Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.
Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.
Rajagopalan
Lakshman E
LE
Department of Discovery Biology, Inflammation Research Unit, Pfizer Global Research and Development, Pfizer Inc., Chesterfield, Missouri 63017, USA. leraja22@gmail.com
Davies
Michael S
MS
Kahn
Larry E
LE
Kornmeier
Christine M
CM
Shimada
Hideaki
H
Steiner
Toni A
TA
Zweifel
Ben S
BS
Wendling
Jay M
JM
Payne
Maria A
MA
Loeffler
Richard F
RF
Case
Brenda L
BL
Norton
Monica B
MB
Parikh
Mihir D
MD
Nemirovskiy
Olga V
OV
Mourey
Robert J
RJ
Masferrer
Jaime L
JL
Misko
Thomas P
TP
Kolodziej
Stephen A
SA
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001549:Benzamides / Q000493:pharmacokinetics / Q000494:pharmacology*; D001682:Biological Availability; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D002465:Cell Movement / Q000187:drug effects; D018925:Chemokines / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000517:prevention & control; D016209:Interleukin-8 / Q000096:biosynthesis; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D018994:Myosin Light Chains / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D054390:Receptors, CCR2 / Q000096:biosynthesis; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D001549:Benzamides; D015815:Cell Adhesion Molecules; D018925:Chemokines; D016209:Interleukin-8; D018994:Myosin Light Chains; C549912:N-methyl-3-(((4-pyridin-4-ylbenzoyl)amino)methyl)benzamide; D047428:Protein Kinase Inhibitors; D054390:Receptors, CCR2; D054460:rho-Associated Kinases