Prevention of Human Interferon-alpha-Induced Thrombocytopenia by a Non-Peptidyl Human Thrombopoietin Receptor Activator, NIP-004.

Human interferon-α (IFN) is useful for the treatment of chronic hepatitis C and neoplastic disorders; however, one major adverse effect is that thrombocytopenia often leads to a dose reduction or discontinuation of therapy. Since around 40% of patients with chronic hepatitis C will develop liver cancer, it is very important to prevent the transition from chronic hepatitis to liver cirrhosis and liver cancer with IFN treatment. However, some patients cannot receive IFN treatment because of thrombocytopenia resulting from liver cirrhosis and autoimmunity. This prompted us to evaluate the effects of a novel non-peptidyl human thrombopoietin (TPO) receptor (c-Mpl) activator, NIP-004, to prevent IFN-induced thrombocytopenia. We recently reported that NIP-004 at 30 μg/kg/day for 2 weeks increased human platelets 4-fold in immunodeficient NOD/Shi-scid, IL-2Rγ^null (NOG) mice transplanted with 1 × 10⁵ human cord blood-derived CD34⁺ cells (Blood, 2006). When PEG-hIFN-α2b (IFN-α2b) at 30 μg/kg was administered three times a week for 3 weeks to NOG mice transplanted with human CD34⁺ cells, the number of human platelets was significantly reduced by 40% compared to the control mice. Moreover, the number of human platelets was restored to the same level as the control mice with daily administration of 30 mg/kg NIP-004 for 2 weeks during the course of IFN treatment. These results indicated that NIP-004 is effective in preventing IFN-α2b-induced thrombocytopenia in vivo. Although IFN is believed to directly inhibit megakaryopoiesis, its mechanisms are not clearly understood. We analyzed the inhibitory effects of IFN-α2b on the colony formation of human megakaryocytes (CFU-MK) and the intracellular signaling induced by IFN and NIP-004. NIP-004 prevented the inhibition of CFU-MK formation by IFN-α2b in a dose dependent manner. IFN-α2b modestly inhibited the proliferation of human leukemia UT-7/EPO cells expressing human c-Mpl, and they were stimulated with TPO and NIP-004, respectively. We analyzed some form of cross-talk between TPO and IFN receptors by Western blot and quantitative RT-PCR analysis. NIP-004 induced the expression of SOCS-3 and CIS in UT-7/EPO-hMpl cells. We suggest that NIP-004 induces inhibitory signaling such as SOCS-3 and CIS and inhibits IFN-induced intracellular signaling. In conclusion, NIP-004 was shown to prevent IFN-α2b-induced thrombocytopenia in vivo and in vitro, suggesting the future clinical application of non-peptidyl c-Mpl activators for the treatment of patients with chronic hepatitis C by IFN.