The development of peptides as therapeutic agents has been hampered by their poor enzymatic stability and bioavailability. Many strategies, such as chemical modification, synthesis of peptidomimetics and formulation, have been employed to overcome these issues. For central nervous system (CNS) active peptides, the blood brain barrier is an added hurdle. Nasal delivery is believed to provide a direct access to the brain via the olfactory nerve, which would bypass the blood brain barrier. This route of administration, however, is dependant on the size and physico-chemical properties of the administered drug. For these reasons, three CNS active peptides were chosen as models. Leu-enkephalin, endomorphin-1 and a-conotoxin MII are three peptides that differ in their size, amino acid sequence and conformation. Using chemical modifications to improve their stability and ability to cross biological membranes, in vitro assessments of derivatives of these peptides were performed and in vivo nasal delivery was attempted on the most promising candidates. The chemical modifications consisted in the addition of lipids and/or sugars to the N- or C-terminus of the peptides. Assessment of the in vivo bioavailability after nasal administration, however, proved to be challenging. The initial method chosen for this purpose was the use of tritiated acetic anhydride which would radiolabel the peptide via acetylation at the N-terminus of the peptide derivatives. Consequently, in vitro stability and permeability of each acetylated derivatives was also studied. Acetylation of the lipidic derivatives, which formed an amide bond, proved to be beneficial for the stability of the lipidic peptides. In contrast, acetylation of the Nterminus sugar derivatives, which formed an ester bond at one or several positions of the sugar, was an unstable modification. Thus, an extraction method for the tested peptides from rat tissues was developed, and LC-MS/MS analyses were conducted to measure the level of peptide in the olfactory bulbs, brain and blood. Leu-enkephalin derivatives were all amide derivatives at the C-terminus of the peptide. The most successful Leu-enkephalinamide derivatives were C8-LeuEnk (2), C12- LeuEnk (3) and Lac-LeuEnk (8), which are the Leu-enphelinamide peptide modified with a C8 lipoamino acid, a C12 lipoamino acid and a lactose moiety respectively. They all exhibited improved permeability across Caco-2 monolayers and stability in Caco-2 cell homogenate and/or plasma. Problems of solubility encountered with C12-LeuEnk (3), however, hampered its testing in vivo after nasal administration. C8-LeuEnk (2) and Lac-LeuEnk (8) were administered intranasally to male Sprague-Dawley rats. Both peptides were found in the olfactory bulbs after 10 minutes administration (2: 49.2 ± 15.6 nM; 8: 40.6 ± 14.6 nM) while blood concentration remained low, showing that the peptide reached the olfactory bulbs directly from the nasal cavity via the olfactory nerve. Brain concentrations were 13.5 ± 10.1 nM for C8-LeuEnk (2) and 13.6 ± 6.9 nM for Lac-LeuEnk (8). These two peptides brain concentrations seemed to be high enough to exhibit analgesic effect when compared to their binding affinity in vitro. This was not statistically significant, however, due to the high standard deviations observed (Kiμ C8-LeuEnk (2) = 7.74 ± 1.15 nM; Kiμ Lac-LeuEnk (8) = 6.69 ± 1.81 nM). Endomorphin-1 was only modified at the N-terminus as previous results have shown that the activity of the peptide is strongly decreased by C-terminus modifications. The most successful modification, regarding permeability across Caco-2 monolayers and water solubility, was shown to be the addition of a lactose moiety to the N-terminus of the peptide. Lac-Endo1 (16) exhibited a permeability of 1.91 ± 0.76 x 10-6 cm/s and was soluble at the concentration used for in vivo nasal administration (2 mg/Kg, 50 μL administration). After 10 minutes administration, Lac-Endo1 (16) was found in the olfactory bulbs (418 ± 410 nM), in the brain (4.01 ± 4.61 nM) and in the blood (1.58 ± 1.85 nM). The large standard deviations observed reflect the difficulties encountered with the extraction process of this peptide. A direct transport for the nasal cavity to the olfactory bulb was observed as illustrated by the low blood concentrations. Brain concentrations, however, were too low to expect a strong analgesic effect from this compound after nasal administration (Kiμ Lac-Endo1 (16) = 11.3 ± 1.2 nM). a-Conotoxin MII is a 16 amino acid long peptide containing two disulfide bonds. The formation of these two disulfide bonds leads to low yields in the synthesis of the derivatives of this peptide. Addition of a lipidic moiety to the peptide did not seem to improve its permeability through biological membranes. This modification resulted in highly lipophilic peptides with dissolution issues in water based media such as those used in the permeability experiments. The most successful a-conotoxin MII derivative was GS-Ctx (25) which exhibited a permeability of 4.22 ± 0.53 x 10-7 cm/s across Caco-2 monolayers. This permeability, however, was too low to consider in vivo administration. In conclusion, we successfully synthesised a series of derivatives of Leu-enkephalin, endomorphin-1 and a-conotoxin MII and screened them through Caco-2 monolayers for permeability and Caco-2 cell homogenates and human plasma for stability. Three derivatives (C8-LeuEnk (2), Lac-LeuEnk (8) and Lac-Endo1 (16)) were intranasally administered and found in the olfactory bulbs 10 minutes after administration. The low blood concentrations observed show that a direct transport from the nasal cavity to the brain occurs. Thus, nasal administration could be an option for delivering to the brain low molecular weight peptides exhibiting increased stability and permeability in vitro.

The development of peptides as therapeutic agents has been hampered by their poor enzymatic stability and bioavailability. Many strategies, such as chemical modification, synthesis of peptidomimetics and formulation, have been employed to overcome these issues. For central nervous system (CNS) active peptides, the blood brain barrier is an added hurdle. Nasal delivery is believed to provide a direct access to the brain via the olfactory nerve, which would bypass the blood brain barrier. This route of administration, however, is dependant on the size and physico-chemical properties of the administered drug. For these reasons, three CNS active peptides were chosen as models. Leu-enkephalin, endomorphin-1 and a-conotoxin MII are three peptides that differ in their size, amino acid sequence and conformation. Using chemical modifications to improve their stability and ability to cross biological membranes, in vitro assessments of derivatives of these peptides were performed and in vivo nasal delivery was attempted on the most promising candidates. The chemical modifications consisted in the addition of lipids and/or sugars to the N- or C-terminus of the peptides. Assessment of the in vivo bioavailability after nasal administration, however, proved to be challenging. The initial method chosen for this purpose was the use of tritiated acetic anhydride which would radiolabel the peptide via acetylation at the N-terminus of the peptide derivatives. Consequently, in vitro stability and permeability of each acetylated derivatives was also studied. Acetylation of the lipidic derivatives, which formed an amide bond, proved to be beneficial for the stability of the lipidic peptides. In contrast, acetylation of the Nterminus sugar derivatives, which formed an ester bond at one or several positions of the sugar, was an unstable modification. Thus, an extraction method for the tested peptides from rat tissues was developed, and LC-MS/MS analyses were conducted to measure the level of peptide in the olfactory bulbs, brain and blood. Leu-enkephalin derivatives were all amide derivatives at the C-terminus of the peptide. The most successful Leu-enkephalinamide derivatives were C8-LeuEnk (2), C12- LeuEnk (3) and Lac-LeuEnk (8), which are the Leu-enphelinamide peptide modified with a C8 lipoamino acid, a C12 lipoamino acid and a lactose moiety respectively. They all exhibited improved permeability across Caco-2 monolayers and stability in Caco-2 cell homogenate and/or plasma. Problems of solubility encountered with C12-LeuEnk (3), however, hampered its testing in vivo after nasal administration. C8-LeuEnk (2) and Lac-LeuEnk (8) were administered intranasally to male Sprague-Dawley rats. Both peptides were found in the olfactory bulbs after 10 minutes administration (2: 49.2 ± 15.6 nM; 8: 40.6 ± 14.6 nM) while blood concentration remained low, showing that the peptide reached the olfactory bulbs directly from the nasal cavity via the olfactory nerve. Brain concentrations were 13.5 ± 10.1 nM for C8-LeuEnk (2) and 13.6 ± 6.9 nM for Lac-LeuEnk (8). These two peptides brain concentrations seemed to be high enough to exhibit analgesic effect when compared to their binding affinity in vitro. This was not statistically significant, however, due to the high standard deviations observed (Kiμ C8-LeuEnk (2) = 7.74 ± 1.15 nM; Kiμ Lac-LeuEnk (8) = 6.69 ± 1.81 nM). Endomorphin-1 was only modified at the N-terminus as previous results have shown that the activity of the peptide is strongly decreased by C-terminus modifications. The most successful modification, regarding permeability across Caco-2 monolayers and water solubility, was shown to be the addition of a lactose moiety to the N-terminus of the peptide. Lac-Endo1 (16) exhibited a permeability of 1.91 ± 0.76 x 10-6 cm/s and was soluble at the concentration used for in vivo nasal administration (2 mg/Kg, 50 μL administration). After 10 minutes administration, Lac-Endo1 (16) was found in the olfactory bulbs (418 ± 410 nM), in the brain (4.01 ± 4.61 nM) and in the blood (1.58 ± 1.85 nM). The large standard deviations observed reflect the difficulties encountered with the extraction process of this peptide. A direct transport for the nasal cavity to the olfactory bulb was observed as illustrated by the low blood concentrations. Brain concentrations, however, were too low to expect a strong analgesic effect from this compound after nasal administration (Kiμ Lac-Endo1 (16) = 11.3 ± 1.2 nM). a-Conotoxin MII is a 16 amino acid long peptide containing two disulfide bonds. The formation of these two disulfide bonds leads to low yields in the synthesis of the derivatives of this peptide. Addition of a lipidic moiety to the peptide did not seem to improve its permeability through biological membranes. This modification resulted in highly lipophilic peptides with dissolution issues in water based media such as those used in the permeability experiments. The most successful a-conotoxin MII derivative was GS-Ctx (25) which exhibited a permeability of 4.22 ± 0.53 x 10-7 cm/s across Caco-2 monolayers. This permeability, however, was too low to consider in vivo administration. In conclusion, we successfully synthesised a series of derivatives of Leu-enkephalin, endomorphin-1 and a-conotoxin MII and screened them through Caco-2 monolayers for permeability and Caco-2 cell homogenates and human plasma for stability. Three derivatives (C8-LeuEnk (2), Lac-LeuEnk (8) and Lac-Endo1 (16)) were intranasally administered and found in the olfactory bulbs 10 minutes after administration. The low blood concentrations observed show that a direct transport from the nasal cavity to the brain occurs. Thus, nasal administration could be an option for delivering to the brain low molecular weight peptides exhibiting increased stability and permeability in vitro.