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Polymer carriers composed of poly(methacrylic acid – grafted – ethylene glycol) (P(MAA-g-EG)) hydrogels modified with poly(butyl acrylate) (PBA) to form IPNs or photopolymerized in the presence of poly(methyl methacrylate) (PMMA) nanoparticles were investigated for their use in the oral delivery of therapeutic agents for cancer treatment. The P(MAA-g-EG) hydrogel provided pH-responsive and hydrophilic properties while PBA or PMMA polymers provided hydrophobic properties. An inulin- doxorubicin conjugate was also synthesized to provide local, direct targeting for the treatment of colon cancer. The pH-responsive behavior of these polymer systems was investigated using equilibrium and dynamic swelling experiments. In gastric conditions (low pH) all materials were in a collapsed state and in intestinal conditions (neutral pH) these material were swollen. The equilibrium swelling ratios decreased with increasing hydrophobic content for both IPNs and compositions of P(MAA-g-EG) containing nanoparticles. The loading efficiencies of doxorubicin, a chemotherapeutic drug, were as high as 56% for IPNs and the IPN structure and hydrophobicity influenced the loading efficiency values. The loading efficiency of doxorubicin using P(MAA-g-EG) containing nanoparticles was as high as 64% and increased with increasing weight percent of PMMA nanoparticles in the P(MAA-g-EG) hydrogel. In gastric conditions (low pH), IPNs released a majority of the encapsulated doxorubicin (up to 70%) as compared to the P(MAA-g-EG) containing nanoparticles (up to 27%). P(MAA-g-EG) containing nanoparticles was used to load and release the inulin-doxorubicin conjugate. Loading efficiency was 54% and release profiles behaved similarly as doxorubicin. Both polymer systems were biocompatible with Caco-2, HT29-MTX, and SW620 cell models over concentration ranging from 1 mg/mL to 5 mg/mL and exposure times lasting from 2 hr to 24 hr. The 75/25 IPN exhibited the highest degree of mucoadhesion and the P(MAA-g-EG)-5.0NP the lowest. Using the same cell lines and cytotoxicity assays, the inulin-doxorubicin conjugate was determined to be more toxic than free doxorubicin at equal doxorubicin concentrations. Doxorobuicin and inulin-doxorubicin conjugate were tested for transport across Caco-2/HT29-MTX cell monolayers with and without the presence of unmodified P(MAA-g-EG) or P(MAA-g-EG)-5.0NP microparticles. The presence of the microparticles did not increase transport across the cell monolayer which is advantageous for local, direct delivery to the colon. / text

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.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

While a number of PEGylated proteins have been studied for injectable applications and reviewers have used this data to speculate possible oral delivery improvements, a detailed investigation of PEGylated insulin for oral delivery and the development of an optimized pH-sensitive carrier for PEGylated insulin conjugates had yet to be accomplished. In order to proceed with oral delivery study, improvements in yield, with respect to previous PEGylation methods were necessary to enable the completion of high throughput drug delivery studies. Subsequently, a reaction scheme for the covalent attachment of PEG to insulin using nitrophenyl carbonate-PEG was developed. It was demonstrated that this reaction occurred at a 1:1 ratio and was site specific at the B29Lys position. A P(MAA-g-EG) hydrogel carrier was developed to optimize loading and release behavior for PEGylated insulin. It was demonstrated that the density and length of polymer grafts affected both loading and release behavior of PEGylated insulin. The best performing grafted polymers had a 3:1 methacrylic acid: ethylene glycol (MAA:EG) ratio and achieved loading efficiencies from 96% to nearly 100%. With respect to release, polymer particles containing fewer, but longer grafts shown to release faster than polymers with shorter grafts with the same MAA:EG ratio. Finally, the effects of PEGylation on intestinal absorption was investigated using an intestinal epithelial model as well as a rat model. It was demonstrated that PEGylated insulin in the presence of P(MAA-g-EG) microparticles did not significantly alter the tight junctions over unmodified insulin. However, the conjugate permeabilities across the membrane were reduced. The pharmacological availability (PA) was then verified by injecting the insulin conjugates subcutaneously in fasted Sprague-Dawley rats. It was determined that PEG 1000 insulin (1KPI) had a PA roughly equivalent to insulin, while it was reduced by 59% for 2KPI and by 81% for 5KPI. The effectiveness of utilizing PEGylated insulin as an oral drug delivery candidate was evaluated with a closed loop intestinal study, in which PEGylated insulin or insulin in solution was delivered directly to the jejunum. It was shown that 1KPI and insulin performed identically; with a pharmacological availability of 0.56%. 2KPI, however improved the pharmacological availability of insulin by 2.8 times. These results demonstrate that PEGylation holds promise for improving the oral delivery of proteins. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Utilization of DNA vaccines in aquaculture has been gaining interest and recent efforts have been focused on methods of delivering DNA vaccines to fish. In the present study, a methodology was sought that could protect DNA vaccines such that they could be orally administered. The main objective of the study was to determine if a DNA vaccine could be effectively compounded into an orally administrable formulation with chitosan or polylactide-co-glycolide (PLG). The immune response of hybrid striped bass (Morone saxatilis x Morone chrysops) following oral delivery of a DNA vaccine containing Mycobacterium marinum Ag85A plasmid in either chitosan or PLG nanoparticle encapsulation was evaluated. Hybrid striped bass were divided into four experimental groups: IM immunization of the DNA vaccine as a positive control, oral delivery of uncomplexed DNA vaccine, oral delivery of chitosan or PLG alone as a negative control, and oral delivery of complexed chitosan or complexed PLG DNA vaccine. Fish were bled at regular intervals and an ELISA was used to evaluate antibody levels in individual fish. While the chitosan /plasmid DNA complex containing the Mycobacterium marinum Ag85A gene failed to produce a significant antibody response, the PLG/plasmid DNA matrix stimulated humoral immune response in the fish. / Master of Science

Ces travaux de thèse ont porté sur le développement de formulations innovantes et nanoparticulaire, destinées à améliorer la prise en charge des patients atteints de leucémie aigüe myéloïdes (LAM). Cette amélioration de la qualité de vie peut passer par le développement d’une formulation orale de décitabine.Trois stratégies de formulations différentes ont été développées : deux formulations de nanocapsules lipides (LNCs) avec encapsulation ou de décitabine, ou d’une prodrogue de décitabine (décitabine(C12)2) . La troisième stratégie a été le développement de particules de type liposomal, dans lesquelles la décitabine a été encapsulée. Après avoir été caractérisée sur des critères physicochimiques, chacune des stratégies basées sur les LNCs a été évaluée par des essais in vitro pour évaluer la perméabilité intestinale de la décitabine lorsqu’elle a été encapsulée. Une des stratégies a permis d’accroitre la perméabilité, in vitro, de la décitabine. L’activité sur la prolifération cellulaire a ensuite été évaluée sur des cellules humaines de LAM. Il a été démontré que l’encapsulation dans les LNCs améliore l’activité de la décitabine et de la décitabine (C12)2. Après l’ensemble de ces essais, en vue d’évaluer le potentiel avantages de ces formulations pour augmenter la demi-vie plasmatique de la décitabine, leurs stabilités dans du plasma humain a été évaluée. La décitabine (C12)2 libre et encapsulée permettent de limiter la dégradation rapide de la décitabine. Finalement, une étude de pharmacocinétique a été mise en place. L’encapsulation de la décitabine, en synthétisant au préalable une prodrogue permet d’augmenter les concentrations maximales atteintes. / The aim of this phD work was to develop nanoparticle formulations to improve patients’quality of life in case of acute myeloid leukemia (AML). These formulations could, for example, allow an oral administration of decitabine. Three different formulations were developed: two were based on lipid nanocapsules (LNCs) with an encapsulation of decitabine or a decitabine prodrug (decitabine(C12)2). The third strategy was aliposomal formulation with a decitabine encapsulation. After being characterized on physico-chemical parameters, in vitro intestinal permeability studies were performed on LNCs strategies. One strategy was able to enhance decitabine permeability. Cell proliferation studies performed on human AMLcell lines showed that encapsulations into LNCs improve decitabine and decitabine(C12)2 activities. In order to evaluate the potential of these formulations to enhance decitabine plasma half-life, their stabilities in human plasma were then assayed. Free decitabine(C12)2 or encapsulated into LNCs has been shown to limit the rapid decitabine degradation. Finally, pharmacokinetic studies were performed. Decitabine encapsulation into LNCs with a previous decitabine prodrug synthesis was able to increase maximal plasma concentrations.

L’objectif général de cette thèse a été d’étudier le potentiel de billes à base de molécules d’α-cyclodextrine et d’huile de soja, pour l’administration orale de principes actifs peu solubles dans l’eau.Nous avons tout d’abord vérifié qu’il était possible d’encapsuler dans les billes des molécules actives (la progestérone et l’indométacine) autres que les rétinoïdes et le diazépam, avec une teneur élevée et un rendement de fabrication satisfaisant. L’étude du comportement des billes nues lyophilisées, en termes de stabilité et de libération dans des milieux digestifs simulés, nous a permis de proposer un mécanisme de libération de la molécule encapsulée qui se déroule en plusieurs étapes: i) hydratation des billes, ii) dissolution de la matrice hydrophile d’α-cyclodextrine, iii) libération de gouttelettes d’huile contenant le principe actif puis de la fraction dissoute dans l’huile par un phénomène de partage, iiii) fragmentation des billes fragilisées et libération totale de l’huile. La présence de sels biliaires dans le milieu, accélère à la fois la libération et la quantité dissoute, en fragilisant les billes et en réduisant la valeur du coefficient de partage du principe actif entre l’huile et le milieu digestif. Nous avons montré in vitro et in vivo qu’il est possible de moduler la libération d’un principe actif à partir d’une même formulation de départ, en jouant sur l’organisation du système (émulsion sèche, billes nues, billes coquées par un nouvel ajout d’α-cyclodextrine sur les billes nues). Les études in vivo chez le rat ont révélé que l’émulsion sèche se comporte comme une forme à libération immédiate, les billes coquées comme une forme à libération prolongée et les billes nues comme une forme à libération intermédiaire. Enfin, la libération du principe actif encapsulé peut également être modulée en modifiant le mode de séchage des billes. Comparativement à la lyophilisation, le séchage à l’étuve modifie les propriétés des billes en augmentant leur résistance dans les milieux digestifs simulés et prolonge la libération de la molécule encapsulée. / The general aim of this thesis was the study of the potential of beads, made of α-cyclodextrin and soybean oil, for the oral delivery of poorly water soluble drugs. We have first verified that it was possible to encapsulate in beads, active molecules (progesterone and indomethacin), other than retinoid and diazepam, with a high drug loading and a satisfying yied. The study of the behaviour of freeze-dried naked beads, in terms of stability and drug release in simulated gastro-intestinal fluids, allowed to propose a mechanism for the release of the encapsulated drug, involving several steps: i) hydration of the freeze-dried beads, ii) dissolution of α-CD hydrophilic matrix, iii) release of oily droplets containing the active drug and then of the fraction of drug dissolved in oil, following a partition phenomenon, iiii) fragmentation of the weakened beads and at last the total release of oil. The presence of bile salts in the medium accelerates both the release and the dissolved amount, by weakening the beads and reducing the partition coefficient value of the active molecule between oil and digestive medium.We have shown in vitro as well as in vivo that it is possible to modulate the release of a model drug from the same initial formulation, according to the degree of organization of the system (dry emulsion, naked beads, coated beads obtained by an additional amount of α-cyclodextrine to the preformed naked beads). In vivo studies in rats have highlighted that dry emulsion behaves as a fast release formulation, the coated beads as a sustained release formulation and the naked beads as an intermediate one. Finally, the release of the encapsulated drug can also be modulated by modifying the drying method of the beads. Compared to freeze-drying, oven-drying modifies the properties of the beads by increasing their resistance in simulated gastro-intestinal fluids and sustaining the release of the encapsulated drug.