Guangwei Yang

Small-molecule Factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Assessment of complement-mediated bacterial killing and the effect of a small molecule factor D inhibitor in vitro

number: 62 XXVI International Complement Workshop, Kanazawa, Ishikawa, Japan, September 4-8, 2016 a. Percentages were calculated relative to NHS incubated at 37oC b. Percentages were calculated relative to the depleted serum fully reconstituted with the missing component(s) at 37oC ACH-4471, fD inhibitor NHS, normal human serum Dpl, depleted serum Neg, negative Pos, positive Heated, NHS heated at 56oC for 30 min INTRODUCTION ACH-4471, fD inhibitor NHS, Normal Human Serum Dpl, Depleted PI, Propidium Iodide FL, Fluorescence Serum/ Treatment % Phagocytic Activity Monocyte Granulocyte 0◦C Neg control 0 a 0a 37◦C Pos control 100 a 100a C1q-Dpl 98b 82b fD-Dpl 99b 144b C1q&fDDpl 31 b 30b C2-Dpl 68b 73b C4-Dpl 55b 80b C5-Dpl 67b 82b Heated 2b 3b 0.1 μM ACH-4471 106 a 90a 1 μM ACH-4471 82 a 82a 10 μM ACH-4471 102 a 91a Monocytes Granulocytes METHODS