Purpose: Hemophilia B is a severe blood clotting disorder caused by the deficiency of factor IX (FIX), and intravenous administration of factor IX is the common treatment. FIX is not bioavailable when given orally due to poor stability and permeability in the gastrointestinal tract. Transferrin (Tf) receptor mediated endocytosis is a potential pathway to enhance the absorption of oral proteins. The purpose of the study was to explore the feasibility of utilizing FIX-Tf fusion proteins to enhance the oral bioavailability of FIX.
Methods: Seven recombinant fusion proteins (rFIX-Tf) with different linkers were constructed by recombinant DNA approach and expressed in HEK293 cells. These fusion proteins were characterized by in vitro transcytosis and transferrin receptor (TfR) binding assay in Caco-2 cells and one-stage clotting assay. The in vivo efficacy study was performed using a tail-bleeding model in hemophilia B mice.The pharmacokinetic study was further investigated.
Results: Fusion proteins rFIX-Tf/G2 (with non-cleavable linker (GGGGS)2) and rFIX-Tf/SVSQ (with cleavable linker SVSQTSKLTRAETVFPDVDGS) were most permeable and showed specific binding ability to TfR in Caco-2 cells. These two fusion proteins retained FIX activity in clotting generation. The in vivo efficacy study showed that both rFIX-Tf/G2 and rFIX-Tf/SVSQ by intravenous injection significantly reduced blood loss in comparison to the vehicle control. Most significantly, rFIX-Tf/G2 demonstrated anti-bleeding activity when administered orally.
Conclusion: Our results showed that the fusion protein technique with Tf could be potentially used for oral delivery of FIX and the linker between FIX and Tf in the fusion protein is crucial. rFIX-Tf/G2 appears to be the most promising fusion protein as a potential oral therapeutics for hemophilia B.
Chen Xie– Western University of Health Sciences
Zhijun Wang– Western University of Health Sciences
Yang Su– Western University of Health Sciences
Sunil Prabhu– Executive Associate Dean and Professor, Western University of Health Sciences
Wei-Chiang Shen– Professor of Pharmaceutical Sciences, University of Southern California