Design of Cyclic-ADT Peptides to Improve Drug Delivery to the Brain via Inhibition of E-Cadherin Interactions at the Adherens Junction

We have developed linear cadherin peptides (i.e., HAV- and ADT peptides) that enhance brain delivery of drug molecules to the central nervous system (CNS). These peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells in the blood-brain barrier (BBB) to increase paracellular drug permeation. In this study, the goal was to design cyclic peptides (ADTC1, ADTC5, and ADTC6) derived from linear ADT6 (Ac-ADTPPV-NH2) to improve their stability and biological activity in improving paracellular delivery of drugs into the brain. The ADTC1 peptide (cyclo(1,8)Ac-CADTPPVC-NH2) was designed by adding two Cys residues at the N- and C-terminus of ADT6 peptide and a disulfide bond from thiol groups of the Cys residues. The ADTC5 peptide (cyclo(1,7)Ac-CDTPPVC-NH2) was derived from ADTC1 by deleting the alanine residue from the N-terminal region of ADTC1 and ADTC6. (Cyclo(1,6)Ac-CDTPPC-NH2) was constructed by deleting the valine residue from the C-terminal region of ADTC5. The results showed that ADTC1 has activity in inhibiting the resealing of the intercellular junctions of the MDCK cell monolayers similar to that of the linear ADT6, indicating that cyclization can maintain the peptide activity. The alanine residue deletion in ADTC5 does not reduce its activity compared to ADTC1 peptide, suggesting that the alanine residue does not have an important role in the activity of the peptide. In contrast, ADTC6 peptide does not have activity in inhibiting the junction resealing, indicating that the valine residue is important for peptide activity. ADTC5 inhibits the junction resealing of MDCK cell monolayers in a concentration-dependent manner with the saturation concentration above 0.4 mM and IC50 around 0.3 mM. Under the current experimental conditions, ADTC5 improves the delivery of 14C-mannitol to the brain about two fold compared to the vehicle negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide does not enhance the BBB passage of large polyethylene glycol molecules (i.e., PEG-1500 and PEG-40000) in an in situ rat brain perfusion model. In conclusion, formation of cyclic peptides can maintain cadherin peptide ability to modulate intercellular junctions of the BBB, and the primary sequence of ADT peptides is important for their biological activity