ABSTRACT
Compound I, a novel small molecule antagonist (Kd=6 nM) of human lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) was tested for activity in a humanized mouse model of delayed-type hypersensitivity (trans vivo delayed-type hypersensitivity). Trans vivo delayed-type hypersensitivity is a model for testing compounds with human targets in mice. Tetanus toxoid and 7-10x10(6) human peripheral blood mononuclear cells from tetanus-sensitized donors were coinjected into footpads of naive mice. Footpads were measured before and 24 h later. Injection of peripheral blood mononuclear cells plus antigen resulted in swelling of 0.178-0.254 mm, significantly greater than peripheral blood mononuclear cells or tetanus toxoid alone (P<0.05). Preincubation of peripheral blood mononuclear cells with anti-human major histocompatibility complex class II (MHCII) or anti-human LFA-1 monoclonal antibody (mAb), but not anti-mouse MHCII or anti-mouse LFA-1 mAb, significantly inhibited the response. Compound I inhibited footpad swelling in a dose related manner (0.1-100 mg/kg, p.o.; ED50 approximately 1 mg/kg), whereas its enantiomer had no effect. These data demonstrate the oral efficacy of a novel antagonist of LFA-1 in trans vivo delayed-type hypersensitivity.
Subject(s)
Hypersensitivity, Delayed/prevention & control , Imidazoles/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocyte Function-Associated Antigen-1/drug effects , Administration, Oral , Animals , Antibodies, Monoclonal , Cell Proliferation , Dose-Response Relationship, Drug , Edema/immunology , Edema/prevention & control , Female , Hypersensitivity, Delayed/immunology , Imidazoles/administration & dosage , Imidazoles/pharmacokinetics , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/pharmacokinetics , In Vitro Techniques , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Major Histocompatibility Complex/immunology , Mice , Mice, Inbred C57BL , Mice, SCID , Models, Animal , Tetanus Toxin/immunologyABSTRACT
The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.
