Assay to determine in vivo receptor occupancy

An assay method for determining drug receptor occupancy in a subject dosed with a drug of interest that covalently binds to a receptor of interest comprising:

• a) contacting a blood sample obtained from the subject with a lysis solution, wherein the subject was treated with a drug of interest, and wherein the lysis solution comprises comprising a quencher that irreversibly binds at the same receptor site as the drug of interest to the blood sample,
• b) isolating drug bound and quencher bound receptor by immunocapture,
• c) digesting the isolated drug bound and quencher bound receptor to generate drug bound and quencher bound surrogate peptides, and
• d) measuring the amount of surrogate peptides using LC-MS/MS and determining the amount of receptor bound to drug by comparing the amount of drug bound surrogate peptides to the total amount of drug bound and quencher bound surrogate peptides.

A method for determining BTK receptor occupancy in a subject dosed with a drug, wherein the drug is shown in FIG. 1A comprising:

• a) adding contacting a blood sample obtained from the subject with a lysis solution containing the comprising a quencher, wherein the quencher is shown in FIG. 1B,
• b) isolating drug bound and quencher bound BTK with anti-BTK biotinylated antibody bound to Streptavidin T1 Beads,
• c) digesting the isolated drug bound and quencher bound BTK-antibody complex with trypsin to generate surrogate peptides, and
• d) measuring the amount of surrogate peptides using LC-MS/MS chromatography and determining the amount of BTK bound to drug by comparing the amount of drug bound surrogate peptide to the total amount of drug bound and quencher bound surrogate peptides.