Screening chemical libraries for the development of oral treatments for bleeding disorders

• A high-throughput screening of 2300 chemical compounds allowed to identify adapalene as a procoagulant on severe hemophilia A plasma. • The mechanism of action of adapalene involves Factor XII activation. Hemophilia is a rare bleeding disorder due to Factor VIII (FVIII) or Factor IX (FIX) deficiency respectively involved in hemophilia A (HA) or B (HB). Treatment has long relied on invasive intravenous infusions of the missing factor for prophylaxis or bleeding treatments. Despite significant progress in patient care, there is still no oral procoagulant drug available for hemophiliacs. Such an oral procoagulant could benefit to other inherited and acquired bleeding disorders, as well as anticoagulants-induced bleedings. To find chemical compounds that could become potential future orally-administered procoagulants, we designed a hierarchical high-throughput screening protocol combining two successive screening filters: a miniaturized fibrin formation assay, followed by a thrombin generation assay, both on severe HA plasma. We screened three chemical collections totalizing more than 2300 chemical compounds; we identified adapalene, a commercialized anti-acneic compound (Differin®) which is strongly hydrophobic. In order to design a drug that could be orally administered, we developed a series of chemical analogs of adapalene, and three of them, with similar procoagulant activities in FVIII deficient plasma, showed improved solubilities. The mechanism of action was thoroughly investigated by a series of thrombin generation and enzymatic assays. These studies allow to conclude that the procoagulant activity of the chemical compounds in FVIII-deficient plasma is due to the activation of Factor XII.