A Novel Non-Peptidyl Human C-Mpl Agonist, NIP-004, Stimulates Human Megakaryopoiesis and Thrombopoiesis.

Thrombopoietin (TPO) is a critical humoral regulator of megakaryopoiesis and thrombopoiesis. TPO induces proliferation and maturation of hematopoietic stem cells and megakaryocytic progenitors by stimulating its cognate receptor, Mpl. We screened 50,000 chemical libraries to find the lead compound which stimulates human leukemia cell line, UT-7/TPO expressing Mpl. NIP-004 is a novel synthetic compound which displays human (Hu) Mpl agonistic activity. NIP-004 stimulated proliferation of HuMpl-expressing cell lines such as UT-7/TPO and UT-7/EPO-HuMpl, but not murine (Mu) Mpl-expressing cell lines. NIP-004 induced megakaryocytic colonies from human CD34⁺ cells, but not mouse or cynomolgus monkey cells. These observations indicate that NIP-004 displays strict species specificity. We thus created a new xenotransplantation model to evaluate in vivo efficacy of NIP-004 for human megakaryopoiesis and thrombopoiesis. We used immunodeficient NOD/SCID/γ_c^null (NOG) mice as recipients, as this line displays high potency to reconstitute human hematopoiesis. NOG mice transplanted human cord blood-derived CD34⁺ cells were treated with NIP-004 at a dose of 30 mg/kg/day s.c. for 2 weeks. After treatment with NIP-004, we observed 1.5-fold increase of HuCD45⁺CD34⁺CD41a⁺ megakaryoblasts and 3-fold increase of HuCD41a⁺ 128N matured polyploid megakaryocytes in murine bone marrow (BM). NIP-004 increased the circulating HuCD41a⁺ platelets by 4-fold at day 14 with statistically significant differences. NIP-004 did not influence the total number of nucleated cells or MuCD41⁺ megakaryocytes, supporting its species specificity in vivo. The percentage of human HuCD45⁺CD34⁺ cells in murine BM was not altered by NIP-004. NIP-004 did not influence the total number (mixed human and murine) of red blood cells, platelets and white blood cells. The number of MuCD41⁺ platelets and chimerism of HuCD45⁺ leucocytes in the peripheral blood were not altered by NIP-004 administration. Furthermore, NIP-004 did not influence the percentage of HuCD19⁺ B lymphoid, HuCD3⁺ T lymphoid or HuCD33⁺ myeloid cells among circulating HuCD45⁺ cells. Immunoelectron microscopic analysis demonstrated that the morphology of HuCD41a⁺ platelets in NIP-004-treated xenotranplanted mice was indistinguishable from normal human peripheral platelets. We also confirmed that ADP stimulation induced the surface expression of CD62P and the active form of gpIIb-IIIa in HuCD41a⁺ platelets from NIP-004-treated xenotransplanted mice at almost the same rate as vehicle-treated mice. In conclusion, NIP-004 possesses HuMpl agonistic activity that was confirmed by the proliferation of various TPO-responsive cell lines and primary human hematopoietic progenitor cells. NIP-004 stimulated human megakaryopoiesis and thrombopoiesis in a xenotransplant animal model. These results indicated NIP-004 has strong potential for clinical development as a new treatment for thrombocytopenia.