Using a Systems Theory approach allows a person to analyze the intertwined elements of the drug development system and the potential influences of the environment. Thomas Hughes's Large Technological Systems (LTS) Theory is one that could be used for this purpose; however, it falls short in its ability to address the complexity of current day regulatory environments. This dissertation provides a critical analysis of Hughes's LTS Theory and his phases of evolution as they apply to the United States (U.S.) system for biologic drug research, development and labeling. It identifies and explains potential flaws with Hughes's LTS Theory and provides suggested improvements. As an alternative approach, this dissertation explores the concept of "techno-regulatory system" where government regulators play an integral part in system innovations and explains why such systems do not always follow Hughes's model. Finally, this dissertation proposes a hybrid version of Hughes's systems approach and uses it to explain the changes that occurred in the drug approval system in response to the push for, opposition, and inclusion of, pediatric research in drug development during the period 1950-2003. / Ph. D.

Despite advancements in the treatment of individuals with Inflammatory Bowel Disease (IBD), many patients will require the need to utilize biological therapies during their disease course. Moreover, some patients with IBD develop disease manifestations outside of the GI (gastrointestinal) tract termed extraintestinal manifestations (EIM). We sought to establish an association between prior EIM exposure and the sequential use of biological therapies in patients with IBD. A retrospective analysis of 555 patients with confirmed IBD and relevant EIM data was performed. EIM exposure was treated as both a dichotomized (ever, never) variable and a categorical (0, 1, 2 or more) variable in our analysis. Crude ratios were established using logistic regression and multinomial regression models. Bivariate analysis was used to test for significant confounding variables and significant confounders were included in the final multivariate regression model. We found female sex (p < 0.001), a disease duration of 13 years or longer (p = 0.001), and an ileocolonic disease location (p = 0.036) to be significantly associated with EIM exposure. We found that a disease duration of 13 years or longer (p = 0.037), diagnosis of Crohn’s Disease (CD) (p < 0.001), corticosteroid use (p < 0.001), and an ileocolonic disease location (p = 0.021) to be significantly associated with use of biologics. Our final adjusted model did not show statistical significance, but did notably indicate that individuals exposed to 2 or more EIM had 1.51 times the odds of progressing to biological therapy (95%CI: 0.67, 3.41; p = 0.32) compared to those patients with no EIM history. As a result, EIM exposure may be an indicator for high-risk IBD patients likely to require biological therapy, especially among particular groups. Our data emphasizes the need for further studies to characterize the association between EIM exposure and specific EIM with the utilization of biologics.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Alison P. Adams / The animal health industry is a growing industry. Owners of pets and other animals want to ensure their animals are healthy. To do this, the animal health industry markets a variety of products from pharmaceutical products, such as antibiotics, to biological products, such as vaccines. These products are developed and marketed after the company provides regulators the necessary information as guided by a set of regulations. Pharmaceutical products follow Title 21 of the Code of Federal Regulations, while biological products follow Title 9 of the Code of Federal Regulations. During the product development process as well as after marketing, regardless of the regulations to follow, each product must go through testing for efficacy, safety, potency, and stability. The regulatory guidelines provide direction to companies on expectations of the testing requirements for each type of product. Different analytical techniques are used to provide the necessary data in support of product development. Discussed in this report, two analytical techniques are well known in the industry, and one is quickly becoming a technique of great value. Mass spectrometry, coupled with liquid chromatography, is an industry standard for testing product potency and purity as well as pharmacokinetics. The enzyme-linked immunosorbent assay (ELISA) is also used to measure potency of products as well as product stability. The newest technique is flow cytometry that characterizes cells within a suspension, most often with the use of cellular biomarkers as targets. By understanding the application of each technique as well as how it relates to regulatory requirements, the industry can provide assurances to regulators that their products are safe and efficacious for the treatment and/or prevention of animal diseases. This report outlines the history, theory, and use of three different analytical techniques currently used for pharmaceutical and biological products in animal health.

Magister Scientiae - MSc (Pharmacy Administration and Policy Regulation) / Technological advancements applied to healthcare may holistically improve the economic burden of prescription medication costs. United States legislative actions requiring utilization of electronic prescribing (e-prescribing) will drive provider utilization to decrease healthcare spending. Federal and state e-prescribe requirements have been met with resistance by the prescribing community, due to claims that the requirements create an economic burden for them. This research intends to demonstrate the long-term economic value of electronic prescribing regulations across the healthcare spectrum.

Le radiomarquage des macro-biomolécules au fluor-18 représente un défi majeur en radiochimie vu leur importance en imagerie moléculaire. Les macro-biomolécules et en particulier les peptides offrent une diversité moléculaire et un ciblage in vivo souvent plus spécifiques et sélectifs que les molécules de plus faibles poids moléculaires. Cependant, les conditions standards de radiomarquage au fluor-18 seraient destructrices pour de tels composés et ne peuvent être utilisées directement. Peu de méthodes directes de radiomarquage existent et présentent certains inconvénients (température encore élevée, activité molaire faible, méthodes peu versatiles…). C’est pourquoi, le radiomarquage par approche prosthétique en deux étapes reste une méthode de choix. Cette stratégie séquentielle implique tout d’abord la préparation d’une molécule radiofluorée, appelée groupe prosthétique, puis sa conjugaison à la macro-biomolécule dans des conditions chimiques biocompatibles. L’objectif de ce travail de thèse a consisté à développer de nouvelles méthodes générales de radiomarquage de macro-biomolécules visant in fine des applications de radiomarquage direct in vivo. Les enjeux principaux ont été la diminution du temps de marquage pour améliorer les procédés de radiosynthèse compte tenu de la demi-vie du fluor-18 (109,8 min), le besoin d’automatisation ainsi que la vitesse et la bioorthogonalité des réactions de conjugaison pour des applications en milieu complexe et dilué. Tout d’abord, l’étude de trois réactions de chimie click, CuAAC, SPAAC et SPSAC, de vitesse et de biocompatibilité croissantes a été considérée. Le développement de groupes prosthétiques spécifiques de chacune d’entre elles ainsi que leur conjugaison sur des composés modèles ont ensuite été étudiés. Par la suite, une étude méthodologique sur la radiofluoration de pyridines substituées a été initiée afin d’obtenir des entités radiomarquables dans des conditions suffisamment douces permettant la « pré-conjuguaison » à la macro-biomolécule avant l’étape de radiofluoration. Enfin, l’utilisation d’un étiquetage enzymatique, le SNAP-tag, a également été explorée et un substrat radiofluoré spécifique synthétisé. Ces différentes approches ont permis d’élargir le panel des méthodes permettant un radiomarquage efficace et biocompatible de macro-biomolécules. / Fluorine-18 radiolabeling of biologics is a challenge in radiochemistry due to their increasing interest in molecular imaging. Biologics, and particularly peptides, offer molecular diversity and often higher specific and selective in vivo targeting compared to low molecular weight molecules. However, fluorine-18-radiolabeling standard conditions could not be used directly because biologics would suffer from these drastic conditions. Only a few direct radiolabeling methods are available and present some drawbacks (high temperature, low molar activity, lack of versatility…). Therefore, a two-steps prosthetic radiolabeling approach remains the method of choice. This sequential strategy involves the preparation of a fluorine-18-labeled molecule, called prosthetic group, which is then conjugated with the macromolecule in biocompatible chemical conditions. The aim of this PhD work was to develop new general methods for the radiolabeling of biologics directed toward in vivo radiolabeling applications. Major issues were time and radiosynthesis processes with regard to the fluorine-18 half life, automatization as well as constant rate and bioorthogonality of conjugation reactions for applications in complex and diluted environment.The first part is devoted to the study of three click chemistry reactions, CuAAC, SPAAC and SPSAC leading to enhanced reaction rates and biocompatibility. Specific prosthetic groups were developed for each reaction and their conjugation was studied with model compounds. Furthermore, a methodological study involving the radiofluorination of substituted pyridines was initiated for the production of entities for mild radiolabeling conditions compatible with a “pre-conjugation” to the biologics before radiofluorination. Finally, an enzymatic labeling approach using the SNAP-tag self-labeling enzyme was explored and a specific radiofluorinated substrate was synthesized. These different approaches allowed an extent of the panel of methods for effective and biocompatible radiolabeling of biologics.

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Biologics are large, complex therapeutic agents generally produced from living organisms. One group of biologics is peptide/protein based. Biological agents offer unique advantages over traditional therapeutics including longer half-lives, higher specificity, greater efficacy, and reduced off-target effects. However, protein/peptide based drugs suffer from both delivery and stability issues. The higher order of protein structures (secondary, tertiary, etc.) derive ~80% of their conformational stability from paltry hydrophobic effects, with net stabilization of 5-15 kcal/mole observed for many proteins. Loss of conformational stability can lead to increased aggregation, precipitation, and degradation; and reduced activity and side effects. To increase stability and improve other properties of the therapeutic agent, additives, referred to as excipients, are included in their formulation. Generally, stabilizing effects from excipients work by imposing enthalpic or entropic penalties on protein/peptide unfolding, increasing the free energy of the denatured state. How excipient stabilizes by what thermodynamic mechanism for a given protein/peptide is not always clear, requiring careful study and optimization for prospective agents. Much effort has gone into understanding excipient protection mechanisms and identifying potential liable regions like amino acid sequence and hydrophobic patches. One area that has received relatively little attention has been the effect of excipients on secondary structure (SS) thermodynamic stabilization/destabilization. SS features are major components of biologic conformation in which deviations, even temporary, can lead to aggregation and precipitation. In this study, an experimental system is proposed to quantify and classify helix stabilization in a model peptide and protein. Thermodynamic stability was evaluated via helix unfolding in the peptide, or protein through use of circular dichromism (CD) and nuclear magnetic resonance (NMR) for model peptide polyL-lysine (PLL) and CD and differential scanning calorimetry (DSC) for model protein bovine serum albumin (BSA). The chosen molecular weight of the PLL polymer, adopts a helical structure, is neutral and a monomeric under tested conditions, making it an ideal model to evaluate excipient effects on helix stability. BSA is largely helical in nature, with most changes and aggregation behavior resulting from loss of helicity, making it a logical extension from the model peptide. Results showed stabilization from mannitol and trehalose being mainly enthalpically driven in both peptide and protein. Enthalpic destabilization was observed for PLL and BSA at low to mid concentrations but stabilizing for PLL and destabilizing for BSA at high concentrations, respectively. Moreover, use of entropy-enthalpy compensation (EEC) plots revealed primary stabilization mechanisms at varying excipient concentrations and types allowing for a classification system to be established under different conditions. Peptide/protein based therapeutics typically exist in a complex milieu of additives designed to enhance stability and performance, or allow novel delivery methods (oral, pulmonary, etc.) not typically available to such agents. Ultimately, this work provides a model for understanding excipient effects on helix stability in a complex system. Further work into other SS, higher order structures, as well as complex formulation systems in the model framework described in this work will help to improve the formulation optimization process.

La polyarthrite rhumatoïde (PR) est le rhumatisme inflammatoire chronique le plus fréquent. Cette maladie est caractérisée par une auto-immunité et une synovite hypertrophique, responsables d'une destruction des articulations périphériques. Les macrophages contribuent aux phénomènes inflammatoires de la PR. Ces cellules peuvent présenter différents états d'activation ou " polarisation ", réversibles, dépendant de leur environnement, notamment cytokinique. Les biothérapies (bDMARDs) ont représenté une avancée majeure dans la prise en charge des manifestations inflammatoires des formes sévères de PR. Toutefois, peu d'études ont évalué si ce bénéfice était lié à une action spécifique sur le macrophage. L'objectif de ce travail de transversal était : (i) d'évaluer in vitro l'effet des principales biothérapies de la PR (anti-TNF : etanercept, adalimumab, certolizumab ; anti-IL- 6R : tocilizumab ; CTLA4-Ig : abatacept) sur la différenciation et l'activation de macrophages dérivés de monocytes issus de patients atteints de PR et de sujets sains, (ii) d'identifier des marqueurs de polarisation du macrophage, corrélés à l'activité de la maladie (DAS28). Parmi les bDMARDs évalués, seuls les anti-TNF ont montré une action sur la polarisation des macrophages. En contexte de différenciation avec M-CSF, les bDMARDs anti-TNF ont induit un biais vers une polarisation non inflammatoire dite alternative. En contexte d'activation inflammatoire, les bDMARDs anti-TNF ont induit une préservation sélective des marqueurs de polarisation liés à l'IL-10 (CD16, CD163, MerTK) et une inhibition des marqueurs inflammatoires (CD40, CD80). Nous avons montré que ce changement phénotypique s'accompagnait : (i) d'un changement fonctionnel concordant avec une polarisation induite par l'IL-10, (ii) d'une inhibition de la production des cytokines de l'inflammation (TNF, IL-6, IL-12), (iii) et d'une majoration de la phagocytose. Nous avons montré que ce mécanisme était dû à une production précoce d'IL-10 et dépendant de STAT3. De plus, nous avons pu montrer que le certolizumab, un anti-TNF, induisait une réponse anti-inflammatoire, impliquant le facteur de transcription NRF2 (nuclear factor erythroid-2-related factor 2), un régulateur central dans la réponse au stress oxydatif. Enfin, nous avons observé que l'expression de CD16, à la surface des macrophages non activés, était corrélée négativement à l'activité de la PR. Ces travaux concourent à montrer l'intérêt du ciblage du macrophage dans la PR et nous ont d'identifier de potentielles cibles théranostiques dans le traitement de la PR par anti-TNF. / Rheumatoid arthritis (RA) is the most frequent chronic inflammatory rheumatism. This disease is characterized by an auto-immunity and a hyperplasic synovitis, both responsible for peripheral joints destruction. Macrophages contribute to inflammatory aspects of RA. This cell type can present various states of activation or "polarization", reversible and dependent on its environment, notably cytokines. Biologics (bDMARDs) represented a revolution in severe RA treatment. However, data regarding their specific action on macrophage are scarce. The aim of our translational work was: (i) to assess the in vitro effect of RA bDMARDs (anti-TNF: etanercept, adalimumab, certolizumab; anti-IL-6R: tocilizumab; CTLA4-Ig: abatacept) on the phenotype of monocytes-derived-macrophages from RA patients and healthy volunteers, during differentiation and activation phases, (ii) to identify polarization markers correlated with disease activity (DAS28). Among bDMARDs, only anti-TNF modulated macrophage polarization. During differentiation, anti-TNF bDMARDs induced a bias toward the so-called alternative non-inflammatory polarization. In inflammatory context, anti-TNF bDMARDs induced a selective preservation of markers associated with IL-10 (CD16, CD163, MerTK) and an inhibition of inflammatory markers (CD40, CD80). We showed that these changes in phenotype were associated with changes in functions consistent with: (i) a polarization induced by IL10, (ii) a decrease in inflammatory cytokines production (TNF, IL-6, IL-12), (iii) and an increase in phagocytosis. We showed that this mechanism was dependant on early IL-10 production and STAT3 signaling. Moreover, we have showed that certolizumab, an anti-TNF agent, induced an anti-inflammatory response, implicating the transcription factor NRF2 (nuclear factor erythroid-2- related factor 2), a central regulator of the response to oxidative stress. We observed that CD16 expression on non-activated macrophages was negatively correlated with RA activity. This work contributes to demonstrate the relevance of macrophage targeting in RA, and enabled us to identify theranostic targets for RA treatment with anti-TNF bDMARDs.

The studies in the present thesis explored the feasibility of using nanocellulose-based filters in virus removal filtration of plasma-derived proteins.   In Paper I, two-step nanofiltration of commercially available human serum albumin (HSA) product, which was diluted to 10 g L-1 by phosphate buffer saline (PBS) and adjusted pH to 7.4, was performed to remove soluble protein aggregates and reduce filter fouling. The two-step filtration of HSA employed nanocellulose-based filters of varying thickness, i.e. 11 μm and 22 μm filters.  The removal of HSA aggregates during filtration through 11 μm pre-filters dramatically improves the flow properties of the 22 μm filter, enabling high protein throughput and high virus clearance. A distribution of pore sizes between 50 nm and 80 nm, which is present in the 11 μm filter and is absent in the 22 μm filter, plays a crucial part in removing the HSA aggregates. With respect to virus filtration, 1 bar constant trans-membrane pressure filtration shows poor removal ability of ΦX174 bacteriophage (28 nm), i.e., log10 reduction value (LRV) ≤ 3.75, while that at 3 bar and 5 bar achieves LRV[MOU1] [LW2]  &gt; 5 model virus clearance and overall rapid filtration. Removal of protein aggregates during bioprocessing of HSA products is key to improving the filtration flux, which makes it possible to apply virus removal filtration for HSA to ensure its virus safety.   In Paper II, nanofiltration of human plasma-derived intravenous immuno-globulin (IVIG) intermediate (11.26 g L-1, pH 4.9) was carried out to demonstrate high product recovery and high model virus clearance. Virus removal filtration of industrial-grade human IVIG was achieved using 33μm filters at both low (60 Lm-2) and high (288 Lm-2) volumetric load. No changes in IVIG structure were detected and high product recovery was recorded. High virus clearance (LRV ≥ 5-6) was achieved for the small-size model viruses (ΦX174 and MS2 bacteriophages) during the load volume of 60 Lm-2. Side-by-side comparisons with commercial virus removal filters suggest that the nanocellulose-based filter paper presents great potential for industrial bioprocessing of plasma-derived IVIG.   In Paper III, process analytical technology (PAT) approach was employed to identify the critical filter parameters, e.g. thickness, basis weight, pore size, and flux, affecting model virus removal efficiency using filters produced by different hot presses.  The quality parameters were analyzed with ANOVA and Shewhart charts. Compared with other studied parameters, the hydraulic flux appears as the most relevant final product quality attribute of the nanocellulose-based filter paper to reflect the virus removal efficiency. In particular, a 15% higher flux may be associated with a 0.5-1.0 log10 reduced virus clearance (p=0.007). The results are highlight the importance of continued systematic studies in quality assurance using statistical process control tools  [MOU1]Define LRV  [LW2]Defined in the line above