Since its initial discovery in 1922 by Banting and Best, the formulation of an oral insulin delivery system has ever been so troublesome. Unfortunately, insulin is indispensable in the treatment of diabetes mellitus, which affects approximately 350 million people worldwide. Various factors contribute to the peptide being such a persistently difficult hormone to be used in an oral formulation. The gastrointestinal tract is home to various protein digestive enzymes such as pepsins in the stomach and trypsin, chymotrypsin and carboxypeptidases in the small intestine, which digests insulin. Also the physical barrier of the gastrointestinal tract, i.e. the columnar epithelial layer which lines the tract, is a tightly bound collection of cells with minimal leakage and is thus a sound barrier for the absorption of peptides and hormones. The aim of this study is to determine whether a dosage form for insulin, entrapped in Pheroid™ vesicles and -micro sponges, can overcome these barriers and successfully deliver insulin at the site of action resulting in a significant therapeutic response. Initial phases of the study consisted of the manufacturing of Pheroid™ vesicles and - microsponges, entrapment of flourescein-isothiocyanate labelled insulin (FITC-insulin) into the Pheroid™. The Pheroid™-insulin complex was analysed with confocal laser scanning microscopy (CLSC) to determine drug loading. In vivo experiment in Sprague - Dawley rats were done where blood glucose levels as well as insulin blood levels were monitored after administration of different Pheroid insulin formulations. Firstly a standard reference was set by subcutaneous injection of insulin (0.5 IU/kg) in rats followed by a comparative study where administration to the stomach, colon and ileum (50.0 IUlkg insulin) were compared by means of blood insulin levels and therapeutic effect between the control and Pheroid™ complexes (Pheroid™ vesicles and microsponges). Each study was done by means of direct injection into the stomach, ileum or colon through which the insulin in saline (control) or insulin-Pheroid™ complex was administered. Nasal administration of 8.0 and 12.0 IU/kg insulin in saline (control) or insulin-Pheroid™ complex was done in the right nostril of Sprague - Dawley rats. Blood samples were taken from the artery carotis communis by means of an inserted cannula. Blood samples were taken just before administration and then at 5, 10, 15, 30, 60, 120 and 180 minutes after administration. Blood glucose levels were measured just after every blood sample was taken and plasma insulin levels were determined with a human insulin specific radioimmunoassay. The results were compared to the reference as well as the control to determine relative bioavailability. Through the results obtained it was discovered that in comparison with the various parts of the or tract, the ileum showed undoubtedly to be the best area of absorption where Pheroid™ vesicles revealed a peak 42.0 % lowering in blood glucose levels after 60 minutes and a peak plasma concentration of 244.0 /uID/ml after 5 minutes together with an 18.7 % lowering in blood glucose levels after just 5 minutes. After nasal administration of Pheroid™ microsponges (8.0 ID/kg insulin) a remarkable lowered blood glucose level of 19.2 % after 10 minutes and 36.5 % after 30 minutes as well as a peak plasma insulin level of220.2 /lID/ml after 3 hours was observed. Insulin entrapped in Pheroid™ microsponges administered at 12.0 ID/kg showed a maximum blood glucose lowering effect of72.4 % after 3 hours with a peak plasma level of 154.8 uID/ml also after 3 hours, thus showing a long acting effect. In conclusion, the delivery system based on Pheroid™ technology shows a sufficient therapeutic effect for insulin and is therefore promising for further in vivo evaluation and ultimately for medicinal use to patients suffering from diabetes mellitus. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Since its initial discovery in 1922 by Banting and Best, the formulation of an oral insulin delivery system has ever been so troublesome. Unfortunately, insulin is indispensable in the treatment of diabetes mellitus, which affects approximately 350 million people worldwide. Various factors contribute to the peptide being such a persistently difficult hormone to be used in an oral formulation. The gastrointestinal tract is home to various protein digestive enzymes such as pepsins in the stomach and trypsin, chymotrypsin and carboxypeptidases in the small intestine, which digests insulin. Also the physical barrier of the gastrointestinal tract, i.e. the columnar epithelial layer which lines the tract, is a tightly bound collection of cells with minimal leakage and is thus a sound barrier for the absorption of peptides and hormones. The aim of this study is to determine whether a dosage form for insulin, entrapped in Pheroid™ vesicles and -micro sponges, can overcome these barriers and successfully deliver insulin at the site of action resulting in a significant therapeutic response. Initial phases of the study consisted of the manufacturing of Pheroid™ vesicles and - microsponges, entrapment of flourescein-isothiocyanate labelled insulin (FITC-insulin) into the Pheroid™. The Pheroid™-insulin complex was analysed with confocal laser scanning microscopy (CLSC) to determine drug loading. In vivo experiment in Sprague - Dawley rats were done where blood glucose levels as well as insulin blood levels were monitored after administration of different Pheroid insulin formulations. Firstly a standard reference was set by subcutaneous injection of insulin (0.5 IU/kg) in rats followed by a comparative study where administration to the stomach, colon and ileum (50.0 IUlkg insulin) were compared by means of blood insulin levels and therapeutic effect between the control and Pheroid™ complexes (Pheroid™ vesicles and microsponges). Each study was done by means of direct injection into the stomach, ileum or colon through which the insulin in saline (control) or insulin-Pheroid™ complex was administered. Nasal administration of 8.0 and 12.0 IU/kg insulin in saline (control) or insulin-Pheroid™ complex was done in the right nostril of Sprague - Dawley rats. Blood samples were taken from the artery carotis communis by means of an inserted cannula. Blood samples were taken just before administration and then at 5, 10, 15, 30, 60, 120 and 180 minutes after administration. Blood glucose levels were measured just after every blood sample was taken and plasma insulin levels were determined with a human insulin specific radioimmunoassay. The results were compared to the reference as well as the control to determine relative bioavailability. Through the results obtained it was discovered that in comparison with the various parts of the or tract, the ileum showed undoubtedly to be the best area of absorption where Pheroid™ vesicles revealed a peak 42.0 % lowering in blood glucose levels after 60 minutes and a peak plasma concentration of 244.0 /uID/ml after 5 minutes together with an 18.7 % lowering in blood glucose levels after just 5 minutes. After nasal administration of Pheroid™ microsponges (8.0 ID/kg insulin) a remarkable lowered blood glucose level of 19.2 % after 10 minutes and 36.5 % after 30 minutes as well as a peak plasma insulin level of220.2 /lID/ml after 3 hours was observed. Insulin entrapped in Pheroid™ microsponges administered at 12.0 ID/kg showed a maximum blood glucose lowering effect of72.4 % after 3 hours with a peak plasma level of 154.8 uID/ml also after 3 hours, thus showing a long acting effect. In conclusion, the delivery system based on Pheroid™ technology shows a sufficient therapeutic effect for insulin and is therefore promising for further in vivo evaluation and ultimately for medicinal use to patients suffering from diabetes mellitus. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Various colloidal delivery systems, including polymeric nanoparticles, metal colloids, liposomes, and microemulsions have been reported to enhance the delivery of therapeutic agents following intranasal administration. However, the mechanisms involved in the uptake of these nanomaterials, especially those in the ultrafine size ranges (diameter < 20 nm) through nasal mucosa and their subsequent biodistribution in the body are not well characterized. The objectives of this study address the knowledge gap regarding ultrafine nanoparticle transfer in the nasal mucosa by quantifying nanoparticle uptake and biodistibution patterns in the presence and absence of known inhibitors of endocytic processes. The uptake of ~ 10 nm fluorescent quantum dots (QDs) was investigated by measuring the concentration of QDs following exposure to bovine respiratory and olfactory mucosal explants. An inductively coupled optical emission spectroscopy method was developed to measure the amount of QDs within the tissues. The results demonstrated that carboxylate-modified QDs (COOH-QDs) show ~2.5 fold greater accumulation in the epithelial and submucosal regions of the olfactory tissues compared to the respiratory tissues. Endocytic inhibitory studies showed that in respiratory tissues clathrin-dependent, macropinocytosis and caveolae-dependent endocytosis process were all involved in the uptake of COOH-QDs. Whereas in olfactory tissues, clathrin-dependent endocytosis was the major endocytic pathway involved in uptake of COOH-QDs. Additional energy-independent pathways appeared to also be active in the transfer of COOH-QDs into the olfactory mucosa. Interestingly, PEGylated quantum dots (PEG-QDs) of similar size ~15 nm were not internalized into the bovine nasal tissues. In vivo fluorescence imaging was used to study the biodistribution of quantum dots following nasal instillation in mice. These studies showed that majority of COOH-QDs remain in the nasal tissues for relatively long periods of time (up to 24 h) whereas PEG-QDs showed no such accumulation. Biodistribution studies of gold nanoparticles (~15 nm) in mice using micro-CT showed that gold nanoparticles were transferred to the posterior turbinate region and a fraction of the administered dose distributed to regions in close proximity to the olfactory bulb. Both NIR imaging and micro-CT imaging were useful tools for visualization of in vivo nanoparticle distribution. A diazepam-containing microemulsion (dispersed phase ~40 nm) was formulated to investigate the uptake mechanisms utilized for fluid-phase colloidal dispersions in the nasal mucosa. The resulting diazepam-containing microemulsion showed enhanced transfer of the drug into the bovine nasal respiratory and olfactory tissues. It is unclear if endocytosis of the fluid-phase nanodispersions played a role in drug absorption from the microemulsions in a manner similar to the uptake of solid-phase nanoparticles, however, since there was significant loss of the epithelial cell layer following exposure to the microemulsion formulation which likely altered the barrier properties of the epithelium. These studies have increased the fundamental understanding of ultrafine nanoparticle uptake in the nasal tissues and the resulting nanoparticle biodistribution patterns. While ultrafine nanoparticles may have limited application in the development of efficient drug delivery systems, an understanding of the size-dependent and tissue-dependent processes responsible for the uptake of particulates into mucosal tissues will contribute to the rational development of nanoparticulate drug delivery strategies investigating the nasal and other routes of administration.

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Approximately 350 million people worldwide suffer from diabetes mellitus (DM) and this number increases yearly. Since the discovery and clinical application of insulin in 1921, subcutaneous injections have been the standard treatment for DM. Because insulin is hydrophilic and has a high molecular weight and low bioavailability, this molecule is poorly absorbed if administered orally. The aim of this study is to evaluate nasal delivery systems for insulin, using Sprague Dawley rats as the nasal absorption model. Pheroid technology and N-trimethyl chitosan chloride (TMC) with different dosages of insulin (4, 8 and 12 IU/kg bodyweight insulin) was administered in the left nostril of the rat by using a micropipette. Pheroid technology is a patented (North-West University) carrier system consisting of a unique oil/water emulsion that actively transports drug actives through various physiological barriers. These formulations were administered nasally to rats in a volume of 100 p/kg bodyweight in different types of Pheroids (vesicles, with a size of 1.7 1 - 1.94 pm and microsponges, with a size of 5.7 1 - 8.25 pm). The systemic absorption of insulin was monitored by measuring arterial blood glucose levels over a period of 3 hours. The TMC formulation with 4 IU/kg insulin produced clinically relevant levels of insulin in the blood and as a result also the maximal hypoglycaemic effect. TMC is a quaternary derivative of chitosan and is able to enhance the absorption of various peptide drugs by opening tight junctions between epithelial cells. Pheroid formulations were also effective in lowering blood glucose levels but only at higher doses (8 and 12 IU/kg) of insulin. This study indicated that Pheroid rnicrosponges had a faster onset of action and a slightly better absorption of insulin when compared to Pheroid vesicles, but many more studies are needed in this field. Although the results of this study with absorption enhancers are encouraging, nasal insulin bioavailability is still very low, and the Pheroid formulations and long-term safety of nasal insulin therapy have yet to be investigated. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

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.