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 × 10−6 cm/s and 1.06 ± 0.62 × 10−6, respectively. C12-Enk-NH2 (2) exhibited an apparent permeability of 2.43 ± 1.26 × 10−6 cm/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): 31.7 min, C12-Enk-NH2 (2): 14.7 min, Ac-C8-Enk-NH2 (3): 83 min, Ac-C12-Enk-NH2 (4): 27 min. 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 = 8.9 min) degrading into Leu-enkephalinamide and then des-Tyr-Leu-Enk-NH2, whereas the d isomer was stable (t1/2 = 120 min). In vivo nasal administration of C8-Enk-NH2 to male rats resulted in concentrations of 5.9 ± 1.84 × 10−2 μM in the olfactory bulbs, 1.35 ± 1.01 × 10−2 μM in the brain and 6.53 ± 1.87 × 10−3 μM in the blood 10 min after administration.