Small Molecule Inhibitors of Phagocytosis for Treatment of Immune Cytopenias D. R. Branch 1, 2, M. K. Purohit 3, I. Scovell 2, A. Neschadim 2, Y. Katsman 2, L. P. Kotra 3 1 Medicine, University of Toronto, Toronto, ON, Canada. 2 Research & Development, Canadian Blood Services, Toronto, ON, Canada. 3 Center for Molecular Design and Preformulations, Toronto General Research Institute, Toronto, ON, Canada. CompoundKB-CodeActivity 5μM SEM 1KB-3-13%10% 3KB-3632%7% 7KB-3145%4% 15KB-5216%11% 14KB-5314%8% 18KB-6920%14% 20KB-634%3% 22KB-6712%8% 24KB-662%4% 28KB-5816%3% 32KB-65-8%17% 37KB-5945%5% 41KB-5417%2% 46KB-6033%2% 47KB-6110%5% 49KB-566%4% 50KB-5758%4% 53KB-6215%14% Biologic activity of second generation derivatives Biologic activity of first generation derivatives at 5μM In vivo activity to ameliorate experimental ITP  We have synthesized a number of compounds based on a pyrazole core structure that have ability to inhibit Fc receptor-mediated phagocytosis.  Thiol compounds were found to be less efficacious then disulfide linked pyrazole derivatives.  Disulfide linked pyrazole derivatives were shown to have some efficacy for amelioration of experimental murine ITP.  Second generation pyrazole derivatives lacking disulfide linkage show promise for inhibition of phagocytosis and are under further study and derivation.ResultsConclusions Immune cytopenias are clinical conditions where patients make antibodies to specific hematopoietic cells in the blood. In these conditions, the cells become coated with antibodies and are subsequently recognized by the Fc  -receptors (FcγR) on the mononuclear phagocyte membrane. Such recognition by monocyte-macrophages (M  ) results in the phagocytosis and intracellular destruction of these cells. This process can create serious and sometimes life-threatening complications for these individuals. Examples include warm autoimmune hemolytic anemias (AIHA; involving the phagocytosis of antibody-coated red blood cells), idiopathic/immune(autoimmune) thrombocytopenia (ITP; the phagocytosis of antibody-coated platelets) and alloimmune hemolytic anemias (involving the phagocytosis of donor red cells transfused into patients having alloantibodies to the donor red cell antigens (HTR) and babies at risk for hemolytic disease of the newborn (HDN) due to the maternal antibodies crossing the placenta). In ITP, platelets are destroyed by autologous antibodies. Current therapies for the treatment of severe cases of ITP include administration of steroids, splenectomy, Rituximab, thrombopoientin receptor agonists and administration of immunoglobulins, IVIg and anti-RhD. IVIg and anti- RhD are a limited resource and costly due to their acquisition from human donations. Furthermore, immunoglobulin therapies haven’t been shown to be efficacious in other immune cytopenias. Thus, finding novel, cheaper, safer and more broadly efficient ways of treating immune cytopenias is warranted. As the destruction of the cells is due to recognition of the Fc portion of the opsonizing antibody by the Fc receptor on phagocytes, a potential treatment modality would be to develop inhibitors of this antibody-mediated Fc receptor-mediated phagocytosis. A collection of molecules carrying sulfhydryl and disulfide functional groups were evaluated for their ability to inhibit the phagocytosis of opsonized blood cells by macrophages. Among these compounds, a pyrazole derivative exhibited weak activity at 1 mM concentration against phagocytosis of the blood cells. A library of derivatives designed based on this prototype molecule has led to the discovery that the homodimers containing a disulfide bridge were more potent inhibitors of the blood cell phagocytosis and that substitution for the disulfide linkage is feasible. A concept of designing structures based on the disulfide bridge but with non-thiol or non-disulfide bridge carrying ligands to inhibit phagocytosis and thus have potential efficacy in the treatment of immune cytopenias has emerged. Compounds lacking thiol or disulfide structures have a higher potential for drug development for clinical use. Thus, a major objective of our work is to design small molecule inhibitors of phagocytosis based on the structure-activity relationships of the pyrazole class of molecules, including non-thiol carrying compounds replacing the disulfide bridge with carbon, nitrogen and oxygen carrying linkers and to evaluate these small molecules as inhibitors of phagocytosis using both in vitro and in vivo tests and to select candidate molecules with high solubility and efficacy for pre-clinical testing.Background Objective Phagocytosis assay (A) and prototype thiol inhibitor (B) From: Rampersad et al., Transfusion 2005;45:384 Chemical structures of first generation derivatives inhibiting Fc receptor mediated phagocytosis of opsonized human red blood cells7 Strategic structural modifications around the hit molecule 7 Synthesis of compound 37Synthesis of compounds Synthesis of disulfide linked pyrazole derivatives Approach & Materials