Received for publication April 24, 2006.
Revised June 13, 2006.
Accepted for publication June 26, 2006.

Utilization of combined chemical modifications to enhance the blood-brain barrier permeability and pharmacological activity of endomorphin-1

Abstract

The endogenous µ-opioid receptor agonist, endomorphin-1 (EM-1), cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit analgesia when given systemically because it is severely restricted by the blood-brain barrier (BBB). To improve the physicochemical characteristics of EM-1 and subsequently achieve greater BBB permeation, we synthesized a series of EM-1 analogues by combining successful chemical modifications including N-terminal cationization, C-terminal chloro-halogenation and unnatural amino acid (D-Ala, Sar and D-Pro-Gly) substitutions in position 2. Presently, their binding and bioassay activity, lipophilicity, stability and antinociceptive activity were determined and compared. Guanidino-addition and chloro-halogenation attenuated the µ-receptor affinity to some extent, but they demonstrated differences in the influence on stability. It appeared that guanidino-addition contributed to brain stability enhancement for the greater part, while chloro-halogenation together with amino acid substitutions in position 2 was of more importance for the stability enhancement in serum than in brain. Determination of the octanol/buffer coefficient revealed that chloro-halogenation did compromise the decreased lipophilicity caused by guanidino-addition, and introduction of D-Ala as well as D-Pro-Gly, but not Sar, in place of L-Pro² also increased the overall lipophilicity to some extent. Among the peptides tested, intracereventricular injection of guanidino-[D-Ala², p-Cl-Phe⁴]EM-1 showed the strongest analgesia, being three times more potent than the parent peptide. We also found that in comparison with EM-1, the four D-Ala containing tetrapeptides and the chloro-halogenated D-Pro-Gly containing pentapeptide elicited significant and prolonged central-mediated analgesia upon subcutaneous administration, indicating that more peptides reached the CNS eliciting greater analgesic effect.

Key words: antinociception, blood-brain barrier permeability, chemical modifications, endomorphin-1, in vitro stability, lipophilicity