Lipophilic derivatives of leu-enkephalinamide: In vitro permeability, stability and in vivo nasal delivery

Leu-enkephalin is an endogenous pain modulating opioid pentapeptide. Its development as a potential pharmaceutic has been hampered by poor membrane permeability and susceptibility to enzymatic degradation. The addition of an unnatural amino acid containing a lipidic side chain at the N-terminus and the modification of the C-terminus to a carboxyamide was performed to enhance the nasal delivery of the peptide. Two lipidic derivatives with varying side chain lengths (C8-Enk-NH2 (1), C12-Enk-NH2 (2)) and their acetylated analogues were successfully synthesised. Caco-2 cell monolayer permeability and Caco-2 cell homogenate stability assays were performed. C8-Enk-NH2 (1) and its acetylated analogue Ac-C8-Enk-NH2 (3) exhibited apparent permeabilities (mean±SD) of 2.51±0.75×10−6cm/s and 1.06±0.62×10−6, respectively. C12-Enk-NH2 (2) exhibited an apparent permeability of 2.43±1.26×10−6cm/s while Ac-C12-Enk-NH2 (4) was not permeable through the Caco-2 monolayers due to its poor solubility. All analogues exhibited improved Caco-2 homogenate stability compared to Leu-Enk-NH2 with t½ values of: C8-Enk-NH2 (1): 31.7min, C12-Enk-NH2 (2): 14.7min, Ac-C8-Enk-NH2 (3): 83min, Ac-C12-Enk-NH2 (4): 27min. However, plasma stability assays revealed that the diastereoisomers of C8-Enk-NH2 (1) did not degrade at the same rate, with the l isomer (t1/2=8.9min) degrading into Leu-enkephalinamide and then des-Tyr-Leu-Enk-NH2, whereas the d isomer was stable (t1/2=120min). In vivo nasal administration of C8-Enk-NH2 to male rats resulted in concentrations of 5.9±1.84×10−2μM in the olfactory bulbs, 1.35±1.01×10−2μM in the brain and 6.53±1.87×10−3μM in the blood 10min after administration.