Differential Effects of a Novel Non-Peptidic Thrombopoietin Mimetic on Proliferation and Differentiation of Human CD34 + Progenitor Cells
2008
Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight ( nd generation agent in preclinical development with promising in vitro potency and aqueous solubility. We have studied the effects of STS-T4 on the growth and differentiation of primary human CD34 + progenitor cells. A dose-dependent proliferation of highly purified human CD34 + cells was observed with EC50 value of less than 1 μM. A bell-shaped dose-response curve was observed, which is consistent with previous reports on compounds stimulating homodimeric cytokine receptors. In addition, the phenotype of the cultured cells was analyzed by flow cytometry and monoclonal antibodies specific for markers of megakaryocyte differentiation. During a culture period of 10 days, 85% of the cells cultured in the presence of STS-T4 differentiated into CD41 + megakaryocytes in the absence of any other cytokines or mimetics. In addition, 30% of these CD41 + cells coexpressed CD42b as a marker of more mature cells. When compared to recombinant human TPO (rhTPO), a similar proportion of the cultured cells expressed CD41 and CD42b in response to STS-T4 and the levels of expression of these antigens on the surface of the megakaryocytes were similar, suggesting that the effects of STS-T4 and rhTPO on human megakaryocyte differentiation are comparable. However, the level of cell proliferation induced by STS-T4 in cultures of CD34 + cells was 50–60% of that induced by rhTPO, supporting the conclusion that the signals mediating proliferation and differentiation of CD34 + progenitor cells are differentially regulated. In addition to the activity profile, the physical properties of STS-T4 are desirable for further development. Aqueous solubility and gastrointestinal permeability are major contributors to oral absorption, and high solubility and permeability generally also reduce the risk of food effects. The aqueous solubility of STS-T4 was measured by a kinetic method with HPLC and visual detection and was determined to be >1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34 + progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.
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