Includes bibliographical references (p. 91-95).

Copy of author's previously published works inserted. Bibliography: leaves 183-220. Three antimicrobial peptides, maculatin 1.1, uperin 3.6 and caerin 4.1 have been isolated from the respective skin glands of the Australian amphibians Litoria genimaculata, Uperoleia mjobergii and Litoria caerulea. To gain a deeper insight into their mechanisms of action, three dimensional structural studies have been conducted using circular dichroism, two-dimensional nuclear resonance and computer modelling techniques. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is also discussed.

This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs. Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control. In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.

Copies of author's previously published articles inserted. / Includes bibliographical references. / xv, 210 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A chemical and pharmacological investigation of compounds derived from amphibian skin. Isolates novel amphibian peptides and further investigates the biological activity of some of the peptides discovered. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2000

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.

Le nombre croissant de médicaments protéiques utilisés en thérapeutique a créé des besoins dans le domaine de leur quantification, principalement dans le plasma, un milieu de composition protéique complexe. Le dosage, essentiel aux études pharmacocinétique/pharmacodynamique, ainsi qu’à l’optimisation de ces traitements, est compliqué par la nature protéique de ces médicaments et par les faibles concentrations auxquelles ils sont attendus dans ces milieux complexes. La méthodologie proposée se démarque des méthodes de dosage usuelles par son caractère universel. Elle fait appel à la spectrométrie de masse adaptée à la quantification des protéines grâce à l’utilisation d’un marquage isotopique différentiel des peptides : après enrichissement et protéolyse, l’échantillon à doser est marqué sur les lysines par la version légère d’un réactif de dérivation. En parallèle, les peptides de la protéine médicament pure marqués par la version lourde du réactif, servent d’étalon interne. La possibilité de quantifier la protéine à partir de plusieurs de ses peptides améliore la fiabilité du dosage. Appliquée à l’epoetin beta aux concentrations attendues en thérapeutique (autour de 0,5 femtomole/µL de plasma), la stratégie proposée permet de situer la limite de quantification à environ 50 attomoles d’epoetin beta/µL de plasma avec une méthodologie de spectrométrie de masse nano-LC-ESI-Q-TRAP fonctionnant en mode MRM. Pour étendre l’universalité de cette approche au champ des protéines médicaments pégylées, une seconde molécule a été étudiée. Il s’agit de l’interféron alfa-2b pégylé qui a permis de mettre en place une stratégie d’extraction spécifique du médicament utilisant sa pégylation. / The growing number of therapeutic proteins has created needs in the field of their quantification, mainly in plasma, which is a complex protein environment. Quantitative analysis of these proteins is essential for pharmacokinetics/pharmacodynamics studies, and for the optimization of treatments. However, the nature itself of the analyte and the low concentrations that are expected in plasma complicate the quantitative analysis. The proposed methodology differs from usual methods on its universal applicability. It relies on mass spectrometry adapted to the quantification of proteins by using peptides differential isotope labelling : after enrichment and proteolysis, the therapeutic protein and the plasmatic proteins are labelled on lysine residues by the light reagent. In parallel, peptides of the pure therapeutic protein, labelled by heavy version of reagent, are used as internal standard. The ability to quantify the protein with several of its peptides improves the reliability of the analysis. When applied to epoetin beta at expected therapeutic concentrations (about 0.5 femtomole/µL of plasma), the proposed strategy leads to a quantification limit close to 50 attomoles of epoetin beta/µL plasma, with a nano-LC-ESI-Q-TRAP mass spectrometry methodology operating in MRM. To extend the universal character of this approach to the field of pegylated protein drugs, a second therapeutic protein model has been studied. This model is a pegylated interferon alfa-2b which allowed developing a strategy for specific extraction of the drug relying on its pegylation.

<p>Pancreatic ductal adenocarcinoma is an especially deadly disease having the lowest 5-year survival rate of any major cancer at just 11%. As in many cancers, systemically-delivered chemotherapy forms the backbone of clinical treatment. However, limitations of systemic delivery exacerbated by the unique desmoplastic and avascular microenvironment surrounding the pancreatic tumor cells result in the failed efficacy of current treatments. The high stromal content in the microenvironment, which is especially overabundant in hyaluronic acid, is thought to physically impede drug perfusion into the tissue. Thus, there is clearly a <strong>critical need</strong> to develop novel treatments for pancreatic ductal adenocarcinoma that can overcome these drug delivery barriers. Long-acting injectable implants offer an attractive drug delivery method that can provide <em><strong>sustained</strong></em> drug release directly at the <em><strong>local</strong></em> targeted site, rather than transient, systemic release. Here we use in situ forming implants (ISFIs), which are a low-viscosity solution outside of the body but transition into a solid drug-eluting depot after injection into an aqueous environment. Our <strong>objective</strong> is to develop and characterize an ISFI that can provide sustained release of bioactive hyaluronidase for use as an intratumoral injection to degrade hyaluronic acid in pancreatic tumors. This work was accomplished in four aims. First, a method was developed using diffusion-weighted MRI for noninvasive characterization of the implants. Second, because hyaluronidase is a protein drug, we studied factors affecting protein release from ISFIs, focusing on external factors of the injection site. Third, we showed that basic salt additives can be used to neutralize the acidic environment created by the implants which may improve protein stability. Finally, we formulated an implant to provide sustained release of hyaluronidase and demonstrated retention of its bioactivity both <em>in vitro</em> and <em>ex vivo</em>.</p>