Effect of guanidino modification and proline substitution on the in vitro stability and blood-brain barrier permeability of endomorphin II

Endomorphin II (ENDII), an endogenous ligand for the μ-opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro⁴ substitution and cationization affected physico-chemical characteristics, blood-brain barrier (BBB) transport, and analgesic profile. END II and its Pro⁴-substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)-addition. MOR was seven times less lipophilic than END II, whereas GU-addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU-addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU-addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t_1/2 compared to END II. GU-addition significantly increased t_1/2 of MOR and END II in both brain (sixfold and 10-fold, respectively) and serum (over 10-fold). Pro⁴-substitution and GU-addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro⁴-substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro⁴-substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs.