Structure-activity relationships of a series of [D-Ala2]deltorphin I and II analogues; in vitro blood-brain barrier permeability and stability

[D-Ala2]deltorphins are enzymatically stable, amphibian heptapeptides that have a higher affinity and selectivity for delta-opioid receptors than any endogenous mammalian compound known. This study investigated the in vitro blood-brain barrier permeability, using primary bovine brain microvessel endothelium culture, and the resistance to enzymatic degradation, in mouse 15% brain membrane homogenates and 100% plasma, of [D-Ala2]deltorphin I, [D-Ala2]deltorphin II and several analogues. Derivatives were designed with the addition of N-terminal neutral and basic Amino acids or with alterations of the Amino acids present within the deltorphin sequences. The results indicated that the N-terminal sequence and the Amino acids in position 4 and 5 are critical to deltorphin analogue BBB permeability and biological stability, i.e., t 1/2 brain; 4.8 hr- [D-Ala2]deltorphin I; > 15 hr- [D-Ala2, Ser4, D-Ala5]deltorphin. Although, no analogue was found to increase the BBB permeability coefficient (PC; x10(-4) cm/min) of the parent compounds ([D-Ala2]deltorphin II, PC = 23.49 +/- 2.42) analogues were identified: [Arg0, D-Ala2]deltorphin II, PC = 19.06 +/- 3.73 and [Pro-1, Pro0, D-Ala2]deltorphin II, PC = 22.22 +/- 5.93; which had similar permeability coefficients, even though they had larger molecular weights and, in the case of the cationic prodrug, a significantly lower lipophilicity. These analogues provide directions in the development of future pro-drugs for the treatment of pain and this study further clarifies the structure-activity relationship of the deltorphins.