A Novel, Small Non-Peptidyl Butyzamide Activates Human Thrombopoietin Receptor and Promotes Megakaryopoiesis.

Butyzamide is a novel non-peptidyl molecule which has agonistic activity to the thrombopoietin (TPO) receptor Mpl. Butyzamide promotes the proliferation of murine pro B cell line Ba/F3 expressing human Mpl (hMpl), and induces phosphorylation of JAK2, STAT3, STAT5 and MAPK. Interestingly, butyzamide does not promote the proliferation of Ba/F3 cells expressing murine Mpl (mMpl). To elucidate the mechanisms for this species specificity, we created stable transfectants such as Ba/F3-hMpl(H499L) and Ba/F3-mMpl(L490H) cells. Butyzamide induced the proliferation of Ba/F3-mMpl(L490H) but not Ba/F3-hMpl(H499L) cells, indicating that a histidine residue in the transmembrane domain of human Mpl is critical for butyzamide-induced signaling. Butyzamide induced the colony-forming unit-megakaryocyte and polyploid megakayocytes from human CD34⁺ hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was orally administered to immunodeficient NOD/Shi-scid/IL-2Rγ_c^null (NOG) mice transplanted with human fetal liver-derived CD34⁺ cells, the human platelet count increased by 6.2- and 22.9-fold at the doses of 10 and 50 mg/kg for 20 days, respectively. Butyzamide also increased the number of reticulated human platelets and human matured megakaryocytes in NOG mice. These results indicate that butyzamide is an orally bioavailable Mpl activator and suggest its potential for clinical development as a therapeutic agent in patients with thrombocytopenia.