In Vitro Metabolic Stabilities and Metabolism of 2′-O-(Methoxyethyl) Partially Modified Phosphorothioate Antisense Oligonucleotides in Preincubated Rat or Human Whole Liver Homogenates

In vitro metabolic stability testing of phosphorothioate 2′-O-methoxyethyl (2′-MOE) partially modified antisense oligonucleotides (ASOs) is not routinely performed to help screen discovery compounds (eg, predict in vivo half-lives), as no suitable in vitro test system currently exists. The aims of this work were to develop, optimize, and evaluate an in vitro whole liver homogenate (rat or human) test system. The test system was used to evaluate in vitro metabolic stabilities (intrinsic clearance) of selected ASOs, with results compared to reported in vivo half-lives, and generated metabolites also identified. Test system optimization involved preincubating whole liver homogenates at 37°C for ≥24 hours, which increased in vitro ASO metabolism rate. From calculated in vitro intrinsic clearance (CLint) values in preincubated rat or human whole liver homogenates, metabolic stabilities of fully phosphorothioated 2′-MOE ASOs (ISIS 104838 and ISIS 301012) were, as expected, greater (ie, lower CLint) than a 2′-MOE ASO containing a single phosphodiester substitution (ISIS 104838PO10). However, comparable-to-lower in vitro metabolic stability for ISIS 301012 was seen compared to ISIS 104838, in contrast to reported ∼2-fold longer in vivo tissue elimination half-lives for ISIS 301012. Identified in vitro metabolic products of ISIS 301012 were consistent with previously reported in vivo observations.