title: Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method.
abstract: FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.1. FC5 and FC44 compared to control VHHs demonstrated significantly (p < 0.01) enhanced transport (50-100-fold) across rat in vitro BBB model as well as in vivo brain targeting assessed by optical imaging. The multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed VHHs demonstrated that while all 4 VHHs have similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF levels, suggesting that they are potential novel carriers for delivery of drugs and macromolecules across the BBB.
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada.
authors.forename: Arsalan S
authors.initials: AS
authors.lastname: Haqqani
authors.forename: Nadia
authors.initials: N
authors.lastname: Caram-Salas
authors.forename: Wen
authors.initials: W
authors.lastname: Ding
authors.forename: Eric
authors.initials: E
authors.lastname: Brunette
authors.forename: Christie E
authors.initials: CE
authors.lastname: Delaney
authors.forename: Ewa
authors.initials: E
authors.lastname: Baumann
authors.forename: Eve
authors.initials: E
authors.lastname: Boileau
authors.forename: Danica
authors.initials: D
authors.lastname: Stanimirovic
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000276:immunology; D001921:Brain / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016503:Drug Delivery Systems; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D036103:Nanotechnology; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D061905:Single-Domain Antibodies / Q000008:administration & dosage / Q000097:blood* / Q000134:cerebrospinal fluid*; D014018:Tissue Distribution
chemical_list: D061905:Single-Domain Antibodies