Biological Activity and Selectivity for Tpo Receptor of the Orally Bioavailable, Small Molecule Tpo Receptor Agonist, SB-497115.

SB-497115 is a selective, low molecular weight, non-peptidyl thrombopoietin receptor (TpoR) agonist. identified by its ability to activate the JAK/STAT signalling pathway. SB-497115 is being developed for the treatment of thrombocytopenias, such ass, immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. SB-497115 requires TpoR to activate the JAK/STAT signalling pathway and stimulates transcription through the STAT based (IRF-1) and megakaryocyte specific (gpIIb) promoters. An analysis of the receptor selectivity of SB-497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon -alpha or Interferon-gamma, were active. SB-497115 was inactive over a three-fold concentration range in proliferation, reporter gene, or STAT activation assays performed on cell lines that did not express TpoR.

To characterize the kinetics and specificity of SB-497115 in cells, multiple molecular markers for Tpo activity were measured. Western blot analysis for activation of the STAT and MAPK pathways was performed using phospho-specific antibodies on lysates of UT7-Tpo cells treated with SB-497115. In addition, mRNA expression of several early response genes associated with proliferation and Tpo activation (i.e., Fos, EGR-1 and thyroid-like receptor 3 ), was measured in response to SB-497115 treatment. The kinetics and level of induction for pathway phosphorylation events and gene expression were similar to that seen with Tpo. A proliferative response in the human Tpo-dependent cell line, UT7-Tpo, by SB-497115 was assayed by thymidine incorporation and an EC50 of 30 nM was demonstrated. An analysis of the receptor selectivity of SB 497115 was undertaken utilizing a panel of various transfected and non-transfected cell lines in which other cytokines, including G-CSF, Epo, IL-3, Interferon-alpha or Interferon-gamma, were active. SB 497115 demonstrated a complete lack of activity over a three-fold concentration range in proliferation, reporter gene or STAT activation assays performed on cell lines that did not express TpoR. SB-497115 was shown to be equal to or better than rhTpo in the ability to induce differentiation of normal human bone marrow progenitors (CD34) into CD41+ cells of the megakaryocyte lineage, with an EC50 of 100 nM. These latter activities of SB-497115 were similar to or greater than the level of maximal activity seen with Tpo in these assays. SB-497115 demonstrates specificity for human and chimpanzee TpoR and increases platelet counts in both chimpanzees and normal human volunteers when dosed orally. In conclusion, SB-497115 is the first non-peptide small molecule TpoR agonist to demonstrate activity in human in vitro bone marrow assays and demonstrate pharmacological activity in humans.