Volume 19, Issue 9, Pages 1142-1151 (September 2012) A Potent and Selective S1P1 Antagonist with Efficacy in Experimental Autoimmune Encephalomyelitis  Jean Quancard, Birgit Bollbuck, Philipp Janser, Daniela Angst, Frédéric Berst, Peter Buehlmayer, Markus Streiff, Christian Beerli, Volker Brinkmann, Danilo Guerini, Paul A. Smith, Timothy J. Seabrook, Martin Traebert, Klaus Seuwen, René Hersperger, Christian Bruns, Frédéric Bassilana, Marc Bigaud  Chemistry & Biology  Volume 19, Issue 9, Pages 1142-1151 (September 2012) DOI: 10.1016/j.chembiol.2012.07.016 Copyright © 2012 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2012 19, 1142-1151DOI: (10. 1016/j. chembiol. 2012 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Schematic Representation of the Optimization Leading to the Discovery of NIBR-0213 (A) Starting points of the optimization. (B) Prototypic compounds used to test the potential of replacing the sulfonamide moiety to improve PK properties. (C) Further optimization of the potency. (D) Discovery of NIBR-0213 and its diastereoisomer. See also Figures S2, S3, S4, S5, and S6. Chemistry & Biology 2012 19, 1142-1151DOI: (10.1016/j.chembiol.2012.07.016) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Pharmacodynamic and Pharmacokinetic Effects of Compounds 1, 7, and NIBR-0213 in the Rat Changes in PBL counts and drug blood levels observed in rats treated orally with compound 1, 7, or NIBR-0213 (n = 3 each, mean ± SEM). (A and B) Compound 1, 7, and NIBR-0213 were given at 100, 30, and 30 mg/kg, respectively to Lewis rats. (C and D) NIBR-0213 was given at 1, 3, 10, and 30 mg/kg to Wistar rats. (E and F) Dose-related reduction in PBL counts (E) and changes in EBD accumulation in lungs (F) observed in Lewis rats at 6 hr after oral treatments with NIBR-0213. Each bar represents an independent treatment group (n = 3–10, mean ± SEM). See also Figure S1. Chemistry & Biology 2012 19, 1142-1151DOI: (10.1016/j.chembiol.2012.07.016) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 In Vitro Characterization of NIBR-0213 (A) S1P receptor profile of NIBR-0213: IC50 values for NIBR-0213 in GTPγS and Ca2+ mobilization assays. Each IC50 value is the average of at least three measurements (mean ± SEM). (B) Schild plot analysis of NIBR-0213 using the S1P1 agonist AUY954 in the GTPγ35S assay. (C) FTY720-phosphate-induced S1P1 receptor internalization. Internalization of S1P1 receptor in CHO cells treated with either FTY720-phosphate alone at 10, 100, or 1,000 nM (n = 3, mean ± SEM), NIBR-0213 alone at 1,000 nM (n = 2), or NIBR-0213 at 1,000 nM followed by FTY720-phosphate at 100 nM (n = 2). (D) GIRK channel activation: representative recording of membrane current from an atrial myocyte after superfusion with 30 μM NIBR-0213 compared to 20 μM Ach. Mean GIRK current amplitudes were normalized to responses evoked by 20 μM ACh in the same cell (n = 4; mean ± SEM). Chemistry & Biology 2012 19, 1142-1151DOI: (10.1016/j.chembiol.2012.07.016) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Efficacy of NIBR-0213 and Fingolimod in a Mouse Therapeutic EAE Model Monitoring of daily EAE clinical scores (A and B) and changes in body weights (C and D) observed in MOG/CFA/Pertussis toxin-treated mice C57BL/6 mice. At peak of disease, the mice were treated orally with either NIBR-0213 (A and C), fingolimod (B and D), or their respective vehicle (n = 10 each, mean ± SEM). Chemistry & Biology 2012 19, 1142-1151DOI: (10.1016/j.chembiol.2012.07.016) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Hematological and Histological Changes Observed in the Mouse EAE Study Peripheral blood lymphocytes, neutrophils, and monocytes counts at termination in all C57BL/6 mice-EAE groups, as measured by an automated hematology analyzer (n = 8 each, mean ± SEM) (A). Histological analysis (representative examples and scorings) of spinal cord samples collected in all C57BL/6 mice-EAE groups: H&E staining for inflammation (B); Iba-1 staining for macrophages/activated microglia (C); solochrome staining for loss of myelin (D). Arrows point to areas of pathology. Scale bar represents 200 μm. Chemistry & Biology 2012 19, 1142-1151DOI: (10.1016/j.chembiol.2012.07.016) Copyright © 2012 Elsevier Ltd Terms and Conditions