Modulation of experimental autoimmune encephalomyelitis by VLA-2 blockade.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Adhesion molecules play important roles in cell-cell and cell-extracellular matrix (ECM) interactions in inflammation. Blocking the interaction between inflammatory cells and vascular endothelia can prevent cell entry into tissues and harmful inflammatory responses, that is, autoimmunity, but could also limit immunosurveillance by anti-viral T cells in sites of infection or latency. Development of progressive multifocal leukoencephalopathy in patients treated with antibody against very late antigen (VLA)-4 prompted us to explore an alternative therapeutic approach. We used an antibody against the integrin alpha2, VLA-2, that interacts with ECM, not vascular endothelium. SJL/J mice were sensitized with myelin proteolipid protein (PLP)(139-151) peptide to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Treatment of mice with VLA-2 antibody suppressed clinical signs and CNS inflammation of EAE, when antibody was given immediately after disease onset. In contrast, VLA-4 or VLA-2 antibody treatment of mice during the priming or remission phase of EAE had minor effects on the disease's clinical course. No differences were found in lymphoproliferative responses to PLP(139-151) among treatment groups. Data suggest that blocking cell-ECM interactions can be an alternative therapy for MS.

An in vivo approach to structure activity relationship analysis of peptide ligands.

PURPOSE: The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif. MATERIALS AND METHODS: The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX(10)C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy. RESULTS: The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion. CONCLUSIONS: Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature.

Synthesis and SAR of N-benzoyl-L-biphenylalanine derivatives: discovery of TR-14035, a dual alpha(4)beta(7)/alpha(4)beta(1) integrin antagonist.

alpha(4)beta(1) and alpha(4)beta(7) integrins are key regulators of physiologic and pathologic responses in inflammation and autoimmune disease. The effectiveness of anti-integrin antibodies to attenuate a number of inflammatory/immune conditions provides a strong rationale to target integrins for drug development. Important advances have been made in identifying potent and selective candidates, peptides and peptidomimetics, for further development. Herein, we report the discovery of a series of novel N-benzoyl-L-biphenylalanine derivatives that are potent inhibitors of alpha4 integrins. The potency of the initial lead compound (1: IC(50) alpha(4)beta(7)/alpha(4)beta(1)=5/33 microM) was optimized via sequential manipulation of substituents to generate low nM, orally bioavailable dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. The SAR also led to the identification of several subnanomolar antagonists (134, 142, and 143). Compound 81 (TR-14035; IC(50) alpha(4)beta(7)/alpha(4)beta(1)=7/87 nM) has completed Phase I studies in Europe. The synthesis, SAR and biological evaluation of these compounds are described.