A Benzothiophene Inhibitor of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Inhibits Tumor Necrosis Factor  Production and Has Oral Anti-Inflammatory Efficacy in Acute and Chronic Models of Inflammation □ S Robert J. Mourey, Barry L. Burnette, Sarah J. Brustkern, J. Scott Daniels, Jeffrey L. Hirsch, William F. Hood, Marvin J. Meyers, Stephen J. Mnich, Betsy S. Pierce, Matthew J. Saabye, John F. Schindler, Sarah A. South, Elizabeth G. Webb, Jian Zhang, and David R. Anderson Departments of Discovery Biology (R.J.M., B.L.B., S.J.B., J.L.H., W.F.H., S.J.M., M.J.S., J.F.S., E.G.W., J.Z.), Medicinal Chemistry (M.J.M., B.S.P., D.R.A.), and Pharmacokinetics, Dynamics, and Metabolism (J.S.D., S.A.S), Inflammation Research Unit, Pfizer Global Research and Development, Chesterfield, Missouri Received January 19, 2010; accepted March 16, 2010 ABSTRACT Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cyto- kines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipo- polysaccharide (LPS)-stimulated tumor necrosis factor  (TNF) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF- 3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12- tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8- one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K i = 3 nM) with good selec- tivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNF production with similar activity (IC 50 = 160 nM). PF-3644022 blocks TNF and IL-6 production in LPS-stimulated human whole blood with IC 50 values of 1.6 and 10.3 M, respectively. Inhibition of TNF in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNF model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNF production in the acute model and inhibition of paw swelling in the chronic model is observed with ED 50 values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C min higher than the EC 50 measured in the rat LPS-induced TNF model. Rheumatoid arthritis (RA) is a chronic inflammatory dis- ease characterized by an imbalance of proinflammatory and anti-inflammatory cytokines, autoimmunity, joint inflamma- tion, and eventual joint destruction (McInnes and Schett, 2007). Evidence supporting the role of the proinflammatory cytokines TNF, IL-1, and IL-6 has been demonstrated in both animal models and human clinical trials (Dayer et al., 2001; Scott and Kingsley, 2006; Hennigan and Kavanaugh, 2008). Use of biologic therapeutics that neutralize these cy- This study was sponsored by Pfizer Inc. Portions of this work were presented as: Daniels JS, Lai Y, Davis JW, South SA, Stevens JC, Mourey RJ, and Anderson DR (2008) Inhibition of hepatobili- ary transporters by a novel kinase inhibitor contributes to liver toxicity in nonclinical species at Great Lakes Drug Metabolism Discussion Group Meet- ing; 2008 May 1–2; Indianapolis, IN. Great Lakes Drug Metabolism Discussion Group, University of Michigan, Ann Arbor, MI. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.110.166173. □ S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: MAPK, mitogen-activated protein kinase; MAPKAP, MAPK-activated protein; MK2, MAPKAP kinase 2; MNK, MAPK-interact- ing kinase; PF-3644022, (10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one; RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL-1, interleukin-1; PRAK, p38-regulated and activated kinase; LPS, lipopolysaccharide; SCW, streptococcal cell wall; rSCW, rat SCW; MSK, mitogen- and stress-activated protein kinase; HSP27, heat shock protein 27; hPBMC, human peripheral blood mononuclear cell; HWB, human whole blood; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PK-PD, pharma- cokinetic-pharmacodynamic; BE, biochemical efficiency; DMSO, dimethyl sulfoxide; JNK, c-Jun N-terminal kinase; ERK, extracellular signal- regulated kinase; AUC, area under the curve; CL, clearance; ff, free fraction. 0022-3565/10/3333-797–807$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 166173/3590513 JPET 333:797–807, 2010 Printed in U.S.A. 797 http://jpet.aspetjournals.org/content/suppl/2010/03/17/jpet.110.166173.DC1.html Supplemental material to this article can be found at: at ASPET Journals on February 18, 2016 jpet.aspetjournals.org Downloaded from