title: Lipophilic derivatives of leu-enkephalinamide: in vitro permeability, stability and in vivo nasal delivery.
abstract: 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 (C(8)-Enk-NH(2) (1), C(12)-Enk-NH(2) (2)) and their acetylated analogues were successfully synthesised. Caco-2 cell monolayer permeability and Caco-2 cell homogenate stability assays were performed. C(8)-Enk-NH(2) (1) and its acetylated analogue Ac-C8-Enk-NH(2) (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-NH(2) (2) exhibited an apparent permeability of 2.43±1.26×10(-6) cm/s while Ac-C12-Enk-NH(2) (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-NH(2) with t(½) values of: C8-Enk-NH(2) (1): 31.7 min, C(12)-Enk-NH(2) (2): 14.7 min, Ac-C8-Enk-NH(2) (3): 83 min, Ac-C(12)-Enk-NH(2) (4): 27 min. However, plasma stability assays revealed that the diastereoisomers of C8-Enk-NH(2) (1) did not degrade at the same rate, with the l isomer (t(1/2)=8.9 min) degrading into Leu-enkephalinamide and then des-Tyr-Leu-Enk-NH(2), whereas the d isomer was stable (t(1/2)=120 min). In vivo nasal administration of C(8)-Enk-NH(2) 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.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Brisbane 4072, Australia.
authors.forename: Cécile D
authors.initials: CD
authors.lastname: Cros
authors.forename: Istvan
authors.initials: I
authors.lastname: Toth
authors.forename: Joanne T
authors.initials: JT
authors.lastname: Blanchfield
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D004743:Enkephalin, Leucine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008055:Lipids / Q000737:chemistry*; D008297:Male; D015394:Molecular Structure; D009297:Nasal Mucosa / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008055:Lipids; D004743:Enkephalin, Leucine; C048614:enkephalinamide-Leu