Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist

Abstract

Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34(+) bone marrow cells into CD41(+) megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

Figure 1

Structure of eltrombopag, an orally bioavailable thrombopoietin receptor agonist.

Figure 2

Cell-based STAT activation. (A): Representative gene activity of eltrombopag on BAF3/interferon regulatory factor (IRF)-1 cells transfected with and without the thrombopoietin receptor (TpoR). (B): Gene activity of eltrombopag and recombinant human thrombopoietin (rhTpo) on 32D-mpl cells transfected with the megakaryocyte-specific glycoprotein IIb promoter. Reprinted from Duffy KJ, Erickson-Miller CL. The discovery of eltrombopag, an orally bioavailable TpoR agonist. In: Metcalf BW, Dillon S, eds. Target Validation in Drug Discovery. Burlington, MA: Academic Press, 2007:241–254, with permission.

Figure 3

Signal transduction induced by eltrombopag. (A): Eltrombopag demonstrated activity on human and chimpanzee (but not other species) platelets by signaling transducers and activators of transcription (STAT) electrophoretic mobility shift assay. Similar kinetics of (B) phospho-STAT5 and (C) mitogen-activated protein kinase (MAPK) expression were detected by Western blot of eltrombopag- or recombinant human thrombopoietin (rhTpo)-treated N2C-Tpo cell lysates. (D): STAT5 activation was also detected in megakaryocytes derived from CD34⁺ cells. (A, B): Reprinted from Duffy KJ, Erickson-Miller CL. The discovery of eltrombopag, an orally bioavailable TpoR agonist. In: Metcalf BW, Dillon S, eds. Target Validation in Drug Discovery. Burlington, MA: Academic Press, 2007:241–254, with permission.

Figure 4

Proliferation and differentiation induced by eltrombopag. (A): Proliferation of BAF3/hTpoR cells induced by eltrombopag after 48 hours of treatment; the dotted line represents the activity of cells treated with 100 ng/ml of recombinant human thrombopoietin. OD, optical density. (B): Representative example of megakaryocyte differentiation of CD34⁺ cells after 10 days of eltrombopag treatment; similar results were obtained with six independent marrow samples. Panel (B) was reprinted from Duffy KJ, Erickson-Miller CL. The discovery of eltrombopag, an orally bioavailable TpoR agonist. In: Metcalf BW, Dillon S, eds. Target Validation in Drug Discovery. Burlington, MA: Academic Press, 2007:241–254, with permission.

Figure 5

Additive effects of eltrombopag and rhTpo. (A): Proliferation, as measured by thymidine incorporation, of N2C-Tpo cells by eltrombopag (0.003–3 μM) in combination with recombinant human thrombopoietin (rhTpo; 1–100 ng/ml). (B): Activation of caspase-3 and caspase-7 by eltrombopag (0–3 μM) in combination with rhTpo (0–100 ng/ml) in N2C-Tpo cells.

Figure 6

Increase in platelet counts in chimpanzees treated orally with eltrombopag for 5 days at 10 mg/kg per day (solid lines; n = 3) or vehicle (dotted lines, n = 2). Data are expressed as the fold increase of each animal over its baseline platelet count. Reprinted from Duffy KJ, Erickson-Miller CL. The discovery of eltrombopag, an orally bioavailable TpoR agonist. In: Metcalf BW, Dillon S, eds. Target Validation in Drug Discovery. Burlington, MA: Academic Press, 2007:241–254, with permission.