Title : Drug discovery for inhibitors of phagocytosis for use in immune cytopenias
Abstract:
Immune cytopenias are characterised by low numbers of certain hematopoietic cells and include immune thrombocytopenia (ITP), autoimmune hemolytic anaemia (AIHA), hemolytic transfusion reactions (HTR), hemolytic disease of the fetus and newborn (HDFN), and autoimmune neutropenia (AIN). Immune cytopenias are caused when the body produces antibodies that target specific hematopoietic cells. Inducing extravascular antibody-mediated phagocytosis by monocyte-macrophages in the spleen and/or liver through their activating Fcγ receptors (FcγRs), causing the destruction of the opsonized blood cells. From a chemical library containing 5000 molecules, 80 different compounds were selected and tested for their ability to inhibit in vitro phagocytosis. The selection criteria for these compounds was based on the pyrazole core structures that had been established from previous research. Compounds were tested for their ability to prevent the phagocytosis of anti-D-opsonized Rh-positive red blood cells (RBCs). IVIG was used as a control inhibitor at a concentration of 1 mg/mL, which corresponds to a concentration of 6.5 µM. In addition to this, dose-inhibitory titration studies were carried out in order to calculate IC50s. Peripheral blood mononuclear cells (PBMCs), liver HEPG2 cells, and kidney HEK293 cell lines were used to measure LDH release, MTT viability, and apoptosis in order to evaluate the toxicity of concentrations up to 250 µM. RBC rosettes (the attachment of antibody-opsonized RBCs' Fcγ to FcγRs) were evaluated in a room temperature test to learn more about the mechanism of phagocytosis suppression. Four molecules at 5 µM inhibited phagocytosis by 69%, whereas IVIG at 6.5 µM inhibited it by 100%. Only two compounds with IC50 values between 3 and 4 µM in the dose-inhibitory response curves showed negligible toxicity across all of the tests they were subjected to. The formation of rosette structures by antibody-opsonized RBCs was prevented by both compounds, as well as by IVIG. There was no evidence that the small molecules blocked FcγR activity, using monoclonal antibodies to FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Subsequently, these lead compounds were chosen to examine their effectiveness in ameliorating experimental ITP in a mouse model. In vivo testing revealed that 1 mg/kg of a single molecule was just as effective as 1000 mg/kg of IVIG in ameliorating the ITP. There was no evidence of cooperation or synergy of the small molecules with IVIG. Thus, we have identified small molecule lead compounds with low toxicity and high in vitro and in vivo efficacy as potential phagocytosis inhibitors that will be further examined in additional pre-clinical tests.
Audience Takeaway:
Replacement of IVIG with a drug that can treat all immune cytopenias would have a dramatic impact on the utilization of human plasma, the cost of continued use of IVIG, and clinical practice. At the very least, a novel therapeutic that can be used in an acute situation until other therapies can work would be useful.
