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Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.
- Source :
-
Analytical chemistry [Anal Chem] 2019 Jul 02; Vol. 91 (13), pp. 8443-8452. Date of Electronic Publication: 2019 Jun 19. - Publication Year :
- 2019
-
Abstract
- We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.
- Subjects :
- Agammaglobulinaemia Tyrosine Kinase immunology
Animals
Antibodies, Immobilized metabolism
Biological Assay
Macaca fascicularis
Agammaglobulinaemia Tyrosine Kinase metabolism
Antibodies, Immobilized immunology
Biomarkers metabolism
Chromatography, Liquid methods
Protein Kinase Inhibitors metabolism
Receptors, Drug metabolism
Tandem Mass Spectrometry methods
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 91
- Issue :
- 13
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31247719
- Full Text :
- https://doi.org/10.1021/acs.analchem.9b01462