Vol. 302, Issue 1, 153-162, July 2002

₄₇/₄₁ Dual Integrin Antagonists Block ₄₇-Dependent Adhesion under Shear Flow

Linda A. Egger, Usha Kidambi, Jin Cao, Gail Van Riper, Ermengilda McCauley, Richard A. Mumford, Suzanne Amo, Russell Lingham, Thomas Lanza, Linus S. Lin, Stephen E. de Laszlo, David N. Young, Ihor E. Kopka, Sharon Tong, Bill Pikounis, Evelyn Benson, Sarah Warwood, Robert F. Bargatze, William K. Hagmann, John A. Schmidt and Patricia A. Detmers

Pharmacology (L.A.E., U.K., P.A.D.), Immunology and Rheumatology (G.V.R., E.M., R.A.M., S.A., R.L.), Medicinal Chemistry (T.L., L.S.L., S.E.d.L., D.N.Y., I.E.K., W.K.H.), Basic Chemistry and Analytical Support (S.T.), and BioMetrics Research (B.P.), Merck, Rahway, New Jersey; LigoCyte Pharmaceuticals (E.B., S.W., R.F.B.), Bozeman, Montana; and Aventis (J.A.S.), Bridgewater, New Jersey

The ₄ integrin, ₄₇, plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of ₄₁ and ₄₇, N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of ₄₇-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human ₄₇ to an ¹²⁵I-MAdCAM-Ig fusion protein with IC₅₀ values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to ₄₁ or ₄₇ on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human ₄₇-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC₅₀ values of 0.1 and 1 µM, respectively. Intravital microscopy was used to quantitate ₄-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn²⁺ to activate ₄₇ binding to ligand, anti-₄ monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-₁ mAb did not block adhesion, demonstrating that this interaction was dependent on ₄₇. TR14035 blocked adhesion to HEVs [ED₅₀ of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, ₄₇/₄₁ antagonists blocked ₄₇-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.