Eltrombopag Olamine (Eltrombopag diethanolamine salt) is the orally active ethanolamine salt of eltrombopag, a small-molecule, nonpeptide thrombopoietin receptor agonist with megakaryopoiesis-stimulating activity. Eltrombopag binds to and stimulates the transmembrane domain of the platelet thrombopoietin receptor (TPO-R or CD110), a member of the hematopoietin receptor superfamily. Activation of TPO-R leads to the proliferation and differentiation of cells in the megakaryocytic lineage and an increase in platelet production.

Eltrombopag demonstrates a half maximal effective concentration (EC50) of 0.27 μM in murine BAF3 cells transfected with the luciferase reporter gene under direction of the STAT-activated IRF-1 promoter and human TpoR (BAF3/IRF-1/hTpoR). Eltrombopag activates the receptor by association with metal ions (i.e., Zn2+) and specific amino acids within the transmembrane and juxtamembrane domains of the TpoR. Eltrombopag (30 μM) results in activation of STAT5 in N2C-Tpo cells, as detected with an antiphospho-STAT5 antibody on Western blots. Eltrombopag stimulates proliferation after a 2-day incubation with an EC50 of 0.03 μM in a BrdU assay conducted in BAF3/hTpoR cells. Eltrombopag also induces differentiation of hematopoietic stem cells into committed megakaryocyte progenitor cells. Eltrombopag increases the differentiation of bone marrow CD34+ cells into CD41+ megakaryocytes in a dose-dependent manner with an EC50 of 0.1 μM. [1] Eltrombopag inhibits N2C-Tpo cell and HEL92.1.7 cell proliferating with IC50 of 20.7 μg/mL and 2.3 μg/mL. [2] Eltrombopag (20 μg/mL) leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Eltrombopag (5 μg/mL) shows clear signs of differentiation, significant changes in the organization of the nuclear contents, and an increase in the cytoplasm/nucleus ratio in HL60 cells. Eltrombopag (5 μg/mL) causes an increase in CD11b, which is consistent with a premacrophage state in U937 cells, and also causes an increase in CD11b in URE cells. Eltrombopag leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner in HL60 cells. [3]

Eltrombopag (10 mg/kg per day) increases platelet counts over twofold approximately 1 week after the last dose for one chimpanzee and approximately 1.5-fold for the other two chimpanzees. [1] Eltrombopag (1 mg/mL) prolongs survival in mouse models of leukemia. [3]

The high-performance liquid chromatography (HPLC) analyses are carried out using a Fast Acid Column (100×7.8 mm) and a HPX-87H Ion Exclusion Column (300 mm×7.8 mm) in series with 2.5 mM H2SO4 in water as the mobile phase at a flow rate of 0.3 mL/min, at 55°C. This method enabled quantification of D-glucose, ethanol, glycerol, D-xylulose, Ribitol, and xylitol. D-ribose, D-ribulose, and D-arabitol coeluted on the Aminex HPX-87H column. The CarboPac MA-1 column of Dionex ICS-3000 is used to analyze representative culture supernatant samples for the presence of arabitol and xylitol. Samples are run at column temperature of 30°C with 480 mM NaOH at flow rate 0.4 mL/min. The CarboPac MA-1 column separated D-arabitol from D-ribose and D-ribulose, but the alkaline conditions degraded D-ribulose interfering with the quantification of D-ribose.Yeast cells are disrupted with glass beads in 100 mM sodium phosphate buffer pH 7.0 containing phenylmethylsulfonyl fluoride and pepstatin A in final concentrations of 0.17 mg/mL and 0.01 mg/mL, respectively.The activity of NAD+-dependent Gdh2p is measured in a reaction buffer of 0.5 M triethanol amine pH 7.7 and 2 mM NADH. After addition of the cell lysate, the reaction is started by adding a mixture of α-ketoglutarate (100 mM) and NH4Cl (200 mM) to a final concentration of 2.4 mM and 4.9 mM, respectively. The GapB activity is measured. Shortly, the reaction mixture is 500 mM triethanol amine pH 7.8, 1 mM ATP, 2 mM MgCl2, 200 μM NADPH, and 10 μg/mL of phosphoglycerate kinase. 3-phosphoglycerate is added to a final concentration of 5 mM to start the reaction. Activity measurements are performed with a Cobas Mira Plus automated analyzer[2].