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Treatment of Inflammatory Diseases Selective Inhibitor of JAK1, for The 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 signal transduction of many type I and type II inflammatory cytokine 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 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: 000–000. he JAKs are cytoplasmic tyrosine kinases 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 kinase inhibitors thritis; Cmax, maximum blood concentration; EPO, erythropoietin; OSM, oncostatin M; 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. Tofacitinib and baricitinib 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 enzyme with compound at RT for 60 min, prior + to the addition of ATP. T cells using a naive CD4 T cell isolation kit II (Miltenyi Biotec). Isolated naive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 mg/ml) K determination. Dissociation constants
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