@article {GuimaraesDMD-AR-2021-000751, author = {Guilherme Guimaraes and Long Yuan and Pei Li}, title = {Antisense Oligonucleotide In Vitro Protein Binding Determination in Plasma, Brain and Cerebral Spinal Fluid Using Hybridization LC-MS/MS}, elocation-id = {DMD-AR-2021-000751}, year = {2021}, doi = {10.1124/dmd.121.000751}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Drug unbound fraction ({\textflorin}u) characterization is a key consideration in pharmacokinetic (PK) and pharmacodynamic (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 LC-MS/MS platform for the direct quantitation of antisense oligonucleotide (ASO) {\textflorin}u. The method provides substantial improvements including minimal matrix effects and high specificity when compared to previously used oligonucleotide {\textflorin}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. While ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO {\textflorin}u in brain or CSF. As ASOs continue to undergo clinical trials for neurological and neuromuscular indications, {\textflorin}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. Significance Statement A novel hybridization LC-MS/MS based approach was successfully developed for the determination of ASO in vitro protein binding in plasma, and for the first time brain and cerebral spinal fluid. Ultrafiltration, equilibrium dialysis, and ultracentrifugation were assessed for the separation of unbound ASO from biological matrices. The hybridization LC-MS/MS platform provided unique advantages, including minimal matrix effects and high specificity, comparing to traditional ligand binding assays or liquid scintillation approaches, which enabled efficient and reliable in vitro protein binding assay.}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/early/2021/12/17/dmd.121.000751}, eprint = {https://dmd.aspetjournals.org/content/early/2021/12/17/dmd.121.000751.full.pdf}, journal = {Drug Metabolism and Disposition} }