Novel Blood-Brain Barrier Shuttle Peptides Discovered through the Phage Display Method.

Petra Majerova,Petra Majerova,Emil Pilipcinec,Dominika Olesova,Jakub Sinsky,Jakub Sinsky,Jozef Hanes,Jozef Hanes,Andrej Kovac,Andrej Kovac,Andrej Kovac

doi:10.3390/molecules25040874

Abstract

Delivery of therapeutic agents into the brain is a major challenge in central nervous system drug development. The blood–brain barrier (BBB) prevents access of biotherapeutics to their targets in the central nervous system and, therefore, prohibits the effective treatment of many neurological disorders. To find blood–brain barrier shuttle peptides that could target therapeutics to the brain, we applied a phage display technology on a primary endothelial rat cellular model. Two identified peptides from a 12 mer phage library, GLHTSATNLYLH and VAARTGEIYVPW, were selected and their permeability was validated using the in vitro BBB model. The permeability of peptides through the BBB was measured by ultra-performance liquid chromatography-tandem mass spectrometry coupled to a triple-quadrupole mass spectrometer (UHPLC-MS/MS). We showed higher permeability for both peptides compared to N–C reversed-sequence peptides through in vitro BBB: for peptide GLHTSATNLYLH 3.3 × 10−7 cm/s and for peptide VAARTGEIYVPW 1.5 × 10−6 cm/s. The results indicate that the peptides identified by the in vitro phage display technology could serve as transporters for the administration of biopharmaceuticals into the brain. Our results also demonstrated the importance of proper BBB model for the discovery of shuttle peptides through phage display libraries.

Highlights

The blood–brain barrier (BBB) represents the bottleneck in brain drug development and is the most critical factor limiting the future growth of neurotherapeutics
Peptides cross the BBB through endocytic mechanisms involving receptor-mediated transcytosis (RMT) and/or adsorptive-mediated transcytosis (AMT) [3], whereby both belong to active mechanisms of transport
Identification of Peptides Binding to Primary Rat Endothelial Cells

Summary

Introduction

The blood–brain barrier (BBB) represents the bottleneck in brain drug development and is the most critical factor limiting the future growth of neurotherapeutics. The BBB is a selective semi-permeable barrier formed by the endothelial cells that shape cerebral microvessels and separate blood from the brain. Therapeutic strategies to deliver drugs into the central nervous system (CNS) are limited by the restrictive tight junctions among the endothelial cells of BBB. Ideal drug candidates are small, lipophilic, hydrophobic, and compact molecules that can cross the BBB [1]. For example, peptides, recombinant proteins, monoclonal antibodies, RNA interference (RNAi)-based drugs, and >98% of small-molecule drugs do not cross the BBB [2]. Peptides cross the BBB through endocytic mechanisms involving receptor-mediated transcytosis (RMT) and/or adsorptive-mediated transcytosis (AMT) [3], whereby both belong to active mechanisms of transport. In the process of transcytosis, the formed vesicles circulate across the cell, bypassing the degradation pathway

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