to perform a meta-analysis of three studies carried out for reagent (Invitrogen). Tissue samples were homogenized using the Precellys GLPG0634. The score of each rat was divided by the average score ob- 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) tained for vehicle in the same readout and study and multiplied by 100. programmed to shake samples three times for 30 s at 5300 rpm with 5-s Relative scores were averaged per readout for all animals present in all pauses between the homogenization steps. Total RNA was isolated as studies that received the same dose. For mouse CIA experiments, the IFA/ recommended by the TRIzol supplier and further purified with NucleoSpin 96 RNA (Macherey-Nagel) according to the manufacturer’s instructions. CII emulsion (0.2 ml) was injected intradermally at the base of the tail at by guest on October 2, 2021 day 1 and again at day 21. This immunization method was modified from RNA yields were determined by spectrophotometry at 260/280 nm. Re- published methods (20). The in vivo efficacy of GLPG0634 was deter- verse transcription of RNA and quantitative PCR were performed as de- mined after daily oral administration for a period of 14 d after onset of scribed above. For statistical analysis, a two-way ANOVA followed by a disease (average clinical score at onset, 2.4 6 0.6; 10 mice/treatment Dunnett post hoc test versus the CIA-vehicle group was performed. group) over the dose range 50 mg/kg twice daily. Administration of eta- Analyte measurement in mouse sera. Quantification of analytes in mouse nercept and pharmacodynamic and pharmacokinetic analyses were essen- sera was performed at Myriad RBM using mouse CytokineMAP A v1.0, tially carried out as described for the rat CIA model. mouse CytokineMAP B v1.0, and mouse CytokineMAP C v1.0. Clinical assessment of arthritis. The individual clinical score was ob- tained by summing the scores recorded for each limb. Arthritis was scored from grade 0 to 4 according to an established method (19). Results Larsen score. X-ray imaging was performed for the hind paws of each To identify new JAK1 inhibitors, a kinase-focused collection of individual animal. An identity number was randomly assigned to each of the ∼10,000 small molecules was screened in a JAK1 biochemical images, and the severity of bone erosion was ranked by two independent assay. From this screen a triazolopyridine series was identified as blinded scorers as described earlier (21). a tractable hit series. Establishment of a detailed structure-activity Histology. For rat CIA studies, the hind paws were collected, fixed in 3.7% relationship in this series led to the identification of GLPG0634 (v/v) formaldehyde at 4˚C for 48 h, and decalcified in RDO solution (Eurobio, Paris, France) for 3 d. Three series of 5-mm sections were made as one of the lead compounds of the series. Characterization of at 100-mm intervals from the paw middle part and stained with Goldner’s GLPG0634 at the biochemical level indicated a selective inhibi- trichrome. The inflammation status and skeletal tissue damage were ex- tion of JAK1 and JAK2 over JAK3 and TYK2 with a rank order amined by light microscope (Provis, Olympus). For mouse CIA studies, potency of JAK1 ∼ JAK2 . TYK2 . JAK3 (Table I). IC values the hind paws were collected, fixed in 3.7% (v/v) formaldehyde at room 50 temperature for 24 h, and decalcified in Osteosoft solution (VWR Inter- determined in inhibition assays correlated with Kd national, Val de Fontenay, France) for 24 d. Each paw was cut in two parts values determined in ligand displacement assays (Table I). according the sagittal axis and embedded in paraffin. Six series of 4-mm Several cellular assay set-ups were applied to elucidate the thick sections were collected. One series of sections was stained with potency and the JAK selectivity profile in a cellular environment. safranin O-light green for morphological examination and disease scoring. Cell lines were preincubated with GLPG0634 and treated with Disease was scored by a double-blinded evaluation as described by Lin et al. (21). Statistical analysis was performed by using a Kruskall–Wallis cytokines that employ different JAK heterodimeric or JAK2 homo- test followed by a Dunn multiple comparison post hoc test with *p , 0.05, dimeric complexes for signaling. GLPG0634 inhibited IL-2– and IL- , , **p 0.01, and ***p 0.001 versus vehicle group. Immunohistochem- 4–induced JAK1/JAK3/gc signaling and IFN-aB2–induced JAK1/ istry was performed with Abs detecting macrophage (anti-F4/80, sc-59171; TYK2 type II signaling most potently. IC values ranged Santa Cruz Biotechnology) and T cell (anti-CD3; Dako) markers. Bio- 50 tinylated horse anti-rabbit Ab and avidin-linked peroxidase (Vectastain from 150 to 760 nM (Table I). IFN-g– and OSM-induced JAK1/ Universal Elite ABC kit; Vector Laboratories) were used to detect the binding JAK2 signaling mediated by type II and gp130 receptor complexes stained in brown color. Immuno-positive cells were quantified by image and IL-3–induced JAK2/bc signaling were inhibited with low 4 JAK1 INHIBITOR GLPG0634 IS EFFICACIOUS IN RODENT CIA MODELS

Table I. Potency and selectivity of GLPG0634 in JAK biochemical dependent signaling in a cellular environment and is more JAK1 assays selective than are tofacitinib and baricitinib. GLPG0634 also inhibited the STAT5 phosphorylation activated by IL-2 and STAT1 6 Recombinant Human Kinase IC50 (nM, SEM; n =2–4) Kd (nM) phosphorylation by IFN-a, although with a lower potency than JAK1 10 6 0.8 11 STAT1 phosphorylation by IL-6. Tofacitinib inhibited IFN-a JAK2 28 6 5.4 32 signaling with similar potency as IL-6/STAT1 signaling but in- JAK3 810 6 180 300 hibited IL-2/STAT5 signaling with higher potency (Table III). These TYK2 116 6 39 ND observations confirm the JAK1/JAK3 inhibition profile of tofacitinib IC50 values for inhibition of recombinant JAK1, JAK2, JAK3, and TYK2 by in cells reported by Meyer et al. (9). The WBA data confirm the GLPG0634 were determined by measuring the incorporation of phosphate into an ULight-JAK-1(Tyr1023) peptide using an europium-labeled anti-phosphotyrosine Ab. observations from the cellular assays showing that GLPG0634 The Kd values of GLPG0634 on JAK1, JAK2, and JAK3 were determined by mea- preferentially inhibits JAK signaling complexes containing JAK1. suring the competition of GLPG0634 with a fluorescently labeled ATP mimetic. IC50 To further explore the consequences of cytokine inhibition, the values corresponding to 50% probe displacement were obtained and Kd values cal- culated according to the Cheng–Prusoff equation. effect of GLPG0634 toward Th1 and Th2 differentiation was ana- ND, Not determined lyzed by measuring IFN-g or IL-13 mRNA expression levels (Fig. 2). As expected, GLPG0634 dose-dependently inhibited the dif- ferentiation of Th2 cells mediated by IL-4, a cytokine that signals micromolar potencies (Table I). JAK2 homodimer–mediated sig- through JAK1 and JAK3. GLPG0634 also inhibited Th1 differen- naling induced by EPO or PRL was inhibited with the lowest tiation with similar potencies of 1 mM or lower. Although Th1 potency. IC50 values could not be determined accurately but were commitment is initiated by IL-12, a cytokine signaling through .10 mM. Inhibition of JAK3 and TYK2 is unlikely to contribute TYK2 and JAK2, the primary effect of GLPG0634 on Th1 differ- Downloaded from to GLPG0634s inhibition of JAK/STAT signaling in view of the entiation is likely through inhibition of JAK1/JAK2-mediated sig- potency difference measured in biochemical assays between JAK1 naling of IFN-g. Inhibition of IFN-g signaling alone was previously and JAK2 versus JAK3 and TYK2 (Table II). Hence, GLPG0634 found to be sufficient to reduce T-bet, the master transcriptional preferentially inhibits JAK/STAT signaling involving JAK1 than regulator critical for Th1 differentiation, and IFN-g in Th1 cells and JAK2 kinase in a cellular context. To test the relative contributions itwasproposedasamechanismexplaining the inhibitory effect of JAK1 versus JAK2 in a physiological relevant model, GLPG0634 of tofacitinib on Th1 cell differentiation (3). GLPG0634 was also http://www.jimmunol.org/ was tested for inhibition of cytokine-induced STAT phosphorylation tested for the ability to inhibit Th17 differentiation driven by a in human WBAs. There was a particular interest in comparing GM- mixture of TGF-b, IL-23, and proinflammatory cytokines (IL-6 CSF/STAT5 versus IL-6/STAT1 signaling in myeloid and T cells, and IL-1b), all shown to be essential for human Th17 differentiation respectively. GM-CSF relies on a JAK2 homodimer for intracellular (18). GLPG0634 inhibited Th17 differentiation under these con- signaling, and IL-6–mediated phosphorylation of STAT1 is JAK1- ditions, although with lower potency than Th1 and Th2 differen- dependent, as shown for T cells by Ghoreschi et al. (22). Similar tiation (Fig. 2). results were obtained by applying an siRNA knockdown approach The pharmacokinetics of GLPG0634 was determined in rats and in HeLa and HCT116 cells. Transfection with siRNAs targeting mice. Following i.v. administration, GLPG0634 displayed a low by guest on October 2, 2021 the individual JAK family members resulted in a knockdown of to moderate plasma clearance, depending on the species tested messenger RNAs for JAK1, JAK2, JAK3, and TYK2 by .70%. (Table IV). In mice, the total clearance represented 58% of the However, at the functional level we could only demonstrate a liver blood flow, and in rats it represented 41%. Steady-state volume significant reduction of IL-6–induced STAT1 phosphorylation of distribution ranged from ∼1.7 l/kg in rats to 6 l/kg in mice, (Fig. 1), indicative for a JAK1-driven signaling event. A large implying a significant species difference in volume of distribution. potency difference was measured for GLPG0634 in the IL-6 Half-life observed after oral administration was 1.7 h in mice and versus GM-CSF WBAs (629 nM versus 17.5 mM; Table III), 3.9 h in rats. Following oral administration, the absolute bioavail- corresponding to an ∼30-fold selectivity for inhibition of JAK1- ability was moderate in rats (45%) and high in mice (∼100%). over JAK2-dependent signaling (Table III). Two clinical JAK Because GLPG0634 was well exposed in several species, in- inhibitors, tofacitinib and baricitinib, were also tested in both assays hibition of JAK signaling in vivo was evaluated in a biomarker assay. and showed high potency. A respective 10- and 3-fold JAK1 over In WBCs, Mx2 mRNA levels are modulated by IFN-a–JAK1/TYK2 JAK2 selectivity was determined even though both compounds signaling (23, 24). Rats were dosed orally with GLPG0634 and both equipotently inhibited JAK1 and JAK2 in biochemical assays (9, basal and ex vivo IFN-a–induced Mx2 mRNA levels were measured. 10). These data indicate that GLPG0634 selectively inhibits JAK1- A statistically significant reduction of normalized Mx2 mRNA

Table II. Potency and selectivity of GLPG0634 in cellular assays

JAKs Involved Cell Type Trigger Readout IC50 (nM) pIC50 6 SEM n JAK1–JAK3 THP-1 IL-4 pSTAT6 154, 203 6.75 6 0.06 2 JAK1–JAK3 NK-92 IL-2 pSTAT5 148, 757, 367 6.46 6 0.12 3 JAK1–TYK2 U2OS IFN-aB2 pSTAT1 494, 436 6.33 6 0.03 2 JAK1–JAK2 HeLa OSM STAT1 reporter 1,045 6.01 6 0.07 4 JAK1–JAK2 U2OS IFN-g pSTAT1 3,364 5.47 1 JAK2 TF-1 IL-3 pSTAT5 3,524 5.45 1 JAK2 BaF3 IL-3 Proliferation 4,546 5.34 6 0.04 3 JAK2 UT7-EPO EPO pSTAT5 .10,000 .52 JAK2 22Rv1 PRL pSTAT5 .10,000 .52

IC50 values in cellular assays were determined by plotting the compound concentration versus the effect on the readouts mentioned. The pIC50 is defined as the negative of the log10 of the compound concentration having a half maximal effect on the readout. The Journal of Immunology 5

mg/kg. Because high efficacy was obtained at 3 mg/kg, two follow- up studies were carried out with lower doses. Even at 0.1 mg/kg, a statistically significant effect was observed in the clinical score from the fifth day of dosing onward (Fig. 4B). Data obtained from each of the three studies were normalized to the corresponding vehicle data, and a meta-analysis of the three studies was per- formed (Fig. 4A, 4C, 4D). A meta-analysis of the steady-state phar- macokinetics from the rat CIA studies showed dose-proportional increases of maximum blood concentration (Cmax) and area under curve between 0.3 and 30 mg/kg and correlated with the dose- dependent efficacy (Fig. 4A). A rapid absorption was observed for GLPG0634, with maximal plasma levels achieved ∼1 h after dosing, for all dose levels tested. Half-life is ∼4–5 h and is in- dependent of dose level. A dose-dependent effect was obtained in all pharmacodynamic readouts. The doses of 1, 3, and 10 mg/kg GLPG0634 reduced the clinical score to the same extent as eta- nercept at endpoint. Different from the GLPG0634 doses tested, the high dose of etanercept normalized the clinical score already from the start of dosing (Fig. 4B). Statistically significant reduc- tion of the clinical score at endpoint was obtained for all doses Downloaded from (Fig. 4C). Protection from bone damage was evidenced by a dose- dependent reduction of the Larsen score obtained after x-ray analysis of the hind paws, with significant effect from 3 mg/kg and onward (Fig. 4D). Similar efficacy was obtained for GLPG0634 as for etanercept. Histological analysis of the rat paws was per- formed in a specific region of interest including the talus, navicular, http://www.jimmunol.org/ and cuneiform bones (Fig. 4E–H). As compared with the vehicle control group (Fig. 4E), etanercept and GLPG0634 groups (Fig. 4E, 4G, 4H) showed a marked reduction of the infiltration of inflam- FIGURE 1. JAK1 silencing inhibits IL-6–induced STAT1 activation. IL- matory cells while protecting the articular cartilage and bone from 6/sIL-6R–induced STAT1 phosphorylation after JAK1, JAK2, JAK3, TYK2, GAPDH (GAPD), or nontargeting (non-T) siRNA transfection in HeLa 1 mg/kg onward. (A) or HCT116 (B) cells was assessed using AlphaScreen technology. Data Confirmation of the therapeutic potential of GLPG0634 in CIA represent the means 6 SD from four data points (duplicate measurements in the rat was provided by studying the compound in the similar for two independent siRNA transfections). RLU, relative luminescence model in the mouse. In addition to the parameters measured in the by guest on October 2, 2021 units. rat, mouse blood and paw samples were taken with the purpose of obtaining detailed insight in the mechanism of action of GLPG0634 levels was measured upon dosing 1 and 10 mg/kg with more pro- in vivo. Dose selection was performed in a dose range–finding nounced reductions at the dose of 10 mg/kg (Fig. 3). For the basal experiment (data not shown), and a dose of 50 mg/kg twice daily Mx2 mRNA levels, a hysteresis between compound peak plasma orally was selected for the mechanistic studies. Fig. 5A shows that levels and maximal inhibition of basal Mx2 mRNA levels was the 50 mg/kg dose provided full protection against inflammation observed as expected. as judged by analysis of the clinical score of paws. Histological The therapeutic potential of GLPG0634 for RA was evaluated in analysis of the mice paws showed that GLPG0634 protected bone a therapeutic setting in the rat collagen-induced arthritis (CIA) model, and cartilage from degradation (Fig. 5B). Immunohistochemistry a well-accepted animal model for RA (19, 25–27). GLPG0634 was performed on the same samples showed that GLPG0634 effectively dosed once daily by oral gavage, initially at doses of 3, 10, and 30 reduced infiltration of T cells (CD3+ cells) and macrophages (F4/

Table III. Potency and selectivity determination of GLPG0634 in human WBAs

Assay IL-6/pSTAT1 IL-2/pSTAT5 IFN-a/pSTAT1 GM-CSF/pSTAT5 JAK involved JAK1 JAK1 . JAK3 JAK1–TYK2 JAK2 Cell type CD4+ CD4+ CD4+ CD33+ JAK1 versus Compounds pIC50 6 SEM (IC50 [nM]; n) JAK2 Selectivity GLPG0634 6.201 6 0.092 5.747 6 0.043 5.948 6 0.021 4.758 6 0.214 28 (629; 7) (1,789; 5) (1,127; 6) (17.453; 7) Tofacitinib 7.130 6 0.119 7.473 6 0.033 7.120 6 0.040 6.130 6 0.179 10 (74; 16) (33; 9) (76; 6) (740; 7) INCB028050 7.631 6 0.169 ND ND 7.195 6 0.055 3 (23.4; 4) (63.8; 6)

+ + IC50 values for inhibition of cytokine-induced STAT phosphorylation were determined by measuring STAT phosphorylation in CD4 cells or CD33 cells using flow cytometry in whole blood. The pIC50 (defined as the negative of the log10 of the compound concentration having a half maximal effect on the readout) was measured for each volunteer and averaged. Data are represented as mean pIC50 6 SEM and IC50s are derived from the mean pIC50 values. JAK1 versus JAK2 selectivity (outer column) was determined by comparing potencies measured in the IL-6/pSTAT1 and GM-CSF/pSTAT5 assays. ND, Not determined. 6 JAK1 INHIBITOR GLPG0634 IS EFFICACIOUS IN RODENT CIA MODELS

80+ cells) in the paw (Fig. 5C). The effects on clinical score, bone and MCP-1 (Fig. 5E). These observations indicate that GLPG0634 and cartilage protection, and cell infiltration were similar to the might affect inflammatory cytokine signaling and chemoattraction results obtained by etanercept. Gene expression studies were of T cells and monocyte/macrophages by reducing these cytokine carried out in WBCs and paws. Expression levels were increased and chemokine levels. At the mechanistic level, the reduction of in arthritic versus healthy animals for all genes tested (Fig. 5D, 5F, Mx1 and Mx2 mRNA levels by GLPG0634 was also observed in 5G). In paws, GLPG0634 reduced the levels of inflammatory and mouse paws (Fig. 5F), as observed in the rat (Fig. 3). Of interest, metalloprotease genes previously linked to disease progression, the changes in Mx1 and Mx2 gene expression in WBCs were not explaining the beneficial role of GLPG0634 in the mouse CIA altered by etanercept treatment (Fig. 5G), showing that GLPG0634 model (Fig. 5D). The mRNA levels of RANKL were also reduced specifically impacts JAK1 signaling. in line with the decrease in the bone lesion score, suggesting that GLPG0634 might protect against bone degradation by reducing Discussion the formation and activity of osteoclasts. Cytokine levels in sera In recent years significant advances have been made in under- from the CIA mice were measured using Luminex technology. As standing the link between the different JAK family members and for the gene expression studies, the levels of the protein markers their involvement in autoimmune, inflammatory, and oncological under study were raised in the serum of diseased versus healthy diseases. The first generation of small-molecule inhibitors has animals, with the exception of stem cell factor (Fig. 5E, Supple- further substantiated the therapeutic potential of JAK inhibitors in mental Table I). GLPG0634 decreased the serum levels of all cyto- the aforementioned diseases. In this study we examined GLPG0634, kines and chemokines measured, including IL-6, IP-10, XCL1, a novel and selective JAK inhibitor that demonstrates selectivity for JAK1 in a cellular environment. GLPG0634 was identified in a kinase-focused library screen and belongs to the triazolopyridine Downloaded from compound class. Characterization of GLPG0634 at the biochemical level indicated a selective inhibition of JAK1 and JAK2 over JAK3 and TYK2, whereas cellular and WBAs revealed a selectivity for JAK1- over JAK2-dependent signaling in a cellular environment. GLPG0634 efficiently blocks cytokine-induced signaling cascades involving JAK1 in several cell lines as well as in human primary http://www.jimmunol.org/ cells. Moreover, Th1, Th2, and Th17 differentiation driven by cy- tokine cocktails, including JAK1-dependent cytokines such as IL-2, IL-4, and IL-6, is also inhibited by GLPG0634 (17, 18). These in vitro findings translate to pharmacodynamic readouts in rodents showing that JAK1 signaling is blocked in vivo as measured by a reduction of Mx2 mRNA levels. Furthermore, GLPG0634 dose- dependently reduces inflammation, cartilage, and bone degrada- tion in the CIA model in rats and mice. by guest on October 2, 2021 The biochemical selectivity profile of GLPG0634 (rank order of potency JAK1 ∼ JAK2 . TYK2 . JAK3) was a poor predictor of the selectivity determined in cellular and WBAs. TYK2 enzyme activity in biochemical assays was inhibited with 11- and 4-fold lower potency versus JAK1 and JAK2, whereas JAK3 enzyme activity was inhibited with a much lower potency. This difference did not translate into a higher cellular potency of inhibiting INF- a/JAK1/TYK2 signaling versus IL-2/JAK1/JAK3 signaling (Tables II, III). It indicates that JAK1 inhibition is in large part responsible for the potency of GLPG0634 in a cellular environment. A JAK inhibitor likely requires equipotent inhibition of JAK1 and TYK2 or JAK3, or even inverse selectivity for the latter to surpass potency derived from JAK1 inhibition. This is demonstrated for tofacitinib, which shows 3-fold selectivity for JAK3 over JAK1 in biochemical assays and inhibits IL-2–induced STAT5 phosphory- lation twice more potently than IL-6–induced STAT1 phosphory- lation (Table III) (9). The observation that GLPG0634 revealed a high selectivity for JAK1 over JAK2 in cellular and human WBAs was unexpected. Interestingly, a similar selectivity shift was FIGURE 2. GLPG0634 inhibits the differentiation of Th1, Th2, and observed for tofacitinib but not for baricitinib when testing these Th17 cells. Naive human CD4+ T cells (N) were stimulated with anti-CD3/ molecules in parallel with GLPG0634 (Table III). This is re- anti-CD28 Abs in the absence (V) or presence of 10, 1, or 0.1 mM markable, as tofacitinib and baricitinib share the same chemical GLPG0634 (10, 1, 0.1) or 10 mM tofacitinib (T) using either Th1, Th2, or scaffold and show similar potencies toward JAK1 and JAK2 in Th17 polarizing conditions as described in Materials and Methods. On day biochemical assays (9, 10, 28). At present no univocal explanation 5, the expression of lineage-specific markers was assessed by quantitative for the discrepancy between the biochemical and cellular/whole RT-PCR. Results were normalized using 18S transcripts and represent relative expression measured on duplicate data points 6 SD. Effect on blood JAK inhibition profiles can be provided. A potential ex- Th1, Th2, and Th17 cell differentiation was evaluated by quantification of planation may be linked to the differences between the bio- IFN-g mRNA, IL-13 mRNA, or IL-17F mRNA expression. Data are chemical and cellular assay formats. First, the biochemical assay representative of independent experiments performed with naive CD4+ relies on kinase activity of a purified truncated protein containing T cells isolated from two donors. the C-terminal quarter of the JAK proteins, including the JH1 The Journal of Immunology 7

Table IV. Rodent pharmacokinetics for GLPG0634

Mouse Rat

Parameter (Unit) 1 mg/kg i.v. 5 mg/kg Orally 1 mg/kg i.v. 5 mg/kg Orally

Cmax or Co (ng/ml) 637 920 1407 (28) 310 (33) Tmax (h) 0.5 2.2 (0.5–5) Area under curve: 0–24 h 347 1893 739 (2) 1,681 (8) (ng/h/ml) T1/2 (h) 2.5 1.7 1.6 (3) 3.9 Cl (l/h/kg) 2.9 1.4 (2) Vss (l/kg) 6 1.8 (3) F (%) ∼100 45 Pharmacokinetics of GLPG0634 in mouse and rat (mean values of n = 3 [CV]) after a single oral (5 mg/kg) or i.v. (1 mg/kg) administration. kinase domain. In contrast, in a cellular environment wild-type induce subtle changes in the three-dimensional space of the ATP- full-length JAKs comprise the regulatory JH2 pseudokinase do- binding pockets of the JAKs, leading to differential kinase activity main, Src homology 2 domains (JH3–JH4), and the amino ter- and/or protein–protein interactions. Crystallography of JAK proteins minal (NH2) FERM domain (JH4–JH7). Additionally, JAKs are harboring more than the JH1 kinase domain in complex with small Downloaded from part of a larger complex, including the cytoplasmic receptor tails, molecule inhibitors will provide more insight here. STATs, and other proteins (2, 4). Second, the endogenous JAKs Oral administration of GLPG0634 in the mouse and rat resulted are subject to posttranslational modifications such as phosphory- in good plasma exposure. GLPG0634 plasma levels in the rat could lation. A differential tyrosine phosphorylation status can give rise be correlated with reduction of Mx2 mRNA levels in WBCs to different IC50 values as exemplified for the non- versus mono- reflecting target engagement and inhibition of JAK/STAT signaling or diphosphorylated forms of the TYK2 kinase domain (29). in vivo. Additional testing of GLPG0634 in vivo in rodent CIA Third, differential negative or positive feedback mechanisms for models in a therapeutic setting revealed that daily oral adminis- http://www.jimmunol.org/ JAK1- versus JAK2-dependent signaling by means of phosphatases, tration resulted in a dose-dependent reduction of inflammation and members of the SOCS or SH2B families, can have a different im- protected bone and cartilage from degradation. In view of the pact on the amplitude and kinetics of JAK enzyme activity and pathological roles of Th1 effector T cells in chronic inflammation signaling output (30–33). Finally, different small molecules might and autoimmune disorders and the dose-dependent reduction of by guest on October 2, 2021

FIGURE 3. Pharmacokinetics/pharmacodynamics modeling of GLPG0634 in healthy male Sprague Dawley rats. Mx2 mRNA levels in WBCs and GLPG0634 plasma levels were determined at various time points after administration of four daily oral doses of 1 mg/kg (A, B) or 10 mg/kg (C, D) GLPG0634 in one experiment. Three animals were used per time point. Mx2 mRNA fold inhibition levels were calculated versus vehicle-treated samples after normalization to b-actin mRNA (left y-axis). GLPG0634 plasma levels are depicted on the right y-axis. (A and C) Fold inhibition of basal Mx2 mRNA levels. (B and D) Fold inhibition of ex vivo IFN-a–induced Mx2 mRNA levels. *0.05 . p . 0.01 versus vehicle, Student t test. 8 JAK1 INHIBITOR GLPG0634 IS EFFICACIOUS IN RODENT CIA MODELS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. GLPG0634 dose-dependently prevents disease progression in the therapeutic rat CIA model. (A) Meta-analysis of plasma exposure of GLPG0634 at steady-state. Total concentrations are provided on the y-axis. (B) Treatment was started 18 d after the first collagen injection and after randomization of animals in treatment groups (day 0) for 15 d. The clinical score readout is shown for the CIA study that included the lowest doses of GLPG0634. (C and D) Meta-analysis of combined data obtained for the three studies after 15 d of treatment. E, etanercept; V, vehicle. GLPG0634 doses are indicated on the x-axis. The numbers of animals used to obtain a meta-analysis score for V, E, 0.1, 0.3, 1, 3, 10, and 30 mg/kg were 29, 10, 20, 20, 29, 20, 10, and 29, respectively. *p , 0.05, **p , 0.01, ***p , 0.001 versus vehicle, Student t test. (C) Difference between the clinical score at day 0 and day 15. (D) Larsen score: bone degradation determined by x-ray analysis at end point. (E–H) Histological analysis of rat hind paws was performed for vehicle (E), etanercept (F), GLPG0634 (1 mg/kg) (G), and GLPG0634 (10 mg/kg) (H) treatment groups. Sagittal sections comprising the talus (T), navicular (N), and the cuneiform (C) bones were stained with Goldner trichrome. Original magnification 320. Despite a remaining inflammation (black asterisk) seen for all animals, the articular cartilage and bone were well preserved (black arrows) in the two groups dosed with GLPG0634 when compared with CIA-vehicle group (red arrow).

Th1 differentiation by GLPG0634 in vitro, its efficacy in the CIA mouse experimental allergic encephalomyelitis model of multiple models is in line with these in vitro observations (34). Quantitative sclerosis (Supplemental Fig. 1). Because high efficacy in this PCR analysis of disease-related inflammatory markers in blood model likely requires passage through the blood–brain barrier, and paws showed a pronounced reduction of these markers by the observation that GLPG0634 is a substrate for P-glycoprotein GLPG0634. This was confirmed at the protein level by measuring (efflux ratio in Caco2 permeability assay decreased from 16 to 6 in cytokine levels in sera of arthritic mice treated with GLPG0634 the presence of verapamil; data not shown) could explain its mod- versus vehicle-treated animals. Histological and x-ray (Larsen erate activity. score) analysis of mice and rat paws revealed reduced bone and Observing efficacy for GLPG0634 in the rat CIA model at doses cartilage degradation coinciding with reduced infiltration by T cells as low as 0.1 and 0.3 mg/kg was surprising. This is unlikely due to and macrophages as shown in the mouse CIA study. The efficacy off-target effects in view of the clean profile of GLPG0634 in kinase of GLPG0634 in the murine CIA models and the changes in the panels. GLPG0634 showed .100-fold selectivity over other kinases disease-related biomarkers are of similar magnitude as the relatively representing the human kinome (175 kinases tested) with the ex- high dose of etanercept. Differing from the efficacy observed in ception of FLT3, FLT4, and CSF1R, for which selectivity still was rodent CIA models, only a moderate efficacy was obtained in the .25-fold (Supplemental Table II). Speculative explanations include The Journal of Immunology 9

and pharmacodynamic data correlated well. The Cmax levels measured for the 1 and 3 mg/kg doses of GLPG0634 in the rat pharmacokinetics/pharmacodynamics and CIA in vivo studies (240–844 nM; Figs. 3A, 3B, 4A) were close to or exceeded the JAK1-dependent IL-6/pSTAT1 human WBA IC50 value (623 nM; Table III). Preliminary evidence obtained by measuring a JAK1 biomarker in a phase I clinical trial for GLPG0634 indicated that target engagement in humans can be measured for doses corre- sponding to the 1 mg/kg dose from the CIA and Mx2 in vivo studies (35). A similar conclusion was reached for baricitinib when com- paring WBA IC50 values with efficacy in a rat arthritis model (10). Also, the clinically relevant dose of 5 mg twice daily for tofacitinib resulted in Cmax levels in human volunteers (41–52 ng/ml) that are ∼2-fold above the JAK1-dependent WBA IC50 value (23 ng/ml or 74 nM; Table III) and will not completely inhibit JAK1 and/or JAK3 for most of the day (36). Hence, incomplete inhibition of JAK1 and the wide range of cytokine signaling it serves can provide therapeutic efficacy, thereby decreasing the risk of potential JAK1-mediated side- effects such as immune suppression. The high selectivity of GLPG0634 for JAK1 versus JAK2 ob- Downloaded from served in vitro is supported by a number of observations made in patient studies. After dosing GLPG0634 for 10 d to healthy vol- unteers up to 450 mg once daily, no relevant findings on hema- tology (including reticulocytes), biochemistry (including cholesterol and lipids), or other safety parameters (electrocardiogram, vital signs) were noted (37). At this dose, JAK1 signaling was suppressed for http://www.jimmunol.org/ 24 h whereas JAK2 signaling was not influenced (37). Moreover, dosing GLPG0634 to 24 RA patients for 4 wk at daily doses of 200 mg showed efficacy on ACR20 scores and on the secondary endpoints of DAS28 and serum C-reactive protein levels without adverse events (38). Instead of anemia, which could have been indicative for inhibition JAK2 signaling impairing hematopoiesis, a small increase in hemoglobin levels was noted as expected with improvement in disease. by guest on October 2, 2021 In conclusion, GLPG0634 is a promising drug candidate for the future treatment of autoimmune and inflammatory disorders such as RA.

Acknowledgments We acknowledge the expert technical or editorial assistance of Ce´cile Belleville, Marie Christine Cecotti, Christelle David, Franc¸ois Gendrot, Didier Merciris, Alain Monjardet, Isabelle Orlans, Isabelle Parent, Laetitia Perret, Emanuelle Wakselmann (affiliated with Galapagos SASU) and of Kara Bortone, Annick Hagers, Annelies Iwens, Daisy Liekens Koen Smits, Maarten Van Balen, Kris Van Beeck (affiliated with Galapagos NV). We FIGURE 5. GLPG0634 is efficacious in a mouse therapeutic CIA model. also thank Sonia Dupont (Galapagos SASU), Filip Beirinckx (Galapagos Mice that developed arthritis were orally treated with vehicle (orange lines NV), Prof. V. Goffin (Faculte´ de Me´decine Necker, Paris, France) and and bars), GLPG0634 twice a day at 50 mg/kg (dark green lines and bars), or collaborators for contributions to cellular assay development and/or com- i.p. with etanercept three times a week at 10 mg/kg (blue lines and bars). (A) pound testing. AbbVie has provided funding to Galapagos for development Clinical score readout. *p , 0.05, **p , 0.01, ***p , 0.001 versus vehicle, of GLPG0634. Student t test. (B) Bone and cartilage lesion scores obtained by histological analysis of hind paws. (C) Immunohistochemical analysis of infiltrating CD3+ Disclosures + TcellsandF4/80 macrophages in hind paws. (D) Expression of inflammation All authors are employees of the Galapagos group (which includes BioFo- markers (IL-6, TNFa,IL-1b), bone degradation marker (RANKL), and in- cus) and are eligible to receive stock options. flammation-linked proteases (MMP3, MMP13) measured 4 h after GLPG0634 administration (intact animals are symbolized by light purple bars). (E) Luminex measurement of serum concentration of several markers of inflam- References mation (IL-6, IP-10/CXCL10, XCL1/lymphotactin, MCP-1/CCL2) 4 h after 1. O’Sullivan, L. A., C. Liongue, R. S. Lewis, S. E. Stephenson, and A. C. Ward. the first compound administration. (F) Quantitative PCR analysis of JAK1- 2007. signaling through the Jak-Stat-Socs pathway in disease. induced genes Mx1 and Mx2 in paws after a single compound administration Mol. Immunol. 44: 2497–2506. G 2. Vainchenker, W., A. Dusa, and S. N. Constantinescu. 2008. JAKs in pathology: and in circulating leukocytes at the end of the 2-wk treatment ( ). role of Janus kinases in hematopoietic malignancies and immunodeficiencies. Semin. Cell Dev. Biol. 19: 385–393. 3. Ghoreschi, K., A. Laurence, and J. J. O’Shea. 2009. Janus kinases in immune tissue-specific compound accumulation, active metabolites, or even cell signaling. Immunol. Rev. 228: 273–287. 4. O’Shea, J. J., M. Pesu, D. C. Borie, and P. S. Changelian. 2004. A new modality other mechanisms, but a final explanation remains to be determined. for immunosuppression: targeting the JAK/STAT pathway. Nat. Rev. Drug When GLPG0634 was dosed at 1 mg/kg or higher pharmacokinetic Discov. 3: 555–564. 10 JAK1 INHIBITOR GLPG0634 IS EFFICACIOUS IN RODENT CIA MODELS

5. O’Shea, J. J., and R. Plenge. 2012. JAK and STAT signaling molecules in im- 21. Lin, H. S., C. Y. Hu, H. Y. Chan, Y. Y. Liew, H. P. Huang, L. Lepescheux, munoregulation and immune-mediated disease. Immunity 36: 542–550. E. Bastianelli, R. Baron, G. Rawadi, and P. Cle´ment-Lacroix. 2007. Anti- 6. Milici, A. J., E. M. Kudlacz, L. Audoly, S. Zwillich, and P. Changelian. 2008. rheumatic activities of histone deacetylase (HDAC) inhibitors in vivo in collagen- Cartilage preservation by inhibition of 3 in two rodent models of induced arthritis in rodents. Br.J.Pharmacol.150: 862–872. rheumatoid arthritis. Arthritis Res. Ther. 10: R14. 22. Ghoreschi, K., M. I. Jesson, X. Li, J. L. Lee, S. Ghosh, J. W. Alsup, J. D. Warner, 7. Kudlacz, E., M. Conklyn, C. Andresen, C. Whitney-Pickett, and P. Changelian. M. Tanaka, S. M. Steward-Tharp, M. Gadina, et al. 2011. Modulation of innate 2008. The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in and adaptive immune responses by tofacitinib (CP-690,550). J. Immunol. 186: a murine model of pulmonary eosinophilia. Eur. J. Pharmacol. 582: 154–161. 4234–4243. 8. Changelian, P. S., M. E. Flanagan, D. J. Ball, C. R. Kent, K. S. Magnuson, 23. Batusic, D. S., T. Armbrust, B. Saile, and G. Ramadori. 2004. Induction of Mx-2 W. H. Martin, B. J. Rizzuti, P. S. Sawyer, B. D. Perry, W. H. Brissette, et al. in rat liver by toxic injury. J. Hepatol. 40: 446–453. 2003. Prevention of organ allograft rejection by a specific in- 24. Pulverer, J. E., U. Rand, S. Lienenklaus, D. Kugel, N. Zietara, G. Kochs, hibitor. Science 302: 875–878. R. Naumann, S. Weiss, P. Staeheli, H. Hauser, and M. Ko¨ster. 2010. Temporal 9. Meyer, D. M., M. I. Jesson, X. Li, M. M. Elrick, C. L. Funckes-Shippy, and spatial resolution of type I and III interferon responses in vivo. J. Virol. 84: J. D. Warner, C. J. Gross, M. E. Dowty, S. K. Ramaiah, J. L. Hirsch, et al. 8626–8638. 2010. Anti-inflammatory activity and neutrophil reductions mediated by the 25. Firestein, G. S. 2003. Evolving concepts of rheumatoid arthritis. Nature 423: JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis. J. 356–361. Inflamm. (Lond.) 7: 41. 26. Smolen, J. S., D. Aletaha, M. Koeller, M. H. Weisman, and P. Emery. 2007. New 10. Fridman, J. S., P. A. Scherle, R. Collins, T. C. Burn, Y. Li, J. Li, M. B. Covington, therapies for treatment of rheumatoid arthritis. Lancet 370: 1861–1874. B. Thomas, P. Collier, M. F. Favata, et al. 2010. Selective inhibition of JAK1 and 27. Hegen, M., J. C. Keith, Jr., M. Collins, and C. L. Nickerson-Nutter. 2008. Utility JAK2 is efficacious in rodent models of arthritis: preclinical characterization of of animal models for identification of potential therapeutics for rheumatoid ar- INCB028050. J. Immunol. 184: 5298–5307. thritis. Ann. Rheum. Dis. 67: 1505–1515. 11. Kremer, J. M., B. J. Bloom, F. C. Breedveld, J. H. Coombs, M. P. Fletcher, 28. Menet, C., L. Van Rompaey, and R. Geney. 2013. Advances in the discovery of D. Gruben, S. Krishnaswami, R. Burgos-Vargas, B. Wilkinson, C. A. Zerbini, selective JAK inhibitors. Prog. Med. Chem. 52: 153–223. and S. H. Zwillich. 2009. The safety and efficacy of a JAK inhibitor in patients 29. Korniski, B., A. J. Wittwer, T. L. Emmons, T. Hall, S. Brown, A. D. Wrightstone, with active rheumatoid arthritis: results of a double-blind, placebo-controlled J. L. Hirsch, J. A. Gormley, R. A. Weinberg, J. W. Leone, et al. 2010. Expression, phase IIa trial of three dosage levels of CP-690,550 versus placebo. Arthritis purification, and characterization of TYK-2 kinase domain, a member of the Rheum. 60: 1895–1905. Janus kinase family. Biochem. Biophys. Res. Commun. 396: 543–548. Downloaded from 12. Greenwald, M. W., R. Fidelus-Grot, R. Levy, J. Liang, K. Vaddi, and W. V. Williams. 30. O’Brien, K. B., J. J. O’Shea, and C. Carter-Su. 2002. SH2-B family members 2010. A randomized dose-ranging, placebo-controlled study of INCB028050, differentially regulate JAK family tyrosine kinases. J. Biol. Chem. 277: 8673–8681. a selective JAK1 and JAK2 inhibitor in subjects with active rheumatoid arthritis. 31. Xu, D., and C. K. Qu. 2008. Protein tyrosine phosphatases in the JAK/STAT Arthritis Rheum. 62(Suppl.): S911. pathway. Front. Biosci. 13: 4925–4932. 13. O’Shea, J. J., S. M. Holland, and L. M. Staudt. 2013. JAKs and STATs in im- 32. Croker, B. A., H. Kiu, and S. E. Nicholson. 2008. SOCS regulation of the JAK/ munity, immunodeficiency, and cancer. N. Engl. J. Med. 368: 161–170. STAT signalling pathway. Semin. Cell Dev. Biol. 19: 414–422. 14. Cox, L., and J. Cools. 2011. JAK3 specific kinase inhibitors: when specificity is 33. Bersenev, A., C. Wu, J. Balcerek, J. Jing, M. Kundu, G. A. Blobel,

not enough. Chem. Biol. 18: 277–278. K. R. Chikwava, and W. Tong. 2010. Lnk constrains myeloproliferative diseases http://www.jimmunol.org/ 15. Haan, C., C. Rolvering, F. Raulf, M. Kapp, P. Dru¨ckes, G. Thoma, I. Behrmann, in mice. J. Clin. Invest. 120: 2058–2069. and H. G. Zerwes. 2011. Jak1 has a dominant role over Jak3 in signal trans- 34. Kopf, M., M. F. Bachmann, and B. J. Marsland. 2010. Averting inflammation by duction through gc-containing cytokine receptors. Chem. Biol. 18: 314–323. targeting the cytokine environment. Nat. Rev. Drug Discov. 9: 703–718. 16. Thoma, G., F. Nuninger, R. Falchetto, E. Hermes, G. A. Tavares, E. Vangrevelinghe, 35. Vanhoutte, F., R. Galien, E. Vets, F. Namour, B. Vayssie`re, L. Van Rompaey, and H. G. Zerwes. 2011. Identification of a potent Janus kinase 3 inhibitor with high B. Smets, R. Brys, P. Wigerinck, and G. van ’t Klooster. 2011. GLPG0634 shows selectivity within the Janus kinase family. J. Med. Chem. 54: 284–288. selective inhibition of JAK1 and maintained JAK-STAT suppression in healthy 17. Newcomb, D. C., M. G. Boswell, W. Zhou, M. M. Huckabee, K. Goleniewska, volunteers. Arthritis Rheum. 63(Suppl.): 2210 (Abstr.). C. M. Sevin, G. K. Hershey, J. K. Kolls, and R. S. Peebles, Jr. 2011. Human 36. van Gurp, E., W. Weimar, R. Gaston, D. Brennan, R. Mendez, J. Pirsch, S. Swan, TH17 cells express a functional IL-13 receptor and IL-13 attenuates IL-17A M. D. Pescovitz, G. Ni, C. Wang, et al. 2008. Phase 1 dose-escalation study of CP- production. J. Allergy Clin. Immunol. 127: 1006–1013, e1–e4. 690 550 in stable renal allograft recipients: preliminary findings of safety, tolera- 18. Volpe, E., N. Servant, R. Zollinger, S. I. Bogiatzi, P. Hupe´, E. Barillot, and bility, effects on lymphocyte subsets and pharmacokinetics. Am.J.Transplant.8:

V. Soumelis. 2008. A critical function for transforming growth factor-b, inter- 1711–1718. by guest on October 2, 2021 leukin 23 and proinflammatory cytokines in driving and modulating human 37. Namour, F., R. Galien, and L. GheyleVanhoutte, F., B. Vayssiere, A. Van der Aa, TH-17 responses. Nat. Immunol. 9: 650–657. B. Smets, and G. van ’t Klooster. 2012. Once daily high dose regimens of 19. Brand, D. D., K. A. Latham, and E. F. Rosloniec. 2007. Collagen-induced ar- GLPG0634 in healthy volunteers are safe and provide continuous inhibition of thritis. Nat. Protoc. 2: 1269–1275. JAK1 but not JAK2. Arthritis Rheum. 64(Suppl.): 1331 (Abstr.). 20. Yang, T., Z. Wang, F. Wu, J. Tan, Y. Shen, E. Li, J. Dai, R. Shen, G. Li, J. Wu, 38. Vanhoutte, F., M. Mazur, A. Van der Aa, P. Wigerinck, and G. van ’t Klooster. et al. 2010. A variant of TNFR2-Fc fusion protein exhibits improved efficacy in 2012. Selective JAK1 inhibition in the treatment of rheumatoid arthritis: proof of treating experimental rheumatoid arthritis. PLOS Comput. Biol. 6: e1000669. concept with GLPG0634. Arthritis Rheum. 64(Suppl.): 2489 (Abstr.).

Supplementary Table 1:

GLPG0634 reduces inflammatory cytokine levels in sera from arthritic mice1

intact CIA/Vehicle CIA/GLPG0634

MIP-1β 156.4 ± 13.8 203.4 ± 20.8 143 ± 13.5

MCP-3 48.0 ± 3.9 224.0 ± 15.3 153.8 ± 9.9

MCP-5 17.0 ± 1.9 62.8 ± 2.4 36.4 ± 1.8

M-CSF1 8.8 ± 0.6 11.1 ± 1.3 7.8 ± 1.1

MDC 348.4 ± 40.7 463.8 ± 32.7 372.4 ± 17.1

SCF 675.2 ± 169.2 672.4 ± 242.2 422.6 ± 130.7

KC/GRO 13.0 ± 0.0 73.6 ± 18.6 33.6 ± 12.6

IL-1α 278.2 ± 69.3 359.0 ± 83.8 123.0 ± 26.2

LIF 528.6 ± 36.5 696.0 ± 108.0 743.8 ± 68.3

1 Legend: Serum concentrations (pg/ml) were measured using Luminex technology for 4 to 5 mice per treatment group (intact, CIA/vehicle and CIA/GLPG0634 indicating vehicle-treated or GLPG634- treated arthritic mice) in one experiment. Data are presented as mean ± SEM. IL-1β, IL-2, IL-3, IL-4, IL-5, IL-7, IL10, IL-11, IL-12p70, IL-17A, GM-CSF, IFNγ, OSM, RANTES, TNFα and FGFβ were below the level of detection. Supplementary Table 2: GLPG0634 kinase selectivity testing

Percentage of IC Performed Kinase 50 inhibition (nM) at at 1µM 2,302 Galapagos ABL(h) N.V. ACK1(h) 17 Millipore ALK(h) -22 Millipore ALK4(h) -12 Millipore Arg(h) 14 Millipore ARK5(h) 1 Millipore ASK1(h) -14 Millipore Aurora-A(h) -13 Millipore >10,000 Galapagos AURORA-B(h) N.V. Axl(h) -5 Millipore Bmx(h) -11 Millipore BRK(h) -2 Millipore BrSK1(h) -5 Millipore >10,000 Galapagos BTK(h) N.V. CaMKI(h) -4 Millipore CaMKII²(h) -19 Millipore CaMKIV(h) -5 Millipore CDK1/cyclinB(h) 7 Millipore >10,000 Galapagos CDK2(h) N.V. CDK5/p25(h) -16 Millipore CDK7/cyclinH/MAT1(h) 12 Millipore CHK1(h) 10 Millipore CHK2(h) 6 Millipore CK2(h) -12 Millipore CK2±2(h) -6 Millipore >10,000 Galapagos cKIT(h) N.V. CLK2(h) 10 Millipore c-RAF(h) -4 Millipore CSK(h) 3 Millipore >10,000 Galapagos CSNK1G2(h) N.V. cSRC(h) -10 Millipore >10,000 Galapagos DAPK1(h) N.V. DCAMKL2(h) 12 Millipore DDR2(h) -17 Millipore DMPK(h) -4 Millipore DRAK1(h) 26 Millipore DYRK2(h) -1 Millipore eEF-2K(h) -4 Millipore EGFR(h) -5 Millipore EphA2(h) -9 Millipore >10,000 Galapagos EPHA5(h) N.V. EphA8(h) 1 Millipore EphB1(h) -44 Millipore EphB4(h) 5 Millipore ErbB4(h) -5 Millipore FAK(h) 23 Millipore Fer(h) -5 Millipore Fes(h) -36 Millipore FGFR4(h) -18 Millipore Fgr(h) 5 Millipore Flt1(h) 37 1,327 Millipore Flt3(h) 58 338 Millipore Flt4(h) 77 274 Millipore 488.9 Galapagos FMS(h) N.V. Fyn(h) 0 Millipore >10,000 Galapagos GCK(h) N.V. GRK5(h) -10 Millipore GRK7(h) -8 Millipore Haspin(h) -6 Millipore Hck(h) -20 Millipore HIPK1(h) -8 Millipore >10,000 Galapagos HIPK2(h) N.V. >10,000 Galapagos ICK(h) N.V. IGF-1R(h) -22 Millipore IR(h) -1 Millipore IRAK1(h) 2 Millipore >10,000 Galapagos IRAK4(h) N.V. IRR(h) -5 Millipore Itk(h) -4 Millipore KDR(h) 28 Millipore >10,000 Galapagos LCK(h) N.V. LIMK1(h) -1 Millipore LKB1(h) -5 Millipore LOK(h) -3 Millipore >10,000 Galapagos LYNA(h) N.V. >10,000 Galapagos MAP3K3(h) N.V. >10,000 Galapagos MAP4K4(h) N.V. MAPK1(h) 1 Millipore >10,000 Galapagos MAPK12(h) N.V. >10,000 Galapagos MAPK14(h) N.V. MAPK2(h) 0 Millipore MARK1(h) 7 Millipore MEK1(h) -3 Millipore MELK(h) -34 Millipore Mer(h) 44 1,274 Millipore Met(h) -3 Millipore >10,000 Galapagos MINK(h) N.V. MINK(h) -2 Millipore MKK6(h) -6 Millipore MKK7²(h) 13 Millipore MLCK(h) -14 Millipore MLK1(h) 1 Millipore >10,000 Galapagos MNK2(h) N.V. MNK2(h) -10 Millipore MRCK±(h) -3 Millipore MRCK²(h) -1 Millipore MSK1(h) 7 Millipore MSK2(h) 2 Millipore MSSK1(h) -7 Millipore MST1(h) -10 Millipore MST2(h) 0 Millipore MST3(h) -7 Millipore mTOR(h) 3 Millipore mTOR/FKBP12(h) -3 Millipore MuSK(h) -2 Millipore NEK11(h) -10 Millipore NEK2(h) -29 Millipore NEK3(h) -4 Millipore NEK6(h) -6 Millipore NLK(h) -6 Millipore p70S6K(h) 3 Millipore PAK2(h) 0 Millipore >10,000 Galapagos PAK4(h) N.V. PAK4(h) -5 Millipore >10,000 Galapagos PAK6(h) N.V. PAR-1B±(h) -7 Millipore PASK(h) 1 Millipore PDGFR±(h) 2 Millipore PDGFR²(h) -10 Millipore PDK1(h) -3 Millipore PhK³2(h) -7 Millipore Pim-1(h) -7 Millipore Pim-2(h) -7 Millipore PKB±(h) -3 Millipore PKB²(h) -14 Millipore PKB³(h) -6 Millipore PKC´(h) -14 Millipore PKC±(h) -2 Millipore PKCµ(h) -10 Millipore PKC¼(h) 2 Millipore PKC¹(h) -4 Millipore PKC²II(h) -1 Millipore PKC³(h) 5 Millipore PKD2(h) -10 Millipore PKG1±(h) 2 Millipore PKG1²(h) -1 Millipore Plk1(h) -13 Millipore Plk3(h) -30 Millipore PRK2(h) -8 Millipore PrKX(h) -14 Millipore PTK5(h) -6 Millipore Pyk2(h) -8 Millipore Ret(h) 9 Millipore >10,000 Galapagos ROCK1(h) N.V. Ron(h) -12 Millipore Ros(h) -9 Millipore Rse(h) 4 Millipore Rsk1(h) 2 Millipore Rsk2(h) 1 Millipore SAPK2b(h) -4 Millipore SAPK4(h) -10 Millipore SGK(h) 7 Millipore SGK2(h) -4 Millipore SIK(h) 1 Millipore Snk(h) 0 Millipore Src(1-530)(h) 32 Millipore SRPK1(h) 4 Millipore SRPK2(h) -10 Millipore STK33(h) -8 Millipore >10,000 Galapagos SYK(h) N.V. >10,000 Galapagos TAK1(h) N.V. TAO1(h) -4 Millipore 1,791 Galapagos TBK1(h) N.V. Tie2(h) -2 Millipore TLK2(h) -6 Millipore TrkB(h) -16 Millipore TSSK1(h) 5 Millipore TSSK2(h) -5 Millipore Txk(h) 14 Millipore ULK2(h) -8 Millipore ULK3(h) 4 Millipore VRK2(h) 2 Millipore WNK2(h) -1 Millipore WNK3(h) -2 Millipore Yes(h) -12 Millipore ZAP-70(h) -12 Millipore ZIPK(h) 3 Millipore 1

1 Legend: GLPG0634 was tested in radioactive biochemical assays at Millipore and Galapagos NV. The incorporation of 33P into a specific substrate was measured. Results of GLPG0634 testing at 1µM at Millipore are indicated as percentage of kinase activity inhibited (second column). For kinases inhibited more than 35%, IC50 values were obtained. IC50 values (nM) determined at Millipore or Galapagos NV are indicated in the third column. Supplementary Figure 1:

GLPG0634 shows moderate efficacy in a mouse experimental allergic encephalomyelitis (EAE) model: the chronic EAE model in C57/BL6 mice was essentially run as described by Wegner et al.1 GLPG0634 was dosed orally at 20 (preventive setting, treatment starts day 1 (d1) after immunization) and 2, 6, 20, 60 mg/kg (therapeutic setting, treatment start at d8) daily and mean clinical scores are shown. The experimental MS therapeutic laquinimod (25 mg/kg) was included as positive control.

See accompanying TIFF file “GLPG0634_Supplementary_Figure1”

1 Wegner C., C. Stadelmann, R. Pförtner, E. Raymond, S. Feigelson, R. Alon, B. Timan, L. Hayardeny and W. Bruck. 2010. Laquinimod interferes with migratory capacity of T cells and reduces IL-17 levels, inflammatory demyelination and acute axonal damage in mice with experimental autoimmune encephalomyelitis. J Neuroimmunol. 227: 133-143.