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

Feyen and Christel MenetFletcher, Reginald Brys, Gerben van 't Klooster, Jean H. M.Vandeghinste, Béatrice Vayssiere, Steve De Vos, Stephen Lepescheux, Thierry Christophe, Katja Conrath, NickPhilippe Clement-Lacroix, Luc Nelles, Bart Smets, Liên Luc Van Rompaey, René Galien, Ellen M. van der Aar,

http://www.jimmunol.org/content/191/7/3568doi: 10.4049/jimmunol.1201348September 2013;

2013; 191:3568-3577; Prepublished online 4J Immunol 

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The Journal of Immunology

Preclinical Characterization of GLPG0634, a SelectiveInhibitor of JAK1, for the Treatment of InflammatoryDiseases

Luc Van Rompaey,* Rene Galien,† Ellen M. van der Aar,* Philippe Clement-Lacroix,†

Luc Nelles,* Bart Smets,* Lien Lepescheux,† Thierry Christophe,* Katja Conrath,*

Nick Vandeghinste,* Beatrice 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 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. The Journal of Immunology, 2013, 191: 3568–3577.

The JAKs are cytoplasmic tyrosine kinases critical for in-tracellular signal transduction of many cytokines, growthfactors, and hormones. Four human JAKs have been de-

scribed: JAK1, JAK2, JAK3, and TYK2. JAKs bind to the intra-cellular moieties of type I and type II receptors, and JAK homo- orheterodimers become activated upon ligand binding. The JAKsphosphorylate each other followed by phosphorylation of tyrosineresidues on the intracellular domains of the receptors. These phos-phorylated residues serve as docking sites for STAT transcriptionfactors. JAK phosphorylation of the STAT proteins results in theirnuclear translocation and provides transcriptional output for thecytokine ligands (1, 2). JAKs play important roles in the functioningof the immune system. Mouse and human genetics studies linkeddeficiencies of JAK1 and JAK3 to severe combined immune defi-ciency and TYK2 to increased susceptibility to infections (3, 4).JAK2 serves signal transduction for inflammatory cytokines such as

IFN-g, IL-12, IL-23, and GM-CSF (2, 5). Hence, JAKs have beentargeted for their therapeutic potential in immune-inflammatorydisorders. In fact, small-molecule JAK inhibitors proved effica-cious in a range of animal disease models and have already shownpromise in the clinic for organ transplant rejection, rheumatoidarthritis (RA), psoriasis, dry eye disease, myelofibrosis, inflamma-tory bowel disease, and asthma (5–10). JAK2-mediated side effectsobserved in clinical trials such as anemia, neutropenia, and throm-bocytopenia appear to be the main cause for not fully exploiting thepharmacodynamic potential of these JAK inhibitors (11–13). Recentfindings suggest that JAK1 dominates JAK1/JAK3/gc signaling,suggesting that JAK1 inhibition might be largely responsible forthe in vivo efficacy of JAK inhibitors in immune-inflammatorydiseases (14–16). These results indicate that a selective JAK1 in-hibitor could provide an increased therapeutic window allowing forhigher 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

immune-inflammatory diseases, we set out to identify selective JAK1inhibitors. One of the lead compounds, GLPG0634, was shown toselectively inhibit JAK1-dependent signaling in cellular and wholeblood assays (WBAs) and showed remarkable efficacy in collagen-induced arthritis (CIA) disease models for RA in both mouse and rat.

Materials and MethodsSmall-molecule kinase inhibitors

Focused kinase collections were sourced from BioFocus (Essex, U.K.).GLPG0634 was synthesized by Galapagos medicinal chemists. Tofacitiniband baricitinib were sourced from Shanghai Haoyuan Chemexpress (Shang-hai, China) and Charnwood Molecular (Loughborough, U.K.), respectively.

*Departement of In Vitro Pharmacology, Galapagos NV, 2800 Mechelen, Belgium;and †Departement of In Vivo Pharmacology and Translational Sciences, GalapagosSASU, 93230 Romainville, France

1Current address: BioFocus, Essex, U.K.

Received for publication May 14, 2012. Accepted for publication July 29, 2013.

Address correspondence and reprint requests to Luc Van Rompaey, Galapagos NV,Generaal de Wittelaan L11 A3, 2800 Mechelen, Belgium. E-mail address: luc.vanrompaey@glpg.com

The online version of this article contains supplemental material.

Abbreviations used in this article: CII, collagen type II; CIA, collagen-induced ar-thritis; Cmax, maximum blood concentration; EPO, erythropoietin; OSM, oncostatinM; PRL, prolactin; RA, rheumatoid arthritis; RT, room temperature; siRNA, smallinterfering RNA; WBA, whole blood assay.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

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Biochemical assays

IC50 determination.Recombinant JAK1, TYK2 (Invitrogen), JAK2, and JAK3(Carna Biosciences) were used to develop activity assays in 50 mM HEPES(pH 7.5), 1 mMEGTA, 10mMMgCl2, 2 mMDTT, and 0.01% Tween 20. Theamount of JAK protein was determined per aliquot, maintaining initial ve-locity and linearity over time. The ATP concentration was equivalent to 43the experimental Km value and the substrate concentration (ULight-conjugatedJAK-1(Tyr1023) peptide; PerkinElmer) corresponded to the experimentallydetermined Km value. After 90 min incubation at room temperature (RT),the amount of phosphorylated substrate was measured by addition of 2 nMeuropium-anti-phosphotyrosine Ab (PerkinElmer) and 10 mM EDTA inLance detection buffer (PerkinElmer). Compound IC50 values were deter-mined by preincubating the enzyme with compound at RT for 60 min, priorto the addition of ATP.

Kd determination. Dissociation constants were determined at ProterosBiostructures (Martinsried, Germany). Proprietary fluorescently labeled ATPmimetics with fast dissociation rates (PRO13, PRO14, and PRO13 for JAK1,JAK2, and JAK3, respectively) were incubated with JH1 domains of purifiedJAKs in 20 mMMOPS (pH 7.5), 1 mMDTT, 0.01% Tween 20, and 500 mMhydroxyectoine (JAK3 only) for 30 min. Compounds (concentrations rangingfrom 520 pM to 1.1 mM) were added in 100% DMSO and time depen-dency of reporter displacement was measured. IC50 values correspondingto 50% probe displacement were obtained and Kd values were calculatedaccording to the Cheng–Prusoff equation.

Cellular assays

STAT6 phosphorylation induced by IL-4. THP-1 cells (ATCC TIB-202)were preincubated with compound at RT for 1 h, incubated with IL-4(10 ng/ml) at RT for 60 min, and processed for flow cytometry. Cellswere fixed in Cytofix/Cytoperm (BD Biosciences) buffer and permeabilizedin Phosflow perm buffer III (BD Biosciences) on ice for 30 min. Afterblocking (Fc blocking reagent; Miltenyi Biotec), pSTAT6 was detected withmouse anti-human PE-labeled anti-pSTAT6 Ab (BD Biosciences).

STAT5 phosphorylation induced by IL-2, IL-3, and erythropoietin. NK-92cells (ATCC CRL-2407) were IL-2 starved overnight, preincubated withcompound 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, catalogno. 93022307) were starved overnight in RPMI 1640 with 0.1% FBS, pre-incubated with compound at RT for 1 h, stimulated with IL-3 (30 ng/ml) atRT for 20 min, and processed for AlphaScreen analysis. UT-7-erythropoietin(EPO) cells (EPO-dependent derivative of UT-7; Centocor) were preincubatedwith compound at RT for 1 h, stimulated with EPO (1 U/ml) for 20 min,and processed for AlphaScreen analysis. pSTAT5 was measured usingAlphaScreen technology essentially according to the manufacturer’s protocol.

STAT1 phosphorylation induced by IFN-a and IFN-g. STAT1 U2OS cells(Invitrogen, catalog no. K1469) were preincubated with compound at 37˚Cfor 1 h, treated with 30,000 U/ml IFN-aB2 (PBL IFN source, catalog no.11115-1) or 20 ng/ml IFN-g (PeproTech, catalog no. 300-02, lot 010827)at 37˚C for 1 h, lysed (lysis buffer containing 2 nM Tb-Ab; Invitrogen) ac-cording to manufacturer’s protocol, and incubated at RT for 60 min. pSTAT1was detected by time-resolved fluorescence resonance energy transfer (Per-kinElmer).

STAT5 phosphorylation induced by prolactin. 22Rv1 cells (ATCC CW22Rv)were starved overnight, preincubated with compound, triggeredwith prolactin(PRL; 500 ng/ml human PRL for 20 min), lysed in 10 mMTris-HCl (pH 7.5),5mMEDTA, 150mMNaCl, 0.5%TritonX-100, 50mMNaF, 30mM sodiumpyrophosphate, 10% glycerol buffer containing phosphatase/protease inhibitorcocktails, and centrifuged. Cell lysate (180 mg) was used for STAT5 im-munoprecipitation (anti-STAT5 polyclonal Abs, Santa Cruz Biotechnology,C-17; protein A-Sepharose beads, GE Healthcare). Total and phosphorylatedSTAT5 were measured by densitometric analysis after Western blotting (anti-pSTAT5 mAb, AX-1, Advantex).

IL-3/JAK2–induced proliferation of Ba/F3 cells. Ba/F3 cells (provided byV. Lacronique, Paris, France), which are dependent on IL-3 and JAK2 sig-naling, were incubated with compound at 37˚C for 40 h, after which cellproliferation was analyzed by measuring ATP content (ATPlite; PerkinElmer).

Oncostatin M-induced STAT1 reporter assay in HeLa cells.HeLa cells (ATCCCCL-2) were transfected with a pSTAT1 reporter construct (Panomics, catalogno. LR0127). After transfection for 24 h, cells were incubated for 1 h withcompound and triggered with oncostatin M (OSM; 33 ng/ml; Peprotech,catalog no. 300-10). After 20 h incubation, the cells were lysed and luciferaseactivity was determined with the luciferase SteadyLite kit according to thesupplier’s recommendations (PerkinElmer, catalog no. 6016759). In parallel,b-galactosidase activity was measured in the presence of 4 mg/ml 2-nitro-phenyl b-D-galactopyranoside (Sigma-Aldrich, catalog no. N1127).

Knockdown experiments.HeLa andHCT116 cells obtained from the AmericanType Culture Collection were transfected with 50 nM ON-TARGETplusSMARTpool small interferingRNA (siRNA) for human JAK1, JAK2, JAK3, orTYK2, or with nontargeting or GAPDHnegative control siRNAs (Dharmacon)using Lipofectamine RNAiMAX transfection reagent from Invitrogen.Four days after transfection cells were starved overnight and stimulatedwith IL-6/sIL-6R (both 250 ng/ml) for 20 min and pSTAT1 levels weredetermined using AlphaScreen technology (PerkinElmer) according to themanufacturer’s protocol.

T cell differentiation studies. PBMCs were isolated from buffy coats ofhealthy donors (Blood Transfusion Center, Red Cross, Leuven, Belgium)using density gradient centrifugation on Lymphoprep. Naive CD4+ T cellswere further isolated by depletion of non–T helper and memory CD4+

T cells using a naive CD4+ T cell isolation kit II (Miltenyi Biotec). Isolatednaive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 mg/ml)and anti-CD28 (5 mg/ml) Abs in the presence of cytokines that drivedifferentiation into Th1, Th2, or Th17 Th subsets. For Th1 cell polarization,cells were cultured in the presence of 10 mg/ml anti–IL-4 Ab (MAB204;R&D Systems), 10 ng/ml IL-2 (R&D Systems), and 10 ng/ml IL-12 (R&DSystems) (17). For Th2 cell polarization, cells were cultured in the presenceof 10 mg/ml anti–IFN-g Ab (Becton Dickinson), 25 ng/ml IL-4 (R&DSystems), and 10 ng/ml IL-2 (17). For Th17 cell polarization, a mix of thefollowing cytokines was used: 10 ng/ml IL-6 (R&D Systems), 10 ng/mlIL-1b (R&D Systems), 1 ng/ml TGF-b (PeproTech), and 100 ng/ml IL-23 (R&D Systems) (18). To monitor effects of compounds on T cell dif-ferentiation, compounds were added at indicated concentrations at the startof T cell differentiation. After 5 d, RNAwas extracted using an RNeasy Minikit (Qiagen), reverse transcribed, and the extent of Th subset differentiationwas monitored by determining expression of IFN-g (Th1 marker), IL-13(Th2 marker), or IL-17F (Th17 marker) using real-time PCR on the ViiA7thermocycler with predesigned TaqMan Assay-on-Demand gene expressionprimer/probe sets (Applied Biosystems). Gene expression was normalized to18S and expressed as DCt values, with DCt = Ctgene 2 Ct18S or expressed asrelative mRNA level of specific gene expression as obtained using the 22DCt

method.

Human WBAs

Human blood was collected from healthy volunteers, who gave informedconsent, into sodium heparin vacutainer tubes by venipuncture. After in-cubation with compounds at 37˚C for 30 min, blood was triggered witheither recombinant human IL-6 (10 ng/ml; R&D Systems), recombinanthuman IL-2 (4 ng/ml; R&D Systems), universal IFN-a (1000 U/ml; PBLBiomedical Laboratories), recombinant human GM-CSF (20 pg/ml; Pepro-Tech), or vehicle (PBS plus 0.1% [w/v] BSA) at 37˚C for 20 min and treatedwith prewarmed 13 lysis/fix buffer (BD Biosciences) to lyse RBCs and fixleukocytes. Cells were permeabilized with 100% methanol and incubatedwith anti-pSTAT1 and anti-CD4 (IL-6– and IFN-a–triggered samples), anti-pSTAT5 and anti-CD4 Abs (IL-2–triggered samples), or anti-pSTAT5 andanti-CD33 Abs (GM-CSF–triggered samples) (all Abs were from BD Bio-sciences) at 4˚C for 30 min, washed once with PBS 13, and analyzed ona FACSCanto II flow cytometer.

Pharmacokinetics

Formulations. GLPG0634 was formulated in polyethyleneglycol 200/0.9%NaCl (60/40; v/v) for i.v. administration and in 0.5% (v/v) methylcellulosefor oral administration for all in vivo studies described. Compound puritywas .95% as measured by HPLC.

Animals. Male Sprague Dawley rats (180–200 g) and CD1 mice (23–25 g)were obtained from Janvier and Harlan (France), respectively. Two daysbefore administration of compound, rats underwent surgery to place a cath-eter in the jugular vein under isoflurane anesthesia. Animals were deprivedof food for at least 16 h before oral dosing until 4–6 h after. Before oraldosing, animals were deprived of food for at least 12 h before compoundadministration until 4 h after administration. All in vivo experiments werecarried out in a dedicated pathogen-free facility (22˚C). Animal care was inaccordance with the French guidelines about the use of animals in scien-tific research. All procedures involving animals, including housing andcare, method of euthanasia, and experimental protocols, were conducted inaccordance with a code of practice established by the local ethical com-mittee (Galapagos).

Pharmacokinetic studies. GLPG0634 was orally dosed as a single esoph-ageal gavage at 5 mg/kg (dosing volume of 5 ml/kg) and i.v. dosed as a bolusvia the caudal vein at 1 mg/kg (dosing volume of 5 ml/kg). In the rat study,each group consisted of three rats and blood samples were collected via thejugular vein. In the mouse study, each group consisted of 21 mice (n = 3/timepoint) and blood samples were collected by intracardiac puncture under

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isoflurane anesthesia. Lithium heparin was used as anticoagulant and bloodwas taken at 0.05, 0.25, 0.5, 1, 3, 5, and 8 h (i.v. route) and 0.25, 0.5, 1, 3, 5, 8,and 24 h (by mouth).

GLPG0634 plasma concentrations were determined by liquid chroma-tography–tandem mass spectrometry with a lower limit of quantification of2 ng/ml. Pharmacokinetic parameters were calculated by noncompartmentalanalysis using WinNonlin software (Pharsight, version 5.2).

In vivo pharmacology

Rodent CIA models. Animals. Dark Agouti rats (females, 7–8 wk old) andDBA/1J mice (male, 6 wk old) were obtained from Janvier (Laval, France).

Materials. CFA and IFA were purchased from Difco (Detroit, MI). Bovinecollagen type II (CII) was obtained from Chondrex (Redmond, WA). Allother reagents used were of reagent grade and all solvents were of analyticalgrade.

CIA.One day before the start of the experiment, CII solution (2 mg/ml) wasprepared 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 homogenizerin a precooled glass bottle in an ice water bath. For rat CIA experiments,the emulsion (0.2 ml) was injected intradermally at the base of the tail atday 1 and again at day 8. This immunization method was modified frompublished methods (19). The in vivo efficacy of GLPG0634 was deter-mined after daily oral administration for a period of 14 d after onset ofdisease (average clinical score at onset, 2.56 0.3; 10 rats/treatment group)over the dose range 0.1–30 mg/kg. The TNF-a blocker etanercept (WyethPharmaceuticals, Taplow, U.K.) was administered three times per week at10 mg/kg by i.p. injection. A fully active dose was reported to requirerepeated dosing in the 3–9 mg/kg range (20). In our model of Dark Agoutifemale rats, disease normalization was reached for 10 mg/kg etanerceptdosed three times a week i.p. as measured by clinical score, inflammation,bone resorption, pannus, and cartilage damage. At day 7 or 11, 200 mlblood was collected by retro-orbital puncture with lithium heparin as an-ticoagulant at predose and 1, 3, and 6 h (n = 2 or 3/time point) for steady-state pharmacokinetics analysis. At sacrifice, hind paws were removed forx-ray analysis and histological examination. A Tukey multiple comparisontest was used to perform a meta-analysis of three studies carried out forGLPG0634. The score of each rat was divided by the average score ob-tained for vehicle in the same readout and study and multiplied by 100.Relative scores were averaged per readout for all animals present in allstudies that received the same dose. For mouse CIA experiments, the IFA/CII emulsion (0.2 ml) was injected intradermally at the base of the tail atday 1 and again at day 21. This immunization method was modified frompublished methods (20). The in vivo efficacy of GLPG0634 was deter-mined after daily oral administration for a period of 14 d after onset ofdisease (average clinical score at onset, 2.4 6 0.6; 10 mice/treatmentgroup) over the dose range 50 mg/kg twice daily. Administration of eta-nercept and pharmacodynamic and pharmacokinetic analyses were essen-tially carried out as described for the rat CIA model.

Clinical assessment of arthritis. The individual clinical score was ob-tained by summing the scores recorded for each limb. Arthritis was scoredfrom grade 0 to 4 according to an established method (19).

Larsen score. X-ray imaging was performed for the hind paws of eachindividual animal. An identity number was randomly assigned to each of theimages, and the severity of bone erosion was ranked by two independentblinded scorers as described earlier (21).

Histology. For rat CIA studies, the hind paws were collected, fixed in 3.7%(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 madeat 100-mm intervals from the paw middle part and stained with Goldner’strichrome. The inflammation status and skeletal tissue damage were ex-amined by light microscope (Provis, Olympus). For mouse CIA studies,the hind paws were collected, fixed in 3.7% (v/v) formaldehyde at roomtemperature for 24 h, and decalcified in Osteosoft solution (VWR Inter-national, Val de Fontenay, France) for 24 d. Each paw was cut in two partsaccording the sagittal axis and embedded in paraffin. Six series of 4-mmthick sections were collected. One series of sections was stained withsafranin O-light green for morphological examination and disease scoring.Disease was scored by a double-blinded evaluation as described by Linet al. (21). Statistical analysis was performed by using a Kruskall–Wallistest followed by a Dunn multiple comparison post hoc test with *p , 0.05,**p , 0.01, and ***p , 0.001 versus vehicle group. Immunohistochem-istry was performed with Abs detecting macrophage (anti-F4/80, sc-59171;Santa Cruz Biotechnology) and T cell (anti-CD3; Dako) markers. Bio-tinylated horse anti-rabbit Ab and avidin-linked peroxidase (VectastainUniversal Elite ABC kit; Vector Laboratories) were used to detect the bindingstained in brown color. Immuno-positive cells were quantified by image

analysis and related to the total number of cells. Statistical analysis wasperformed by using an ANOVA unpaired test with *p , 0.05, **p , 0.01,and ***p , 0.001 versus vehicle group.

Gene expression and pharmacokinetic/pharmacodynamic modeling in rodentblood. Fed male Sprague Dawley rats (180–200 g) received four daily oraldoses of vehicle or GLPG0634 at 1 or 10 mg/kg. Blood samples were takenon 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 byliquid chromatography–tandem mass spectrometry. Each blood sample (2 ml)was divided in two tubes and either left untreated (control) or treated with520 U/ml rat IFN-a at 37˚C for 1 h. RBCs were lysed (buffer EL; Qiagen)and the WBCs pelleted, dissolved, and homogenized in 350 mL buffer RLT(Qiagen). Total RNA was extracted using the QIAamp RNA Blood Mini kit(Qiagen) and 500 ng was reverse transcribed using TaqMan RT (AppliedBiosystems) with oligo(dT) priming. Five microliters of 53 diluted cDNApreparations was used for real-time quantitative PCR (TaqMan technology,using a StepOnePlus thermocycler; Applied Biosystems) with gene-specificprobes and primers designed according to standard procedures.

For the analysis of gene expression in mouse whole blood cells (cir-culating leukocytes), blood was sampled in RNAprotect tubes (Qiagen) andprocessed using the RNeasy protect animal blood kit (Qiagen). Total RNA(300 ng) was reverse transcribed using a high-capacity cDNA synthesis kit(Applied Biosystems) with random hexamers. Quantitative PCR reactionswere performed using QuantiFast SYBR Green PCR Master mix (Qiagen)and gene-specific primer pairs for b-actin (Eurogentec) and QuantiTectprimer assays for all other genes analyzed (Qiagen). Reactions were car-ried out with a denaturation step at 95˚C for 5 min followed by 40 cycles(95˚C for 10 s, 60˚C for 1 min) in a ViiA7 real-time PCR system (AppliedBiosystems). Real-time PCR data for each target gene were expressed asDCt, corresponding to Ct obtained for the gene of interest normalized withthe Ct of the b-actin gene.

Gene expression analysis in mouse paws. Hind paws were dissected bycutting above the ankle joint and removing the digits. The remaining tissuewas transferred into 2 ml homogenization tubes (Quality Scientific Plastics)containing 1 mm zirconium beads (BioSpec Products) and 750 ml TRIzolreagent (Invitrogen). Tissue samples were homogenized using the Precellys24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France)programmed to shake samples three times for 30 s at 5300 rpm with 5-spauses between the homogenization steps. Total RNA was isolated asrecommended by the TRIzol supplier and further purified with NucleoSpin96 RNA (Macherey-Nagel) according to the manufacturer’s instructions.RNA yields were determined by spectrophotometry at 260/280 nm. Re-verse transcription of RNA and quantitative PCR were performed as de-scribed above. For statistical analysis, a two-way ANOVA followed by aDunnett post hoc test versus the CIA-vehicle group was performed.

Analyte measurement in mouse sera. Quantification of analytes in mousesera was performed at Myriad RBM using mouse CytokineMAP A v1.0,mouse CytokineMAP B v1.0, and mouse CytokineMAP C v1.0.

ResultsTo identify new JAK1 inhibitors, a kinase-focused collection of∼10,000 small molecules was screened in a JAK1 biochemicalassay. From this screen a triazolopyridine series was identified asa tractable hit series. Establishment of a detailed structure-activityrelationship in this series led to the identification of GLPG0634as one of the lead compounds of the series. Characterization ofGLPG0634 at the biochemical level indicated a selective inhibi-tion of JAK1 and JAK2 over JAK3 and TYK2 with a rank orderpotency of JAK1 ∼ JAK2 . TYK2 . JAK3 (Table I). IC50 valuesdetermined in tyrosine kinase inhibition assays correlated with Kd

values determined in ligand displacement assays (Table I).Several cellular assay set-ups were applied to elucidate the

potency and the JAK selectivity profile in a cellular environment.Cell lines were preincubated with GLPG0634 and treated withcytokines that employ different JAK heterodimeric or JAK2 homo-dimeric complexes for signaling. GLPG0634 inhibited IL-2– and IL-4–induced JAK1/JAK3/gc signaling and IFN-aB2–induced JAK1/TYK2 type II receptor signaling most potently. IC50 values rangedfrom 150 to 760 nM (Table I). IFN-g– and OSM-induced JAK1/JAK2 signaling mediated by type II and gp130 receptor complexesand IL-3–induced JAK2/bc signaling were inhibited with low

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micromolar potencies (Table I). JAK2 homodimer–mediated sig-naling induced by EPO or PRL was inhibited with the lowestpotency. IC50 values could not be determined accurately but were.10 mM. Inhibition of JAK3 and TYK2 is unlikely to contributeto GLPG0634s inhibition of JAK/STAT signaling in view of thepotency difference measured in biochemical assays between JAK1and JAK2 versus JAK3 and TYK2 (Table II). Hence, GLPG0634preferentially inhibits JAK/STAT signaling involving JAK1 thanJAK2 kinase in a cellular context. To test the relative contributionsof JAK1 versus JAK2 in a physiological relevant model, GLPG0634was tested for inhibition of cytokine-induced STAT phosphorylationin human WBAs. There was a particular interest in comparing GM-CSF/STAT5 versus IL-6/STAT1 signaling in myeloid and T cells,respectively. GM-CSF relies on a JAK2 homodimer for intracellularsignaling, and IL-6–mediated phosphorylation of STAT1 is JAK1-dependent, as shown for T cells by Ghoreschi et al. (22). Similarresults were obtained by applying an siRNA knockdown approachin HeLa and HCT116 cells. Transfection with siRNAs targetingthe individual JAK family members resulted in a knockdown ofmessenger RNAs for JAK1, JAK2, JAK3, and TYK2 by .70%.However, at the functional level we could only demonstrate asignificant reduction of IL-6–induced STAT1 phosphorylation(Fig. 1), indicative for a JAK1-driven signaling event. A largepotency difference was measured for GLPG0634 in the IL-6versus GM-CSF WBAs (629 nM versus 17.5 mM; Table III),corresponding to an ∼30-fold selectivity for inhibition of JAK1-over JAK2-dependent signaling (Table III). Two clinical JAKinhibitors, tofacitinib and baricitinib, were also tested in both assaysand showed high potency. A respective 10- and 3-fold JAK1 overJAK2 selectivity was determined even though both compoundsequipotently inhibited JAK1 and JAK2 in biochemical assays (9,10). These data indicate that GLPG0634 selectively inhibits JAK1-

dependent signaling in a cellular environment and is more JAK1selective than are tofacitinib and baricitinib. GLPG0634 alsoinhibited the STAT5 phosphorylation activated by IL-2 and STAT1phosphorylation by IFN-a, although with a lower potency thanSTAT1 phosphorylation by IL-6. Tofacitinib inhibited IFN-asignaling with similar potency as IL-6/STAT1 signaling but in-hibited IL-2/STAT5 signaling with higher potency (Table III). Theseobservations confirm the JAK1/JAK3 inhibition profile of tofacitinibin cells reported by Meyer et al. (9). The WBA data confirm theobservations from the cellular assays showing that GLPG0634preferentially inhibits JAK signaling complexes containing JAK1.To further explore the consequences of cytokine inhibition, the

effect of GLPG0634 toward Th1 and Th2 differentiation was ana-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 signalsthrough JAK1 and JAK3. GLPG0634 also inhibited Th1 differen-tiation with similar potencies of 1 mM or lower. Although Th1commitment is initiated by IL-12, a cytokine signaling throughTYK2 and JAK2, the primary effect of GLPG0634 on Th1 differ-entiation is likely through inhibition of JAK1/JAK2-mediated sig-naling of IFN-g. Inhibition of IFN-g signaling alone was previouslyfound to be sufficient to reduce T-bet, the master transcriptionalregulator critical for Th1 differentiation, and IFN-g in Th1 cells andit was proposed as a mechanism explaining the inhibitory effectof tofacitinib on Th1 cell differentiation (3). GLPG0634 was alsotested for the ability to inhibit Th17 differentiation driven by amixture of TGF-b, IL-23, and proinflammatory cytokines (IL-6and IL-1b), all shown to be essential for human Th17 differentiation(18). GLPG0634 inhibited Th17 differentiation under these con-ditions, although with lower potency than Th1 and Th2 differen-tiation (Fig. 2).The pharmacokinetics of GLPG0634 was determined in rats and

mice. Following i.v. administration, GLPG0634 displayed a lowto moderate plasma clearance, depending on the species tested(Table IV). In mice, the total clearance represented 58% of theliver blood flow, and in rats it represented 41%. Steady-state volumeof distribution ranged from ∼1.7 l/kg in rats to 6 l/kg in mice,implying a significant species difference in volume of distribution.Half-life observed after oral administration was 1.7 h in mice and3.9 h in rats. Following oral administration, the absolute bioavail-ability was moderate in rats (45%) and high in mice (∼100%).Because GLPG0634 was well exposed in several species, in-

hibition of JAK signaling in vivo was evaluated in a biomarker assay.InWBCs, Mx2 mRNA levels are modulated by IFN-a–JAK1/TYK2signaling (23, 24). Rats were dosed orally with GLPG0634 and bothbasal and ex vivo IFN-a–induced Mx2 mRNA levels were measured.A statistically significant reduction of normalized Mx2 mRNA

Table I. Potency and selectivity of GLPG0634 in JAK biochemicalassays

Recombinant Human Kinase IC50 (nM, 6SEM; n = 2–4) Kd (nM)

JAK1 10 6 0.8 11JAK2 28 6 5.4 32JAK3 810 6 180 300TYK2 116 6 39 ND

IC50 values for inhibition of recombinant JAK1, JAK2, JAK3, and TYK2 byGLPG0634 were determined by measuring the incorporation of phosphate into anULight-JAK-1(Tyr1023) peptide using an europium-labeled anti-phosphotyrosine Ab.The Kd values of GLPG0634 on JAK1, JAK2, and JAK3 were determined by mea-suring the competition of GLPG0634 with a fluorescently labeled ATP mimetic. IC50

values corresponding to 50% probe displacement were obtained and Kd values cal-culated according to the Cheng–Prusoff equation.

ND, Not determined

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 2JAK1–JAK3 NK-92 IL-2 pSTAT5 148, 757, 367 6.46 6 0.12 3JAK1–TYK2 U2OS IFN-aB2 pSTAT1 494, 436 6.33 6 0.03 2JAK1–JAK2 HeLa OSM STAT1 reporter 1,045 6.01 6 0.07 4JAK1–JAK2 U2OS IFN-g pSTAT1 3,364 5.47 1JAK2 TF-1 IL-3 pSTAT5 3,524 5.45 1JAK2 BaF3 IL-3 Proliferation 4,546 5.34 6 0.04 3JAK2 UT7-EPO EPO pSTAT5 .10,000 .5 2JAK2 22Rv1 PRL pSTAT5 .10,000 .5 2

IC50 values in cellular assays were determined by plotting the compound concentration versus the effect on the readoutsmentioned. The pIC50 is defined as the negative of the log10 of the compound concentration having a half maximal effect on thereadout.

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levels was measured upon dosing 1 and 10 mg/kg with more pro-nounced reductions at the dose of 10 mg/kg (Fig. 3). For the basalMx2 mRNA levels, a hysteresis between compound peak plasmalevels and maximal inhibition of basal Mx2 mRNA levels wasobserved as expected.The therapeutic potential of GLPG0634 for RAwas evaluated in

a therapeutic setting in the rat collagen-induced arthritis (CIA)model,a well-accepted animal model for RA (19, 25–27). GLPG0634 wasdosed once daily by oral gavage, initially at doses of 3, 10, and 30

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 scorefrom the fifth day of dosing onward (Fig. 4B). Data obtained fromeach of the three studies were normalized to the correspondingvehicle 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-proportionalincreases of maximum blood concentration (Cmax) and area undercurve between 0.3 and 30 mg/kg and correlated with the dose-dependent efficacy (Fig. 4A). A rapid absorption was observed forGLPG0634, with maximal plasma levels achieved ∼1 h afterdosing, for all dose levels tested. Half-life is ∼4–5 h and is in-dependent of dose level. A dose-dependent effect was obtained inall pharmacodynamic readouts. The doses of 1, 3, and 10 mg/kgGLPG0634 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 alreadyfrom the start of dosing (Fig. 4B). Statistically significant reduc-tion of the clinical score at endpoint was obtained for all doses(Fig. 4C). Protection from bone damage was evidenced by a dose-dependent reduction of the Larsen score obtained after x-rayanalysis of the hind paws, with significant effect from 3 mg/kgand onward (Fig. 4D). Similar efficacy was obtained for GLPG0634as for etanercept. Histological analysis of the rat paws was per-formed in a specific region of interest including the talus, navicular,and cuneiform bones (Fig. 4E–H). As compared with the vehiclecontrol group (Fig. 4E), etanercept and GLPG0634 groups (Fig. 4E,4G, 4H) showed a marked reduction of the infiltration of inflam-matory cells while protecting the articular cartilage and bone from1 mg/kg onward.Confirmation of the therapeutic potential of GLPG0634 in CIA

in the rat was provided by studying the compound in the similarmodel in the mouse. In addition to the parameters measured in therat, mouse blood and paw samples were taken with the purpose ofobtaining detailed insight in themechanism of action of GLPG0634in vivo. Dose selection was performed in a dose range–findingexperiment (data not shown), and a dose of 50 mg/kg twice dailyorally was selected for the mechanistic studies. Fig. 5A shows thatthe 50 mg/kg dose provided full protection against inflammationas judged by analysis of the clinical score of paws. Histologicalanalysis of the mice paws showed that GLPG0634 protected boneand cartilage from degradation (Fig. 5B). Immunohistochemistryperformed on the same samples showed that GLPG0634 effectivelyreduced infiltration of T cells (CD3+ cells) and macrophages (F4/

FIGURE 1. JAK1 silencing inhibits IL-6–induced STAT1 activation. IL-

6/sIL-6R–induced STAT1 phosphorylation after JAK1, JAK2, JAK3, TYK2,

GAPDH (GAPD), or nontargeting (non-T) siRNA transfection in HeLa

(A) or HCT116 (B) cells was assessed using AlphaScreen technology. Data

represent the means 6 SD from four data points (duplicate measurements

for two independent siRNA transfections). RLU, relative luminescence

units.

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 JAK2Cell type CD4+ CD4+ CD4+ CD33+

Compounds pIC50 6 SEM (IC50 [nM]; n)JAK1 versus

JAK2 Selectivity

GLPG0634 6.201 6 0.092(629; 7)

5.747 6 0.043(1,789; 5)

5.948 6 0.021(1,127; 6)

4.758 6 0.214(17.453; 7)

28

Tofacitinib 7.130 6 0.119(74; 16)

7.473 6 0.033(33; 9)

7.120 6 0.040(76; 6)

6.130 6 0.179(740; 7)

10

INCB028050 7.631 6 0.169(23.4; 4)

ND ND 7.195 6 0.055(63.8; 6)

3

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 onthe readout) was measured for each volunteer and averaged. Data are represented as mean pIC506 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.

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80+ cells) in the paw (Fig. 5C). The effects on clinical score, boneand cartilage protection, and cell infiltration were similar to theresults obtained by etanercept. Gene expression studies werecarried out in WBCs and paws. Expression levels were increasedin arthritic versus healthy animals for all genes tested (Fig. 5D, 5F,5G). In paws, GLPG0634 reduced the levels of inflammatory andmetalloprotease genes previously linked to disease progression,explaining the beneficial role of GLPG0634 in the mouse CIAmodel (Fig. 5D). The mRNA levels of RANKL were also reducedin line with the decrease in the bone lesion score, suggesting thatGLPG0634 might protect against bone degradation by reducingthe formation and activity of osteoclasts. Cytokine levels in serafrom the CIA mice were measured using Luminex technology. Asfor the gene expression studies, the levels of the protein markersunder study were raised in the serum of diseased versus healthyanimals, with the exception of stem cell factor (Fig. 5E, Supple-mental Table I). GLPG0634 decreased the serum levels of all cyto-kines and chemokines measured, including IL-6, IP-10, XCL1,

and MCP-1 (Fig. 5E). These observations indicate that GLPG0634might affect inflammatory cytokine signaling and chemoattractionof T cells and monocyte/macrophages by reducing these cytokineand chemokine levels. At the mechanistic level, the reduction ofMx1 and Mx2 mRNA levels by GLPG0634 was also observed inmouse paws (Fig. 5F), as observed in the rat (Fig. 3). Of interest,the changes in Mx1 and Mx2 gene expression in WBCs were notaltered by etanercept treatment (Fig. 5G), showing that GLPG0634specifically impacts JAK1 signaling.

DiscussionIn recent years significant advances have been made in under-standing the link between the different JAK family members andtheir involvement in autoimmune, inflammatory, and oncologicaldiseases. The first generation of small-molecule inhibitors hasfurther substantiated the therapeutic potential of JAK inhibitors inthe aforementioned diseases. In this study we examined GLPG0634,a novel and selective JAK inhibitor that demonstrates selectivity forJAK1 in a cellular environment. GLPG0634 was identified in akinase-focused library screen and belongs to the triazolopyridinecompound class. Characterization of GLPG0634 at the biochemicallevel indicated a selective inhibition of JAK1 and JAK2 over JAK3and TYK2, whereas cellular and WBAs revealed a selectivity forJAK1- over JAK2-dependent signaling in a cellular environment.GLPG0634 efficiently blocks cytokine-induced signaling cascadesinvolving JAK1 in several cell lines as well as in human primarycells. 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). Thesein vitro findings translate to pharmacodynamic readouts in rodentsshowing that JAK1 signaling is blocked in vivo as measured bya reduction of Mx2 mRNA levels. Furthermore, GLPG0634 dose-dependently reduces inflammation, cartilage, and bone degrada-tion in the CIA model in rats and mice.The biochemical selectivity profile of GLPG0634 (rank order of

potency JAK1 ∼ JAK2 . TYK2 . JAK3) was a poor predictor ofthe selectivity determined in cellular and WBAs. TYK2 enzymeactivity in biochemical assays was inhibited with 11- and 4-foldlower potency versus JAK1 and JAK2, whereas JAK3 enzymeactivity was inhibited with a much lower potency. This differencedid not translate into a higher cellular potency of inhibiting INF-a/JAK1/TYK2 signaling versus IL-2/JAK1/JAK3 signaling (TablesII, III). It indicates that JAK1 inhibition is in large part responsiblefor the potency of GLPG0634 in a cellular environment. A JAKinhibitor likely requires equipotent inhibition of JAK1 and TYK2 orJAK3, or even inverse selectivity for the latter enzymes to surpasspotency derived from JAK1 inhibition. This is demonstrated fortofacitinib, which shows 3-fold selectivity for JAK3 over JAK1 inbiochemical 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 ahigh selectivity for JAK1 over JAK2 in cellular and human WBAswas unexpected. Interestingly, a similar selectivity shift wasobserved for tofacitinib but not for baricitinib when testing thesemolecules in parallel with GLPG0634 (Table III). This is re-markable, as tofacitinib and baricitinib share the same chemicalscaffold and show similar potencies toward JAK1 and JAK2 inbiochemical assays (9, 10, 28). At present no univocal explanationfor the discrepancy between the biochemical and cellular/wholeblood JAK inhibition profiles can be provided. A potential ex-planation may be linked to the differences between the bio-chemical and cellular assay formats. First, the biochemical assayrelies on kinase activity of a purified truncated protein containingthe C-terminal quarter of the JAK proteins, including the JH1

FIGURE 2. GLPG0634 inhibits the differentiation of Th1, Th2, and

Th17 cells. Naive human CD4+ T cells (N) were stimulated with anti-CD3/

anti-CD28 Abs in the absence (V) or presence of 10, 1, or 0.1 mM

GLPG0634 (10, 1, 0.1) or 10 mM tofacitinib (T) using either Th1, Th2, or

Th17 polarizing conditions as described in Materials and Methods. On day

5, the expression of lineage-specific markers was assessed by quantitative

RT-PCR. Results were normalized using 18S transcripts and represent

relative expression measured on duplicate data points 6 SD. Effect on

Th1, Th2, and Th17 cell differentiation was evaluated by quantification of

IFN-g mRNA, IL-13 mRNA, or IL-17F mRNA expression. Data are

representative of independent experiments performed with naive CD4+

T cells isolated from two donors.

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kinase domain. In contrast, in a cellular environment wild-typefull-length JAKs comprise the regulatory JH2 pseudokinase do-main, Src homology 2 domains (JH3–JH4), and the amino ter-minal (NH2) FERM domain (JH4–JH7). Additionally, JAKs arepart of a larger complex, including the cytoplasmic receptor tails,STATs, and other proteins (2, 4). Second, the endogenous JAKsare subject to posttranslational modifications such as phosphory-lation. A differential tyrosine phosphorylation status can give riseto different IC50 values as exemplified for the non- versus mono-or diphosphorylated forms of the TYK2 kinase domain (29).Third, differential negative or positive feedback mechanisms forJAK1- versus JAK2-dependent signaling by means of phosphatases,members of the SOCS or SH2B families, can have a different im-pact on the amplitude and kinetics of JAK enzyme activity andsignaling output (30–33). Finally, different small molecules might

induce subtle changes in the three-dimensional space of the ATP-binding pockets of the JAKs, leading to differential kinase activityand/or protein–protein interactions. Crystallography of JAK proteinsharboring more than the JH1 kinase domain in complex with smallmolecule inhibitors will provide more insight here.Oral administration of GLPG0634 in the mouse and rat resulted

in good plasma exposure. GLPG0634 plasma levels in the rat couldbe correlated with reduction of Mx2 mRNA levels in WBCsreflecting target engagement and inhibition of JAK/STAT signalingin vivo. Additional testing of GLPG0634 in vivo in rodent CIAmodels in a therapeutic setting revealed that daily oral adminis-tration resulted in a dose-dependent reduction of inflammation andprotected bone and cartilage from degradation. In view of thepathological roles of Th1 effector T cells in chronic inflammationand autoimmune disorders and the dose-dependent reduction of

Table IV. Rodent pharmacokinetics for GLPG0634

Parameter (Unit)

Mouse Rat

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(ng/h/ml)

347 1893 739 (2) 1,681 (8)

T1/2 (h) 2.5 1.7 1.6 (3) 3.9Cl (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.

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.

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Th1 differentiation by GLPG0634 in vitro, its efficacy in the CIAmodels is in line with these in vitro observations (34). QuantitativePCR analysis of disease-related inflammatory markers in bloodand paws showed a pronounced reduction of these markers byGLPG0634. This was confirmed at the protein level by measuringcytokine levels in sera of arthritic mice treated with GLPG0634versus vehicle-treated animals. Histological and x-ray (Larsenscore) analysis of mice and rat paws revealed reduced bone andcartilage degradation coinciding with reduced infiltration by T cellsand macrophages as shown in the mouse CIA study. The efficacyof GLPG0634 in the murine CIA models and the changes in thedisease-related biomarkers are of similar magnitude as the relativelyhigh dose of etanercept. Differing from the efficacy observed inrodent CIA models, only a moderate efficacy was obtained in the

mouse experimental allergic encephalomyelitis model of multiplesclerosis (Supplemental Fig. 1). Because high efficacy in thismodel likely requires passage through the blood–brain barrier,the observation that GLPG0634 is a substrate for P-glycoprotein(efflux ratio in Caco2 permeability assay decreased from 16 to 6 inthe presence of verapamil; data not shown) could explain its mod-erate activity.Observing efficacy for GLPG0634 in the rat CIA model at doses

as low as 0.1 and 0.3 mg/kg was surprising. This is unlikely due tooff-target effects in view of the clean profile of GLPG0634 in kinasepanels. GLPG0634 showed.100-fold selectivity over other kinasesrepresenting the human kinome (175 kinases tested) with the ex-ception of FLT3, FLT4, and CSF1R, for which selectivity still was.25-fold (Supplemental Table II). Speculative explanations include

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).

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tissue-specific compound accumulation, active metabolites, or evenother mechanisms, but a final explanation remains to be determined.When GLPG0634 was dosed at 1 mg/kg or higher pharmacokinetic

and pharmacodynamic data correlated well. The Cmax levelsmeasured for the 1 and 3 mg/kg doses of GLPG0634 in the ratpharmacokinetics/pharmacodynamics and CIA in vivo studies(240–844 nM; Figs. 3A, 3B, 4A) were close to or exceeded theJAK1-dependent IL-6/pSTAT1 human WBA IC50 value (623 nM;Table III). Preliminary evidence obtained by measuring a JAK1biomarker in a phase I clinical trial for GLPG0634 indicated thattarget 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 tofacitinibresulted in Cmax levels in human volunteers (41–52 ng/ml) that are∼2-fold above the JAK1-dependent WBA IC50 value (23 ng/ml or74 nM; Table III) and will not completely inhibit JAK1 and/or JAK3for most of the day (36). Hence, incomplete inhibition of JAK1 andthe wide range of cytokine signaling it serves can provide therapeuticefficacy, thereby decreasing the risk of potential JAK1-mediated side-effects such as immune suppression.The high selectivity of GLPG0634 for JAK1 versus JAK2 ob-

served in vitro is supported by a number of observations made inpatient 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 cholesteroland lipids), or other safety parameters (electrocardiogram, vital signs)were noted (37). At this dose, JAK1 signaling was suppressed for24 h whereas JAK2 signaling was not influenced (37). Moreover,dosing GLPG0634 to 24 RA patients for 4 wk at daily doses of200 mg showed efficacy on ACR20 scores and on the secondaryendpoints of DAS28 and serum C-reactive protein levels withoutadverse events (38). Instead of anemia, which could have beenindicative for inhibition JAK2 signaling impairing hematopoiesis,a small increase in hemoglobin levels was noted as expected withimprovement in disease.In conclusion, GLPG0634 is a promising drug candidate for the

future treatment of autoimmune and inflammatory disorders suchas RA.

AcknowledgmentsWe acknowledge the expert technical or editorial assistance of Cecile

Belleville, Marie Christine Cecotti, Christelle David, Francois 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

also thank Sonia Dupont (Galapagos SASU), Filip Beirinckx (Galapagos

NV), Prof. V. Goffin (Faculte de Medecine Necker, Paris, France) and

collaborators for contributions to cellular assay development and/or com-

pound testing. AbbVie has provided funding to Galapagos for development

of GLPG0634.

DisclosuresAll authors are employees of the Galapagos group (which includes BioFo-

cus) and are eligible to receive stock options.

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FIGURE 5. GLPG0634 is efficacious in a mouse therapeutic CIA model.

Mice that developed arthritis were orally treated with vehicle (orange lines

and bars), GLPG0634 twice a day at 50 mg/kg (dark green lines and bars), or

i.p. with etanercept three times a week at 10 mg/kg (blue lines and bars). (A)

Clinical score readout. *p, 0.05, **p, 0.01, ***p, 0.001 versus vehicle,

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and in circulating leukocytes at the end of the 2-wk treatment (G).

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