Guanidino cationization effects on the blood-brain barrier permeability, in vitro stability and receptor binding of endomorphins

Cationization has been shown to improve the blood-brain barrier (BBB) passage of peptides via adsorptive endocytosis. Guanidino (GU) addition to the amino group of tyrosine is one way to cationize peptides containing tyrosine. Endomorphins (END), a possible endogenous ligand for the μ opioid receptor have an N-terminal tyrosine and thus can be used to study the effect of cationization. Structural relationships of opioids in drug delivery will direct future directions in analgesia research. In this study we investigated GU effects on in vitro BBB transport, stability and receptor binding of END II, [Pro⁴] END II by comparing non-GU peptides and their GU analogs. An in vitro bovine brain microvessel endothelial cell model (BBMEQ was used to study BBB permeability. In vitro stability was studied via incubating peptide with mouse serum and mouse brain homogenate resuspended at 15% protein. Receptor binding was done using rat whole brain tissue incubated at 25°C for 180 minutes. BBMEC: BBMEC confluent monolayer membrane filters were placed in side-by-side diffusion chambers. Peptides were placed in the huminal chamber and sampling done from the abluminal chamber over time-points (0-120 min). GU addition to END II increased BBB permeability from 31.9 ± 0.90 x 10^-4 to 32.5 ± 0.53 x 10^-4 while GU addition of [Pro⁴] END II increased BBB permeability significantly from 30.6 ± 1.2 x 1^-4 to 40.2 ± 0.5 x 10⁴.In vitro stability: Peptides were added to samples time-course incubated at 37°C and analyzed by HPLC. END II half-life increased from 13.2 min to 140.2 min in brain and from 3.2 min to 26.6 min in serum with GU addition. [Pro⁴] END II half-life increased from 24.2 min to 149.09 min in brain and from 2.92 min to 34.33 min in serum with GU addition. Receptor Binding: END II was found to have an IC₅₀ δ/μ ratio of 0.71 whereas both GU END II and GU [PrO⁴] END II increased to >300. These data provide evidence that increased cationization with GU modification improves both stability and permeability and increases 1C₅₀ δ/μ ratios.