Abstract

The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Drug unbound fraction (ƒ_u) characterization is a key consideration in pharmacokinetic and pharmacodynamic (PK/PD) modeling, assuming only unbound drug can interact with the target, and therefore has direct implications in the efficacy and potential toxicity of the drug. The current study describes the implementation of a hybridization liquid chromatography–tandem mass spectrometry (LC-MS/MS) platform for the direct quantitation of antisense oligonucleotide (ASO) ƒ_u. The method provides substantial improvements, including minimal matrix effects and high specificity when compared with previously used oligonucleotide ƒ_u detection methods such as ligand binding assays or liquid scintillation. The hybridization LC-MS/MS platform was integrated with ultracentrifugation, ultrafiltration, and equilibrium dialysis, and method performance for each technique was evaluated. Although ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO ƒ_u in brain or cerebral spinal fluid (CSF). As ASOs continue to undergo clinical trials for neurologic and neuromuscular indications, ƒ_u characterization in brain and CSF can provide invaluable information about ASO distribution and target engagement in the central nervous system, therefore providing support for in vivo PK/PD data characterization.