Development and validation of a novel reporter gene assay for determination of recombinant human thrombopoietin.

Recombinant human thrombopoietin (rhTPO) was approved by the National Medical Products Administration in 2010 for the treatment of thrombocytopenia in patients with immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. Nevertheless, no method for determining rhTPO bioactivity has been recorded in different national/regional pharmacopoeia. Novel methods for lot release and stability testing are needed that are simpler, quicker, and more accurate. Here, we developed a novel reporter gene assay (RGA) for rhTPO bioassay with Ba/F3 cell lines that stably expressed human TPO receptor and luciferase reporter driven by sis-inducible element, gamma response region, and gamma-interferon activated sequence. During careful optimization, the RGA method demonstrated high performance characteristics. According to the International Council for Harmonization Q2 (R1) guidelines and the Chinese Pharmacopoeia 2020 edition, the validation results demonstrated that this method is highly time-saving, sensitive, and robust for research, development, manufacture, and quality control of rhTPO.

Development of Romiplostim for Treatment of Primary Immune Thrombocytopenia From a Pharmacokinetic and Pharmacodynamic Perspective.

Romiplostim is a novel thrombopoiesis-stimulating peptibody consisting of a carrier Fc domain and a peptide domain that binds to the thrombopoietin receptor (TPOR) on platelets and platelet precursors. Similar to endogenous thrombopoietin, romiplostim activates the TPOR to stimulate the growth and maturation of megakaryocytes, resulting in increased production of platelets in the circulation. Binding of romiplostim to TPOR on the platelets and megakaryocytes presumably triggers subsequent internalization and degradation. Therefore, increased platelet counts following romiplostim treatment results in increased elimination of the drug. The TPOR target-mediated process is saturable, resulting in nonlinear volume of distribution and clearance of romiplostim. Therefore, target-mediated disposition plays a decreasing role in drug elimination with increasing romiplostim serum concentration. Conversely, nonspecific elimination processes such as renal clearance play an increasing role with increasing romiplostim serum concentration. Limited pharmacokinetics data demonstrated that the exposure to romiplostim was lower after multiple dose administrations than after the first dose, although large inter-subject variability was observed. Large inter- and intra-subject variability in the platelet response was also observed at a given dose. These findings suggest considerable heterogeneity of disease in patients with primary immune thrombocytopenia and support the need for individual dose adjustments based on platelet counts.

Oral small-molecule tyrosine kinase inhibitor midostaurin (PKC412) inhibits growth and induces megakaryocytic differentiation in human leukemia cells.

Midostaurin (PKC412), a small-molecule multiple tyrosine kinase inhibitor, has been shown to suppress the growth of various tumor cells. Since kinases inhibited by midostaurin are involved in megakaryocytic differentiation, we hypothesized that this novel target therapeutic might have a role in the treatment of human leukemia cells. Hence, we examined the effect of midostaurin on human erythroleukemia cells and evaluated potential mechanisms. Midostaurin inhibited the growth of both K562 and HEL cells in dose- and time-dependent manner. Morphological changes such as enlarged contours, multipolarity of mitotic spindles, and multinucleation of megakaryocytes were noted in both cell lines treated by midostaurin. A smaller population of apoptotic cells was also observed. DNA histogram revealed polyploid and hypoploid populations of midostaurin-treated cells. Midostaurin treatment enhanced the surface expression of the megakaryocytic marker CD61; in contrast, the erythroid marker glycophorin A expression was decreased. At optimal conditions for inducing megakaryocytic differentiation, midostaurin upregulated the expression and signaling of c-Mpl, a thrombopoietin receptor-encoding gene, in HEL cells. These results indicate that midostaurin can inhibit growth; induce megakaryocytic differentiation; and to a lesser extent, cause apoptosis in HEL cells. This effect might involve the expression and signaling of c-Mpl.

Characterization of novel non-peptide thrombopoietin mimetics, their species specificity and the activation mechanism of the thrombopoietin receptor.

A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells. To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain. Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should be helpful for structure-activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.

Thrombopoietin mimetic agents in the management of immune thrombocytopenic purpura.

Thrombopoietin (TPO) is a potent endogenous cytokine and the principal regulator of platelet production. Advances in the understanding of the structure of TPO enabled development of the first generation of thrombopoietic growth factors, recombinant human thrombopoietin (rhTPO) and pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF). Clinical results showed that these agents were effective in promoting increases in platelet counts in a variety of thrombocytopenic disorders. However, clinical development was halted when studies demonstrated risk for autoantibody formation with cross-reactivity to endogenous TPO. A second generation of thrombopoietic growth factors, including TPO peptide and nonpeptide mimetics and TPO agonist antibodies, utilizing different mechanisms from recombinant growth factors to promote platelet production, are currently in development. The TPO peptide mimetic AMG 531 and the nonpeptide mimetic eltrombopag are in advanced clinical trials and have both resulted in dose-dependent increases in platelets in healthy subjects and in significant increases in platelets in patients with chronic immune thrombocytopenic purpura (ITP). Clinical trials are also being conducted to examine the efficacy and safety of eltrombopag to treat thrombocytopenia in hepatitis C virus (HCV)-infected individuals. These agents appear to be well tolerated and the formation of autoantibodies appears to be limited to first-generation growth factors. Increases in marrow reticulin have been demonstrated with some growth factors, but this appears to be a reversible phenomenon and is not associated with formation of collagen fibrosis. There appears to be no increased incidence of thrombotic events in patients who achieve high platelet counts with growth factor treatments, and although occasional thrombotic events have been reported, their association to the treatment is uncertain. While there is evidence that activation of signaling pathways involved in platelet production may result in reduction in the threshold for platelet activation, this appears to have minimal clinical relevance for the use of thrombopoietin growth factors.