Manuel Aivado

Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome

Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 microg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.

Comparative analyses of the small molecule thrombopoietin receptor agonist eltrombopag and thrombopoietin on in vitro platelet function

OBJECTIVE The thrombopoietin receptor (TPOR) is a therapeutic target for treatment of thrombocytopenia because stimulation of this receptor results in enhanced megakaryocyte proliferation, differentiation, and ultimately platelet production. In addition to effects on megakaryocytes, TPOR stimulation also impacts platelet function. The present study examined platelet function following stimulation with the small molecule TPOR agonist eltrombopag. MATERIALS AND METHODS Platelets were obtained from healthy volunteers, and signal transduction pathway activation was examined in washed platelet preparations. Platelet aggregation was examined in both washed platelet preparations and platelet-rich plasma. Platelet alpha-granule release was determined via fluorescein-activated cell sorting measurement of CD62P. RESULTS In signal transduction studies of washed human platelets, eltrombopag induced the phosphorylation signal transducers and activators of transcription (STAT) proteins with no phosphorylation of Akt, whereas recombinant human TPO (rhTPO) induced the phosphorylation of Akt as well as STAT-1, -3, and -5. In studies conducted at subthreshold/submaximal concentrations of adenosine diphosphate (ADP) or collagen, eltrombopag pretreatment did not result in platelet aggregation. In contrast, rhTPO acted in synergy with submaximal concentrations of ADP or collagen to induce maximal aggregation under all conditions examined. Similarly, platelet activation as examined via surface expression of CD62P was not enhanced by eltrombopag pretreatment as compared to rhTPO. CONCLUSIONS These results demonstrate that the nonpeptidyl TPOR agonist eltrombopag stimulates platelet signal transduction with little or no effect on overall platelet function, in contrast to TPO, which significantly primes platelet activation. These data demonstrate that effects of TPOR ligands on platelet function can vary depending on the specific mechanism utilized to stimulate the TPOR.

Platelet counts and haemorrhagic diathesis in patients with myelodysplastic syndromes

Objectives:  Most patients with myelodysplastic syndromes (MDS) present with single or multiple lineage cytopenias in peripheral blood despite a hypercellular bone marrow. Thrombocytopenia, attributable to ineffective platelet production by dysfunctional megakaryocytes, has been estimated to occur in 40–65% of patients. However, there are hardly any studies on the clinical relevance of low platelet counts in MDS.

Reduced proliferation of non-megakaryocytic acute myelogenous leukemia and other leukemia and lymphoma cell lines in response to eltrombopag

Leukemia cell lines were treated with eltrombopag or thrombopoietin and their proliferative response was determined. Eltrombopag did not increase proliferation of cell lines that did not express high levels of megakaryocyte markers. Instead, treatment with eltrombopag alone inhibited proliferation of many cell lines (IC(50) range=0.56-21 microg/mL). The addition of other cytokines, such as G-CSF, Epo or Tpo, did not affect the decrease in proliferation. The decrease in proliferation appears to be through a TpoR-independent, nonapoptotic mechanism. These findings suggest that eltrombopag does not enhance, but rather inhibits, proliferation of leukemia cell lines in vitro.