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 deltaopioid 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 1/4 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; x 10^-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 pro-drug, 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.