Endocrine Abstracts

Introduction

Nowadays thrombopoietin receptor agonists (TPO-RAs) represent a promising approach for the patients with immune thrombocytopenia (ITP). Usage of two licensed TPO-RAs, romiplostim and eltrombopag, provide efficient support of ITP patients´ platelet count and, as a consequence, better control of disease. Both agents have an acceptable toxicity profile, but there are several another disadvantages in terms of usage of these drugs such as cost (especially for romiplostim), daily dosing (for eltrombopag) and specific adverse events. That´s why discovery of new TPO-Ras or development of similar products of existing TPO-Ras are emergent approaches to improve the life quality of ITP patients. The non-clinical development plan of both similar and new TPO-Ras should contain sensitive and specific in vitro pharmacology assays. Using two known TPO-Ras (recombinant human thrombopoietin (rhTPO) and romiplostim) in this study we have performed validation of the two in vitro assays to provide robust functional and binding data.

Methods

Romiplostim-TPO-R binding affinity was monitored with BLI technique using Octet RED96 instrument (ForteBio, Pall). In this study recombinant human thrombopoietin receptor with a C-terminal 6-His-tag (R&D Systems Inc.) was immobilized to anti-penta-HIS biosensors. Kinetic binding constants of romiplostim-TPO-R were determined through global fits using ForteBio Data Analysis. Association and dissociation rates were simultaneously fit to 1:1 ligand binding model to determine the affinity constant (KD) value. To evaluate functional effects of rhTPO murine 32D cell line expressed human TPO-R was developed. After transfection and during the development of stable and functional 32D-hTPO-R cell line clonal selection was performed. Presence of human TPO-R was determined using fluorescence-activated cell sorting analysis, Western blot analysis was used to evaluate TPO-R phosphorylation after rhTPO stimulation. Functional characterization of generated 32D-hTPO-R was performed in rhTPO-induced proliferation assay based on the CellTiter-Glo Luminescent Assay (Promega Corp.). EC50 values were analysed to measure functional proliferation response.

Results

In binding assay KD of romiplostim-TPO-R interaction was in 0.1228–0.134 nM range. In proliferation assay in 32D cells stable expressed functional human TPO-R for rhTPO EC50 was determined at 688 pg/ml. Both assays were validated in terms of main parameters such as linearity, specificity, precision. Acceptable degrees determined.

Conclusions

In this study our data demonstrated that binding and functional assays were well validated to receive robust data. Thus, in vitro pharmacology assays mentioned above can be used for the purpose of similar and new TPO-Ras drug development.