Cassandra Yun Kazuki Fukami Rongrong Jiang ^* Raku Shinkyo ^*

• Eisai, Cambridge, United States

Plasma protein binding can have a profound effect on the pharmacokinetic-pharmacodynamic relationship. In this study, two pairs of sequence-matched phosphorodiamidate morpholino oligomer (PMO) and 2'-O-methoxyethyl/phosphorothioate (MOE/PS)-modified antisense oligonucleotides (ASO) were characterized using an ultrafiltration method coupled with hybridization electrochemiluminescence to measure the unbound fraction (fu) in both mouse and human plasma. The fu,plasma differed greatly with MOE/PS demonstrating a significantly higher binding than PMO. Our findings support the current understanding that the fu is largely driven by their chemistries, with no significant species difference between mouse and human plasma.Furthermore, saturation of fu,plasma for MOE/PS may occur when concentrations are increased beyond 1 µM, whereas the binding of PMO does not appear to saturate upon increasing concentrations up to 10 µM. Additionally, individual binding measurements of five prominent proteins: human serum albumin, α1-acid glycoprotein, human γ-globulin, low-density lipoprotein, and high-density lipoprotein, were included to further characterize the interaction between protein and ASO in human plasma. The results indicate, for the first time, that human γ-globulins exhibit predominant binding affinity to both MOE/PS and PMO ASO at physiological concentrations, more notably than human serum albumin which is the most dominant protein in human plasma.Our new findings may shed light on overlooked contributions from human γ-globulins not only to plasma protein binding of ASO but also the disposition of ASO to tissues.