ABSTRACT
Mitsunobu reactions were employed to link t-butyl esters of α4 integrin inhibitors at each of the termini of a three-arm, 40 kDa, branched PEG. Cleavage of the t-butyl esters using HCO2H provided easily isolated PEG derivatives, which are potent α4 integrin inhibitors, and which achieve sustained levels and bioactivity in vivo, following subcutaneous administration to rats.
Subject(s)
Integrin alpha4/chemistry , Polyethylene Glycols/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Esters , Half-Life , Humans , Injections, Subcutaneous , Integrin alpha4/immunology , Integrin alpha4/metabolism , Jurkat Cells , RatsABSTRACT
A series of (S)-2-(2-(diethylamino)-5-(N-alkyl-N-sulfonamido)pyrimidin-4-ylamino)-3-(4-(carbamoyloxy)phenyl)propanoic acid is discovered as orally available VLA-4 antagonists. Representative compounds 11b and 11p showed efficacy in multiple in vivo animal models. The in vitro selectivity of 11p is also described.
Subject(s)
Antirheumatic Agents/pharmacology , Integrin alpha4beta1/antagonists & inhibitors , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Animals , Antirheumatic Agents/administration & dosage , Antirheumatic Agents/chemical synthesis , Arthritis, Experimental/drug therapy , Asthma/drug therapy , Cell Adhesion/drug effects , Collagen Type II/antagonists & inhibitors , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Fibronectins/chemistry , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Conformation , Multiple Sclerosis/drug therapy , Pyrimidines/administration & dosage , Pyrimidines/chemical synthesis , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/chemical synthesisABSTRACT
Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4ß7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg(2+) was greater than Ca(2+) at promoting cell adhesion. This homogeneous and label-free format exhibited low variability with a calculated Z' of 0.6. In addition to measuring α4ß7-mediated 8866 cell adhesion to MadCAM, the authors also demonstrate that this platform can measure adhesion of Jurkat cells expressing α4ß1 to the vascular cell adhesion molecule. Thus, the SRU BIND platform is widely applicable to measuring cell adhesion events mediated by other integrins binding to their receptors in an assay format that is amenable to high-throughput screening.
