Assessment of an in vitro blood-brain barrier model using several [Met⁵]enkephalin opioid analogs

Confluent monolayers of primary and continuous passaged cultures of bovine brain microvessel endothelial cells (BMEC) have been suggested to model the blood-brain barrier (BBB). Increased lipophilicity has been previously suggested to increase BBB penetration. The intent of this study was to examine the effect that structural modifications of the [Met⁵]enkephalin analog DPDPE had on lipophilicity and passage across the BMEC. The BMEC consisted of a monolayer of confluent primary BMEC grown on polycarbonate (10 μm) filters. Permeability coefficients were calculated on the basis of the diffusion of peptides across the BMEC in a Side-Bi-Side® diffusion chamber. Lipophilicity of the peptides examined was determined by using reversed- phase HPLC and calculating the capacity factor (k). Diffusion across the BMEC (for all peptides examined) was linear from 15 to 120 min; therefore, these time points were used to calculate permeability coefficients. Permeability coefficients ranged from 14.34 to 92.00 cm/min (x 10^-4), with [ρ- CIPhE(4,4')]biphalin the highest. Analysis of variance coupled with the Newman-Keuls test showed significantly greater (P < .01) passage of select peptide analogs across the BMEC, including [ρ-CIPhe(4,4')]biphalin, [ρ- CIPHe⁴]DPDPE and reduced DPDPE. Interestingly, upon passage across the confluent monolayer, reduced DPDPE was converted to cyclized DPDPE. Calculated HPLC capacity factors ranged from 3.82 to 12.50. The most lipophilic peptide (highest) examined was acetylated Phe⁰-DPDPE. Analysis of the regression line of permeability coefficients plotted against capacity factors yielded a correlation coefficient of 0.745 (P < .01). The data provided in this study offer strong evidence that increasing peptide lipophilicity enhances passage across the BMEC. The greatest BMEC permeability coefficients, though not the greatest capacity factors, were obtained with peptides having a chlorohalogenation at the Phe⁴ residue, suggesting that factors other than lipophilicity may play a role in BMEC passage. Comparison of the permeability coefficients obtained from the BMEC system with those obtained from in vivo BBB studies suggest that the BMEC system may be very useful in predicting peptide (analog) passage across the in vivo BBB.