Journal
MOLECULAR PHARMACEUTICS
Volume 10, Issue 5, Pages 1542-1556Publisher
AMER CHEMICAL SOC
DOI: 10.1021/mp3004995
Keywords
blood-brain barrier; single-domain antibodies; CSF; pharmacokinetics; MRM; SRM; mass spectrometry; drug delivery
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FC5 and FC44 are single-domain antibodies (V(H)Hs), 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 V(H)Hs, EG2 and A20.1. FC5 and FC44 compared to control, V(H)Hs 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 V(H)Hs demonstrated that while all 4 V(H)Hs 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.
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