A novel liposaccharide system for the delivery of endomorphin-1 across the blood-brain barrier

Koda, Yasuko (2007). A novel liposaccharide system for the delivery of endomorphin-1 across the blood-brain barrier PhD Thesis, School of Pharmacy, University of Queensland.

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Author Koda, Yasuko
Thesis Title A novel liposaccharide system for the delivery of endomorphin-1 across the blood-brain barrier
School, Centre or Institute School of Pharmacy
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Dr Joanne Blanchfield
Abstract/Summary The endogenous opioid peptide endomorphin-1 (Endo-1; Tyr-Pro-Trp-Phe-NH2) was isolated from mammalian brain tissue by Zadina et.al. in 1997. This naturally occurring peptide exhibits high selectivity and affinity for the µ-opioid receptor and its efficacy as a µ-opioid agonist has been shown in vivo and in vitro as similar to or higher than that of morphine. The ability to use these endogenous opioid ligands as biologically available pain relief could redefine pain management by offering potent analgesia with potentially minimal side effects. However, the significant therapeutic potential of endogenous opioid peptides is hampered by their poor metabolic stability and permeability cross the blood-brain barrier (BBB) and gastrointestinal (GI) tract in the case of oral administration. In order to improve bioavailability, chemical modifications of Endo-1 bearing a variety of lipids and sugars at the N-/C-terminus and unique peptides incorporating 2',6'- dimethyl-L-tyrosine (DMT) in place of the Tyr residue of Endo-1 were designed and synthesized. These analogues were evaluated for bioavailability using Caco-2 cells and pharmacological efficacy using SH-SY5Y cells in in vitro studies. The most promising compound was selected for in vivo biodistribution studies which were performed using intra venous (i.v.) injection and oral administration in adult male Sprague-Dawley (S.D.) rats. All results were compared to the native peptide Endo-1. Enzymatic stability assays using Caco-2 cell homogenates showed lipoamino acids and glucose succinate conjugated at N-terminus of Endo-1 analogues exhibited great stability with approximately 5-15 fold longer half lives than Endo-1 (t1/2=5.5 minutes). The stable in metabolic enzymes, lipophilic analogue C8-lipoamino acid conjugate (C8Laa-Endo-1) exhibited high apparent permeability (Papp= 3.54 × 10-6 cm/s) through Caco-2 cell monolayers. Conjugates with increased alkyl chain length Laas (carbon atom number of C12 and C18) conjugated to Endo-1 exhibited poor solubility, thus C12Laa-Endo-1 was subjected to various formulation studies achieving 87% liposome encapsulation using hydration of freeze-dried monophase method of preparation. Liposome formulated C12Laa-Endo-1 showed improved water solubility and exhibited significant enzymatic stability and high permeability. Chemically modified Endo-1 analogues were also assessed for opioid receptor binding affinity and functional efficacy using human neuroblastoma SH-SY5Y cells that express µ- and s-opioid receptors. Modification of the C-terminus of Endo-1 caused a severe drop in affinity for the µ- and s-opioid receptors, whereas N-terminal modified compounds exhibited small decreases in affinity. Analogues bearing both DMT and C8Laa conjugated at N-terminus exhibited 20-25 fold greater binding affinity than the native peptide Endo-1. The rank order of inhibition of forskolin-stimulated c-AMP accumulation was similar to receptor binding affinities. The IC50 of (DMT)1-Endo-1 and C8Laa-(DMT)1-Endo-1 was 0.43 and 0.32 nM respectively compared to 14 nM for Endo-1. The substitution of DMT in the place of Tyr residue exhibits great enhancement of these opioid activities. An in vivo study of the biodistribution of C8Laa-Endo-1 was carried out with i.v. injection and oral administration. The distribution study required N-terminal [3H]- acetylation of both Endo-1 as control and C8Laa-Endo-1. A variety of organs were taken at certain time points (15, 30 and 60 minutes) and the [3H] levels measured. C8Laa-Endo-1 showed approximately 10% uptake in the brain at 15 minutes post i.v. injection. After oral administration, C8Laa-Endo-1 accumulated in the small intestine more than Endo-1. Endo-1 was observed more in the liver than C8Laa-Endo-1 over experimental time points following i.v. injection and oral administration suggesting that Endo-1 may be metabolized via first pass metabolism in the liver. In conclusion, N-terminal modifications improved enzymatic stability and cell monolayer permeability in Caco-2 cells assays. Opioid agonist activities in SHSY5Ycells confirmed that an unencumbered C-terminus was essential for Endo-1 pharmacological activity. Furthermore, DMT incorporation at Tyr residue of Endo-1 enhanced opioid agonist activity.

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Created: Fri, 21 Nov 2008, 21:08:15 EST